Objective To establish an indirect enzyme-linked immunosorbent assay(ELISA)method for testing the concentration of a monoclonal antibody target tumor necrosis factor-α(TNF-α)in animal serum.Methods The critical parameters of the method including coating concentration of human TNF-α,source,concentration and stability of HRP-labeled goat anti-human immunoglobulin G(IgG)were investigated.The specificity,accuracy,precision,linearity and Limited of Determination of the method were investigated.Results The critical parameters of the method were confirmed as below:TNF-α was coated at 400 ng·mL-1;HRP labeled goat anti-human IgG antibody was diluted at 1:3.0 ×105;the diluted horseradish peroxidase labeled goat anti-human IgG antibody is well stored at 4 ℃ for 3 days.Meanwhile the method was confirmed to have good specificity,the recovery rate ranged from 84.00％to 106.82％,the coefficient of variation of different antibody concentration levels were no more than 10％;the method had a good linearity and the standard curve was y=(-8.37×103-2.37 × 106)/[1+(x/29.80)106]+2.37 × 106(R2=0.999);the limit of quantification was 1 ng·mL-1,all of which met the requirements.Conclusion A accurate and robust ELISA method was developed to test the concentration of anti-TNF-α monoclonal antibody in serum.

Objective: To analyze the clinical phenotype and screen the genetic mutations of hereditary deafness in three deaf families to clarify their molecular biology etiology. Methods: From January 2019 to January 2020, three deaf children and family members were collected for medical history, physical examination, audiology evaluation, electrocardiogram and cardiac color Doppler ultrasound, temporal bone CT examination, and peripheral blood DNA was obtained for high-throughput sequencing of deafness genes. Sanger sequencing was performed to verify the variant sites among family members. The pathogenicity of the variants was evaluated according to the American College of Medical Genetics and Genomics. Results: The probands in the three families had deafness phenotypes. In family 1, proband had multiple lentigines, special facial features, growth retardation, pectus carinatum, abnormal skin elasticity, cryptorchidism and other manifestations. In family 2, proband had special facial features, growth retardation and abnormal heart, and the proband in family 3 had growth retardation and abnormal electrocardiogram. Genetic testing of three families detected three heterozygous mutations in the PTPN11 gene: c.1391G>C (p.Gly464Ala), c.1510A>G (p.Met504Val), c.1502G>A (p.Arg501Lys). All three sites were missense mutations, and the mutation sites were highly conserved among multiple homologous species. Based on clinical manifestations and genetic test results, proband 1 was diagnosed with multiple lentigines Noonan syndrome, and probands 2 and 3 were diagnosed with Noonan syndrome. Conclusion: Missense mutations in the PTPN11 gene may be the cause of the disease in the three deaf families. This study enriches the clinical phenotype and mutation spectrum of the PTPN11 gene in the Chinese population.
Deafness/genetics* ; Genetic Testing ; Hearing Loss/genetics* ; Humans ; Male ; Mutation ; Phenotype ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics*

Objective:To explore the clinical efficacy of myofascial trigger point electric stimulation based on mirror therapy on phantom limb pain after lower limb amputation. Methods:From May to November, 2020, 50 patients with phantom limb pain after lower limb amputation were randomly divided into control group (n = 25) and experiment group (n = 25). Both groups accepted mirror therapy, while the experiment group received myofascial trigger point electric stimulation before mirror therapy, for four weeks. They were assessed with short-form of McGill Pain Questionnaire (SF-MPQ), Pittsburgh Sleep Quality Index (PSQI), Hamilton Anxiety Scale (HAMA), Timed 'Up & Go' Test (TUGT) and 6-minute walk test (6MWT) before and after treatment. Results:All the indexes improved in both groups after treatment (|t| > 8.210, P < 0.001), and improved more in the experiment group than in the control group (|t| > 5.103, P < 0.001), except the present pain intensity of SF-MPQ. Conclusion:Mirror therapy is effective on phantom limb pain after lower limb amputation in terms of pain, sleep, anxiety and walking, and the effect could be stronger after myofascial trigger point electric stimulation.

