Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.
Humans ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms/pathology* ; Lysine/metabolism* ; NF-kappa B/metabolism* ; TNF Receptor-Associated Factor 6/metabolism* ; Transcription Factor AP-1/metabolism* ; Ubiquitin/metabolism*

OBJECTIVES: To study the genetic polymorphism and population genetic parameters of 16 X-STR loci in Xinjiang Uygur population. METHODS: The Goldeneye^® DNA identification system 17X was used to amplify 16 X-STR loci in 502 unrelated individuals (251 females and 251 males). The amplified products were detected by 3130xl genetic analyzer. Allele frequencies and population genetic parameters were analyzed statistically. The genetic distances between Uygur and other 8 populations were calculated. Multidimensional scaling and phylogenetic tree were constructed based on genetic distance. RESULTS: In the 16 X-STR loci, a total of 67 alleles were detected in 502 Xinjiang Uygur unrelated individuals. The allele frequencies ranged from 0.001 3 to 0.572 4. PIC ranged from 0.568 8 to 0.855 3. The cumulative discrimination power in females and males were 0.999 999 999 999 999 and 0.999 999 999 743 071, respectively. The cumulative mean paternity exclusion chance in trios and in duos were 0.999 999 997 791 859 and 0.999 998 989 000 730, respectively. The genetic distance between Uygur population and Kazakh population was closer, and the genetic distance between Uygur and Han population was farther. CONCLUSIONS The 16 X-STR loci are highly polymorphic and suitable for identification in Uygur population, which can provide a powerful supplement for the study of individual identification, paternity identification and population genetics.
Female ; Humans ; Male ; DNA, Ribosomal ; Ethnicity/genetics* ; Gene Frequency ; Paternity ; Phylogeny ; Polymorphism, Genetic ; Microsatellite Repeats ; Chromosomes, Human, X/genetics*

OBJECTIVES: To investigate the genetic polymorphism of InDel loci in SifalnDel 45plex system in the Han population in Jiangsu Province and the Mongolian population in Inner Mongolia, and to evaluate the effectiveness of the system in forensic medicine. METHODS: SifaInDel 45plex system was used for genotyping in blood samples of 398 unrelated individuals from the above two populations, and allele frequencies and population genetic parameters of the two populations were calculated respectively. Eight intercontinental populations in the gnomAD database were used as reference populations. The genetic distances between the two studied populations and eight reference populations were calculated based on the allele frequencies of 27 autosomal-InDels (A-InDels). The phylogenetic trees and multidimensional scaling (MDS) analysis diagrams were constructed accordingly. RESULTS: Among two studied populations, the 27 A-InDels and 16 X-InDels showed no linkage disequilibrium between each other and the allele frequency distributions were in Hardy-Weinberg equilibrium. The CDP of the 27 A-InDels in two studied populations were all higher than 0.999 999 999 9, and the CPE_trio were all less than 0.999 9. The CDP of the 16 X-InDels in Han in Jiangsu and Mongolian in Inner Mongolia female and male samples were 0.999 997 962, 0.999 998 389, and 0.999 818 940, 0.999 856 063, respectively. The CMEC_trio were all less than 0.999 9. The results of population genetics showed that the Jiangsu Han nationality, Inner Mongolia Mongolian nationality and East Asian population clustered into one branch, showing closer genetic relationship. The other 7 intercontinental populations clustered into another group. And the above 3 populations displayed distant genetic relationships with the other 7 intercontinental populations. CONCLUSIONS The InDels in the SifaInDel 45plex system have good genetic polymorphism in the two studied populations, which can be used for forensic individual identification or as an effective complement for paternity identification, and to distinguish different intercontinental populations.
Humans ; Phylogeny ; Gene Frequency ; Polymorphism, Genetic ; Genetics, Population ; Asian People/genetics* ; China ; INDEL Mutation

