Objective To establish the high-throughput screening fluorescence polarization assay for HSP90 inhibitor.Methods E.coli strain BL21 ( DE3) competent cells were transformed with pET24α( +)-HSP90αplasmid.The cell lysate supernatant was induced to product the soluble protein and purified with Ni-NTA agarose.Western blot analysis was used to identify whether the purified protein is HSP90α.The fluorescence polarization assay for screening HSP90 inhibitors was established and optimized using varying concentrations of recombinant HSP90 protein and molecular probe VER00051001.Meanwhile, the binding activity of GA and NVP-AUY922 for HSP90αwas measured by fluorescence polarization assay.Results HSP90αwas induced expression and purified successfully.The fluorescence polarization assay was performed using 80 nM probe VER00051001 and 2.01μg/mL HSP90α, with the Z factor of 0.83.GA and NVP-AUY922 competed with the probes VER00051001 for binding sites of HSP90, with IC50 of 55 nM and 13 nM, respectively.Conclusion A reliable model was established using fluorescence polarization assay for screening HSP90 inhibitors.

Objective To investigate the effect of different stimulants on the LAG3 expression and function of spleen lymphocytes in mice. Methods The spleen lymphocytes from mice were isolated by density centrifugation.The LAG3 expressions in T cell subsets after exposure to conA, PMA, PHA or anti-CD3/28 antibodies for 24 h or 72 h were analyzed by Flow cytometry.The IFN-γsecretions of conditional medium were detected by ELISA kit.The proliferation of lymphocytes was examined by MTT analysis.Results Treatment with conA for 24 h or 72 h dose-dependently increased LAG3 +CD3 +and LAG3 +CD4 +CD3 +T cell percentages.Similarly, an exposure of anti-CD3/28 antibodies for 72 h significantly increased LAG3 +CD3 +and LAG3 +CD4 +CD3 + T cell percentages.Meanwhile, conA and anti-CD3/28 antibodies increased the IFN-γsecretion of lymphocytes in a time-dependent manner.Furthermore, Treatment with conA, PMA, PHA or anti-CD3/28 antibodies for 72 h could enhance the proliferation of lymphocyte. Conclusion conA and anti-CD3/28 antibodies are effective activators of T cells, and both of them could promote the expression of LAG3 and IFN-γsecretion of lymphocytes.

BACKGROUND:Previous studies have demonstrated that icariin has important roles in promoting bone formation and inhibiting bone resorption,but its effects on osteoporosis-mediated bone pain have not been reported. OBJECTIVE:To investigate the possible mechanism of icariin alleviating bone pain in postmenopausal senile osteoporosis. METHODS:(1)Animal experiment:200 C57BL/6 mice were randomly divided into 4 groups:sham group(n=50),model group(n=50),icariin treatment group(n=50),and carbonic anhydrase Ⅱ inhibitor(Brinzolamide)treatment group(n=50).Ovariectomy was performed on C57BL/6 mice to establish a postmenopausal osteoporosis model in all groups except the sham group.The icariin group was given icariin on the second day after modeling,and pain behavior tests(Von Frey,Hot Plate,and Tail Flick tests)were performed every 2 weeks for 20 weeks.After sampling,bone mass was detected by microCT,osteoclast activity was detected by hematoxylin-eosin and tartrate-resistant acid phosphatase staining,and neuronal morphology and related ion channel expression were detected by tissue immunofluorescence staining.(2)Cell experiment:Osteoclast precursor cells derived from mouse bone marrow were extracted and induced to differentiate into osteoclasts using the RANKL/M-CSF system in vitro and supplemented with icariin of different concentrations(1 and 10 μmol/L).Tartrate-resistant acid phosphatase staining was used to detect osteoclast differentiation,ghost pen cyclic peptide staining was used to detect osteoclast actin ring,bone plate absorption assay was used to detect osteoclast osteophagy function,pH value of the system was detected by pH meter,and expression of osteoclast differentiation-related proteins was detected by western blot.In addition,mouse dorsal root ganglion-derived nerve cells were extracted and treated with icariin.Cell counting kit-8 was used to detect neuronal activity and CGRP staining was used to detect neuronal morphology. RESULTS AND CONCLUSION:Compared with the model group,the icariin treatment group had higher bone mineral density,fewer tartrate-resistant acid phosphatase-positive osteoclasts in bone tissue,decreased neuronal activity,and decreased neuronal transient receptor potential vanilloid subtype 1 channel and carbonic anhydrase Ⅱ expression.Behavioral results showed that the icariin treatment group was less sensitive to pain than the model group.Icariin inhibited osteoclast differentiation and bone phagocytosis in vitro.Icariin enhanced the activity of dorsal root ganglion neurons and inhibited the expression of calcitonin gene-related peptide and transient receptor potential vanilloid subtype 1 channels in dorsal root ganglia in a relatively non-toxic pH range.To conclude,icariin alleviates bone pain caused by postmenopausal osteoporosis by regulating the acidic microenvironment through its effect on osteoclasts.