BACKGROUND: Zinc finger and BTB domain-containing protein 46 (Zbtb46) is a transcription factor identified in classical dendritic cells, and maintains dendritic cell quiescence in a steady state. Zbtb46 has been reported to be a negative indicator of acute myeloid leukemia (AML). We found that Zbtb46 was expressed at a relatively higher level in hematopoietic stem and progenitor cells (HSPCs) compared to mature cells, and higher in AML cells compared to normal bone marrow (BM) cells. However, the role of Zbtb46 in HSPCs and AML cells remains unclear. Therefore, we sought to elucidate the effect of Zbtb46 in normal hematopoiesis and AML cells. METHODS: We generated Zbtb46 and Zbtb46Mx1-Cre mice. The deletion of Zbtb46 in Zbtb46Mx1-Cre mice was induced by intraperitoneal injection of double-stranded poly (I). poly (C) (poly(I:C)), and referred as Zbtb46 cKO. After confirming the deletion of Zbtb46, the frequency and numbers of HSPCs and mature blood cells were analyzed by flow cytometry. Serial intraperitoneal injection of 5-fluorouracil was administrated to determine the repopulation ability of HSCs from Zbtb46 and Zbtb46 cKO mice. The correlation between Zbtb46 expression and prognosis was analyzed using the data from the Cancer Genome Atlas. To investigate the role of Zbtb46 in AML cells, we knocked down the expression of Zbtb46 in THP-1 cells using lentiviral vectors expressing small hairpin RNAs targeting Zbtb46. Cell proliferation rate was determined by cell count assay. Cell apoptosis and bromodeoxyuridine incorporation were determined by flow cytometry. RESULTS: The percentages and absolute numbers of HSPCs and mature blood cells were comparable in Zbtb46 cKO mice and its Zbtb46 littermates (Zbtb46vs. Zbtb46 cKO, HPC: 801,310 ± 84,282 vs. 907,202 ± 97,403, t = 0.82, P = 0.46; LSK: 86,895 ± 7802 vs. 102,210 ± 5025, t = 1.65, P = 0.17; HSC: 19,753 ± 3116 vs. 17,608 ± 3508, t = 0.46, P = 0.67). The repopulation ability of HSCs from Zbtb46Mx1-Cre mice was similar to those from Zbtb46 control (P = 0.26). Zbtb46 had elevated expression in AML cells compared to total BM cells from normal control. Knockdown of Zbtb46 in THP-1 cells led to a significant increase in cell apoptosis and reduced cell growth and proliferation. CONCLUSION Collectively, our data indicate that Zbtb46 is essential for survival and proliferation of AML cells, but dispensable for normal hematopoiesis.

Objective To investigate the protective effects of Salvianolic acid on myocardial ischemic injury of rats from the aspect of inhibiting inflammatory reaction.Methods Sixty Sprague Dawley (SD) male rats were randomly divided into four groups (n =15per group):sham operation group,model group,experimental-high (36 mg · kg-1) group,experimental-low (18mg · kg-1) group with intraperitoneal injection dosing.Same amount of distilled water was injected to sham operation group and model group,lasting for 4 days.Myocardial ischemia model was established by coronary artery ligation of left anterior descending (LAD) branch after the last administration.Six hours after the operation,the blood of eye canthus was collected to assay the levels of creatine kinase(CK),MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) in serum.The heart was collected 24 h after the operation,stained by hematoxylin eosin (HE)and checked the pathological change of the cardiac muscle tissue,assayed the cardiac troponin (cTnT),tumor necrosis factor α (TNF-α) in serum and myeloperoxidase (MPO) was measured in myocardial tissue homogenate.Results After 6 hours' myocardial ischemia,the serum CK in sham operation group was (529.61 ± 141.93) U · L-1,CK-MB was (708.12 ±385.93) U · L-1,LDH was (330.12 ±158.38) U · L-1 and cTnT was (294.12 ±55.10)pg· mL-1 after 24 hours' myocardial ischemia.The serum CK in model group was (996.42 ± 413.42) U · L-1,CK-MB was (1346.11 ±558.30) U · L-1,LDH was (520.12 ±154.76) U · L-1and cTnT was (513.00 ±69.71)pg · mL-1after 24 hours' myocardial ischemia.The serum CK in experimental-low and experimental-high groups were (499.45 ±159.33),(514.91 ±98.82)U · L-1,CK-MB were (831.42 ±385.11),(592.10 ±206.32)U·L-1,LDH were (462.62 ±229.68),(437.72 ± 175.80)U· L-1,and cTnT were (431.12 ±106.00),(338.80 ± 76.92)pg · mL-1after 24 hours' myocardial ischemia.Compared with the sham operation group,the CK,CK-MB and LDH value in the serum of model group increased significantly (P ＜ 0.05).Compared with the model group,the salvianolic acid significantly both can decrease the CK and CK-MB in serum (P ＜ 0.05).Compared with sham operation group,the cTnT value in the serum of model group increased significantly (P ＜ 0.05).Compared with model group,high dosage of salvianolic acid could significantly decrease the cTnT value (P ＜ 0.05).After 24 hours'myocardial ischemia,the TNF-α in serum of sham operation group was (118.90 ± 17.58) pg · mL-1,the MPO in heart tissue was (32.25 ± 3.75) U · L-1.The TNF-α in serum of model group was (156.00 ± 28.24) pg · mL-1,the MPO in heart tissue was (104.83 ±22.87) U · L-1.The TNF-α in serum of experimental-low and experimental-high groups were (136.05 ± 34.00),(125.06 ± 30.13) pg · mL-1,the MPO in heart tissue were (91.70 ±21.57),(68.00 ± 18.47) U · L-1.Compared with sham operation group,the TNF-α values in the serum and the MPO value in model group increased significantly (P ＜ 0.05).Compared with model group,these values of experimental-high group decreased with significantly (all P ＜ 0.05).Conclusion Salvianolic acid showed the protection of myocardial tissue,which also could significantly reduce the myocardial cell injury and inflammatory cell infiltration of cardiac myocytes,which could be related with the decreased levels of TNF-α and MPO.