Background::Mounting evidence, consistent with our previous study, showed that γ-aminobutyric acid type A receptor (GABAAR) played an indispensable role in airway inflammation and mucus hypersecretion in asthma. Monocyte chemotactic protein-inducing protein 1 (MCPIP1) was a key negative regulator of inflammation. Recent studies showed that inflammation was largely suppressed by enhanced MCPIP1 expression in many inflammatory diseases. However, the role and potential mechanism of MCPIP1 in airway inflammation and mucus hypersecretion in asthma were still not well studied. This study was to explore the role of MCPIP1 in asthmatic airway inflammation and mucus hypersecretion in both mice and BEAS-2B cells, and its potential mechanism.Methods::In vivo, mice were sensitized and challenged by ovalbumin (OVA) to induce asthma. Airway inflammation and mucus secretion were analyzed. In vitro, BEAS-2B cells were chosen. Interleukin (IL)-13 was used to stimulate inflammation and mucus hypersecretion in cells. MCPIP1 Lentiviral vector (LA-MCPIP1) and plasmid-MCPIP1 were used to up-regulate MCPIP1 in lung and cells, respectively. MCP-1, thymic stromal lymphopoietin (TSLP), mucin 5AC (MUC5AC), MCPIP1, and GABAARβ2 expressions were measured in both lung and BEAS-2B cells. Immunofluorescence staining was performed to observe the expression of GABAARβ2 in cells. Results::MCPIP1 was up-regulated by LA-MCPIP1 ( P < 0.001) and plasmid-MCPIP1 ( P < 0.001) in lung and cells, respectively. OVA-induced airway inflammation and mucus hypersecretion, OVA-enhanced MCP-1, TSLP, MUC5AC, and GABAARβ2 expressions, and OVA-reduced MCPIP1 were significantly blunted by LA-MCPIP1 in mice (all P < 0.001). IL-13-enhanced MCP-1, TSLP, MUC5AC, and GABAARβ2 expressions, and IL-13-reduced MCPIP1 were markedly abrogated by plasmid-MCPIP1 in BEAS-2B cells (all P < 0.001). Conclusion::The results of this study suggested that OVA and IL-13-induced airway inflammation and mucus hypersecretion were negatively regulated by MCPIP1 in both lung and BEAS-2B cells, involving GABAAR signaling pathway.

In recent years, micro/nano-scaled separation technique has attracted increasing attention due to its inherent advantages. The porous layer open tubular ( PLOT) capillary column is an important microcolumn form. Comparied to wide-bore PLOT capillary columns ( inner diameter >25 μm ) , the PLOT capillary columns with a narrow inner diameter yield higher separation efficiency and lower reagent consumption. In this paper, the preparation methods for porous layer open tubular capillary columns with narrow inner diameter, less than or equal to 25 μm, are reviewed. Detection techniques combined with mass spectrometry and their applications in liquid chromatography are also disussed.

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5 x 10(8), 1 x 10(8), 1 x 10(7), and 1 x 10(6) tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5 x 10(8) and 1 x 10(8) tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10(8) of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.
Animals ; Antibodies, Protozoan/blood ; Cat Diseases/parasitology ; Cats ; Chickens ; Poultry Diseases/blood/mortality/*parasitology/pathology ; Swine ; Swine Diseases/parasitology ; Toxoplasma/genetics/growth & development/*pathogenicity/physiology ; Toxoplasmosis, Animal/blood/mortality/*parasitology/pathology ; Virulence

Animals ; Cellular Reprogramming ; Dentate Gyrus ; cytology ; Fibroblasts ; cytology ; Mice ; Neural Stem Cells ; cytology ; transplantation ; Swine

OBJECTIVETo investigate the in vitro protective effect of Pinus massoniana bark extracts (PMBE) against cisplatin-induced nephrotoxicity in human embryonic kidney cells (HEK293), and preliminarily study its mechanism.

METHODHuman embryonic kidney cells (HEK293) were cultured in vitro. The MTT assay was adopted to test the effect of PMBE and cisplatin on growth of HEK293 cells, and the protective effect of PMBE on cisplatin-induced nephrotoxicity of HEK293, and then detect the intracellular reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) content, catalase (CAT), superoxide dismutase (SOD) and activity of thioredoxin reductase (TrxR).

RESULTPMBE could promote growth of HEK293 cells at low concentrations, but generate slight nephrotoxicity at high concentration. Cisplatin could inhibit growth of HEK293 cells, increase ROS and MDA content, while reducing SOD, CAT and TrxR. The pre-protective PMBE was added to reduce cisplatin's injury to HEK293 cells, ROS, MDA and GSH content, SOD, CAT and TrxR within certain range.

CONCLUSIONPMBE at specific concentration has the protective effect in cisplatin-induced nephrotoxicity in HEK293 cells. Its mechanism may be related to PMBE's antioxidant activity.

Animals ; Antioxidants ; metabolism ; Cisplatin ; toxicity ; Epithelial Cells ; drug effects ; enzymology ; metabolism ; Glutathione ; metabolism ; Glutathione Peroxidase ; metabolism ; HEK293 Cells ; Humans ; Kidney ; drug effects ; enzymology ; metabolism ; Malondialdehyde ; metabolism ; Mice ; Pinus ; chemistry ; Plant Bark ; chemistry ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Thioredoxin-Disulfide Reductase ; metabolism

Background Q-value is used to express the asphricity of the anterior corneal surface.As a optical surface,the optial morphology of cornea is composed of a series of tangential sections that cut-off through the optical axial.Therefore,tangential section could represent the true optical morphology of the corneal surface,and Q-value calculation by tangential radius can better represent the asphericity.Objective This study was to calculate Q-value of meridian section of the anterior corneal surface by tangential radius of the tangential topography and analyze the corneal asphericity in horizontal interval.Methods Ninety right eyes of 90 myopia subjects aged 16-30years and with mean spherical equivalent of (-5.45 ± 2.75) D received corneal topography examination using Orbscan Ⅱ system.The subjects were assigned to low myopia group,moderate myopia group and high myopia group based on their diopter and 30 eyes for each group.The tangential curvatures on meridian section at a 0.1 mm interval from apex to periphery of the anterior surface were accepted and the Q value of the semimeridian section was calculated by linear regression mathematical formulas of the tangential radius.Mydriatic optometry,intraocular pressure,keratometer and fundus examinations were performed on the subjects.Written informed consent was obtained from each subject before any medical examination.Results The coefficients of determination in all the semimeridians were over 0.5.The average calculated Q-values in the nasal and temporal horizontal interval were -0.32±0.11 and-0.30±0.12,with a significant difference between them (t =2.009,P＜0.05).The vertex radius of curvature was (7.78±0.27)mm and (7.72±0.25)mm respectively in the nasal and temporal horizontal interval,showing a significant difference(t=-1.016,P＞0.05).No significant difference was seen in Q values of both nasal and temporal areas among three myopic groups (nasal:F =0.192,P =0.825 ; temporal:F =0.912,P =0.406).The average Q value of the nasal and temporal principal meridian was-0.33±0.14 and-0.30±0.13 respectively,and the r0 was 7.76±0.30 and 7.74±0.24 respectively.A weak positive correlation was found between r0 and mean Q value of nasal meridian (r=0.320,P＜0.05),but no significant correlation was found between r0 and mean Q-value of temporal meridian (r=0.104,P＞0.05).No significant differenees were seen in the Q values between nasal meridian and nasal zone (t=0.349,P＞0.05) as well as between temporal meridian and temporal zone(t=-0.373,P＞0.05).Conclusions The study analyzes the calculated Q-value of the semimeridian section in borizontal area with myopia by linear regression mathematical formulas of tangential radius on tangential topography.The anterior surface of the cornea is proved to be prolate ellipse in shape in the subjects with myopia.

OBJECTIVETo study neck and shoulder work-related muscle fatigue of female sewing machine operators.

METHODS18 health female sewing machine operators without musculoskeletal disorders work in Beijing garment industry factory as volunteers in participate of this study. The maximal voluntary contraction (MVC) and 20% MVC of bilateral upper trapezium and cervical erectors spinae was tested before sewing operations, then the whole 20 time windows (1 time window = 10 min) sewing machine operations was monitored and the surface electromyography (sEMG) signals simultaneously was recorded after monitoring the 20%MVC was tested. Use amplitude analysis method to reduction recorded EMG signals.

RESULTSDuring work, the median load for the left cervical erector spinae (LCES), right cervical erector spinae (RCES), left upper trapezium (LUT) and right upper trapezium (RUT) respectively was 6.78 ± 1.05, 6.94 ± 1.12, 5.68 ± 2.56 and 6.47 ± 3.22, work load of right is higher than the left; static load analysis indicated the value of RMS(20%MVC) before work was higher than that value after work, the increase of right CES and UT RMS(20%MVC) was more; the largest 20%MVE of bilateral CES occurred at 20th time window, and that of bilateral UT happened at 16th.

CONCLUSIONSThe work load of female sewing machine operators is sustained "static" load, and work load of right neck-shoulder is higher than left, right neck-shoulder muscle is more fatigable and much serious once fatigued.

Adult ; Electromyography ; Female ; Humans ; Muscle Fatigue ; physiology ; Posture ; Shoulder ; physiology ; Textile Industry ; Work ; Young Adult