BACKGROUNDCollagen VI-related myopathies are autosomal dominant and recessive hereditary myopathies, mainly including Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM). Muscle magnetic resonance imaging (MRI) has been widely used to diagnosis muscular disorders. The purpose of this study was to evaluate the diagnostic value of thigh muscles MRI for collagen VI-related myopathies.

METHODSEleven patients with collagen VI gene mutation-related myopathies were enrolled in this study. MRI of the thigh muscles was performed in all patients with collagen VI gene mutation-related myopathies and in 361 patients with other neuromuscular disorders (disease controls). T1-weighted images were used to assess fatty infiltration of the muscles using a modified Mercuri's scale. We assessed the sensitivity and specificity of the MRI features of collagen VI-related myopathies. The relationship between fatty infiltration of muscles and specific collagen VI gene mutations was also investigated.

RESULTSEleven patients with collagen VI gene mutation-related myopathies included six UCMD patients and five BM patients. There was no significant difference between UCMD and BM patients in the fatty infiltration of each thigh muscle except sartorius (P = 0.033); therefore, we combined the UCMD and BM data. Mean fatty infiltration scores were 3.1 and 3.0 in adductor magnus and gluteus maximus, while the scores were 1.3, 1.3, and 1.5 in gracilis, adductor longus, and sartorius, respectively. A "target" sign in rectus femoris (RF) was present in seven cases, and a "sandwich" sign in vastus lateralis (VL) was present in ten cases. The "target" and "sandwich" signs had sensitivities of 63.6% and 90.9% and specificities of 97.3% and 96.9% for the diagnosis of collagen VI-related myopathies, respectively. Fatty infiltration scores were 2.0-3.0 in seven patients with mutations in the triple-helical domain, and 1.0-1.5 in three of four patients with mutations in the N- or C-domain of the collagen VI genes.

CONCLUSIONSThe "target" sign in RF and "sandwich" sign in VL are common MRI features and are useful for the diagnosis of collagen VI-related myopathies. The severity of fatty infiltration of muscles may have a relationship with the mutation location of collagen VI gene.

Adolescent ; Adult ; Child ; Child, Preschool ; Collagen Type VI ; genetics ; metabolism ; Female ; Humans ; Infant ; Infant, Newborn ; Magnetic Resonance Imaging ; Male ; Muscle, Skeletal ; pathology ; Muscular Diseases ; genetics ; metabolism ; pathology ; Mutation ; genetics ; Sensitivity and Specificity ; Thigh ; pathology ; Young Adult

Acrylamide ; toxicity ; Animals ; Behavior, Animal ; drug effects ; Blotting, Western ; Cytoskeletal Proteins ; blood ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Gait Ataxia ; blood ; chemically induced ; Male ; Motor Activity ; drug effects ; Neurotoxicity Syndromes ; blood ; etiology ; Rats ; Rats, Wistar

BACKGROUNDAcute kidney injury (AKI) has been recognized as a major healthcare problem affecting millions of patients worldwide. However, epidemiologic data concerning AKI in China are still lacking. The objectives of this study were to characterize AKI defined by RIFLE criteria, assess the association with hospital mortality, and evaluate the impact of AKI in the context of other risk factors.

METHODSThis prospective multicenter observational study enrolled 3,063 consecutive patients from 1 July 2009 to 31 August 2009 in 22 ICUs across mainland China. We excluded patients who were admitted for less than 24 hours (n = 1623), younger than 18 years (n = 127), receiving chronic hemodialysis (n = 29), receiving renal transplantation (n = 1) and unknown reasons (n = 28). There were 1255 patients in the final analysis. AKI was diagnosed and classified according to RIFLE criteria.

RESULTSThere were 396 patients (31.6%) who had AKI, with RIFLE maximum class R, I, and F in 126 (10.0%), 91 (7.3%), and 179 (14.3%) patients, respectively. Renal function deteriorated in 206 patients (16.4%). In comparison with non AKI patients, patients in the risk class on ICU admission were more likely to progress to the injury class (odds ratio (OR) 3.564, 95% confidence interval (CI) 1.706 - 7.443, P = 0.001], while patients in the risk class (OR 5.215, 95% CI 2.798-9.719, P < 0.001) and injury class (OR 13.316, 95% CI 7.507-23.622, P < 0.001) had a significantly higher probability of deteriorating into failure class. The adjusted hazard ratios for 90-day mortality were 1.884 for the risk group, 3.401 for the injury group, and 5.306 for the failure group.

CONCLUSIONSThe prevalence of AKI was high among critically ill patients in Chinese ICUs. In comparison with non-AKI patients, patients with RIFLE class R or class I on ICU admission were more susceptibility to progression to class I or class F. The RIFLE criteria were robust and correlated well with clinical deterioration and mortality.

Acute Kidney Injury ; epidemiology ; etiology ; pathology ; Adult ; Aged ; China ; epidemiology ; Female ; Humans ; Intensive Care Units ; statistics & numerical data ; Male ; Middle Aged ; Prospective Studies ; Risk Factors

OBJECTIVETo establish a bcr-abl(+) cell line resistance to nilotinib, and to investigate the possible mechanisms of resistance.

METHODSK562 cells were treated with gradually increasing concentrations of nilotinib to generate resistance cell line K562-RN. The folder of drug-resistance was evaluated by MTT assay. Cells apoptosis rate was detected by flow cytometry, the mRNA level of bcr-abl fusion gene by FISH, and the expression of apoptosis relative gene mRNA and protein (such as bcr-abl, HO-1, mdr1, Bcl-2 and caspase-3) by RQ-PCR and western blot.

RESULTSThe resistant cell line K562-RN was successfully established, with 2.01 fold resistant to nilotinib compared with K562 cell line \[the IC(50) value of nilotinib to K562 and K562-RN were (12.320 ± 1.720) µmol/L and (24.742 ± 2.310) µmol/L, respectively\]. It also had the cross resistance to adriamycin, homoharringtonine, etoposide and imatinib. Treated with different concentrations of nilotinib, cell apoptosis rate of K562-RN was significantly lower than that of K562 cells. The rate of bcr-abl gene positive cells was 92% in K562-RN by FISH assay. The mRNA and protein levels of bcr-abl, HO-1 and mdr1 expression up-regulated in K562-RN cells, while those of caspase-3 expression down-regulated, being significantly statistical difference when compared with K562 cells (P < 0.05).

CONCLUSIONHuman leukemic cell line resistance to nilotinib, K562-RN is established successfully by gradually increasing concentrations of drug. The mechanisms of resistance in K562-RN is probably associated with increasing expression of bcr-abl, HO-1, mdr1 and decreasing expression of caspase-3 mRNA and protein levels.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Caspase 3 ; metabolism ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl ; metabolism ; Gene Expression Regulation, Leukemic ; Heme Oxygenase-1 ; metabolism ; Humans ; K562 Cells ; drug effects ; Pyrimidines ; pharmacology