BACKGROUND:Recombinant bovine basic fibroblast growth factor is a manifold effect cytokine which can promote angiogenesis, wound healing, tissue repair and bone regeneration. Recombinant bovine basic fibroblast growth factor with good histocompatibility is easy to operate and has been widely used in oral and maxil ary surgery.
OBJECTIVE:To evaluate the effect of recombinant bovine basic fibroblast growth factor against dry socket syndrome after tooth extraction.
METHODS:A total of 160 patients who had been extracted mandibular third molar were selected and randomly divided into two groups. In the experimental group, recombinant bovine basic fibroblast growth factor was put into the sockets after mandibular third molars were extracted, while in the control group, we let the wounds to be healed natural y without any materials. The incidence of dry socket syndrome was observed and compared between two groups at 3 days, 5 days and 1 week after tooth extraction.
RESULTS AND CONCLUSION:One patient had dry socket after operation in the experimental group, and the incidence was 1.25%. In the control group, 10 patients suffered from dry socket, and the incidence was 12.5%. There was a significant difference in the incidence of dry socket between the two groups (P<0.01). There was visible granulation tissue within the tooth socket after tooth extraction in the experimental group, and extraction sockets narrowed and were fil ed with granulation tissues, which was 1-2 days earlier than the control group. No al ergies, tissue hyperplasia and other local and systemic reactions occurred in patients receiving implantation of recombinant bovine basic fibroblast growth factor gel. These findings indicate that local implantation of recombinant bovine basic fibroblast growth factor gel after mandibular tooth extractions can speed up the healing of dental extraction wounds.

Objective:To characterize the injury to the ipsilateral ankle joint after low energy spiral fracture of the distal tibia.Methods:A retrospective study was conducted to analyze the 80 patients with distal tibial spiral fracture who had been treated at Department of Traumatic Orthopedics, Affiliated Hospital of Jining Medical University from March 2010 to March 2021. There were 61 males and 19 females with an age of (43.5±12.5) years. Their mean follow-up time was 67.0 (38.5, 90.0) months. All patients underwent preoperative X-ray examination, 64 ones preoperative CT examination, and 30 ones preoperative MRI examination. Of the 80 patients, 3 received conservative treatment with plaster external fixation, 3 closed reduction and intramedullary nail fixation, and 74 plate fixation. Statistically analyzed were incidence of posterior malleolus fracture, characteristics of posterior malleolus fracture lines, normal matching rate of the ipsilateral ankle joint, positive rate of intraoperative Cotton test or stress external rotation test of ipsilateral ankle joint, positive rates of passive pronation-external rotation and supination-external rotation stress tests during follow-up, incidences of short-term ankle pain (≤2 years) and medium-long term pain (>2 years) after operation, injuries to the anterior inferior tibiofibular ligament, the deep medial malleolus deltoid ligament and the posterior malleolus, and incidence of ankle injury.Results:The diagnostic rate for posterior malleolus fracture was 16.3% (13/80) on X-ray film, 60.9% (39/64) on CT and 76.7% (23/30) on MRI. 74.5% (35/47) of the posterior malleolus fracture lines opened on the lateral side. The normal matching rate of the ipsilateral ankle joint was 96.3% (77/80). The positive rates of intraoperative Cotton test and stress external rotation test were 34.8% (8/23) and 7.1% (1/14), respectively. The positive rates of passive pronation-external rotation and supination-external rotation stress tests during follow-up were 46.2% (12/26) and 34.6% (9/26). The incidences of postoperative short term (≤2 years) and medium-long term (>2 years) ankle pain were 37.7% (29/77) and 20.8% (16/77). MRI examination showed that the rates of injury to the anterior inferior tibiofibular ligament, deep medial malleolus deltoid ligament and posterior malleolus were 80.0% (24/30), 80.0% (24/30) and 76.7% (23/30). The incidence of ankle injury was 88.8% (71/80).Conclusions:It is highly probable that spiral fracture of the distal tibia is complicated with ipsilateral ankle injury. The medial malleolus, lateral malleolus, and posterior malleolus are prone to the following hidden injuries while the ankle joint is normally matched in the most cases: injury to the deep deltoid ligament in different degrees, rupture of the inferior tibiofibular anterior ligament and posterior malleolus fracture. Therefore, the ankle injury is likely to be missed in diagnosis. The secondary torsion injuries to the pronation-external rotation and supination-external rotation at the leg are likely to cause ipsilateral ankle injury.

Epigenetic modifications, including DNA methylation, histone modifications, RNA epigenetics and nucleosome remodeling, are important for gene transcription, but such modifications do not change the coding sequence of the gene. Although these events are heritable, they are potentially reversible, thus opening up opportunities for the therapy of cancer. So epigenetic modifications have been thrusted into the forefront of new drug discoveries. Current knowledge suggests that the agents that intervene epigenetics by "turning back on" silenced genes may represent a significant advancement in treating many forms of cancer. In this review, we summarized the research progress of epigenetic gene regulation and the proteins which could read epigenetic codes. And the relationship between epigenetics and cancer was discussed comprehensively.
DNA Methylation ; Epigenesis, Genetic ; genetics ; Gene Expression Regulation ; genetics ; Histones ; metabolism ; Humans ; Neoplasms ; genetics ; therapy

Studies have shown that the clinical manifestation of patients with neuropsychiatric disorders might be related to the abnormal connectivity of brain functions. Psychogenic non-epileptic seizures (PNES) are different from the conventional epileptic seizures due to the lack of the expected electroencephalographically epileptic changes in central nervous system, but are related to the presence of significant psychological factors. Diagnosis of PNES remains challenging. We found in the present work that the connectivity between the frontal and parieto-occipital in PNES was weaker than that of the controls by using network analysis based on electroencephalogram (EEG) signals. In addition, PNES were recognized by using the network properties as linear discriminant nalysis (LDA) input and classification accuracy was 85%. This study may provide a feasible tool for clinical diagnosis of PNES.
Brain ; physiopathology ; Electroencephalography ; Epilepsy ; Humans ; Seizures ; diagnosis

OBJECTIVETo investigative the influencing factors of metastatic lymph node staging (N staging), and the effect of number of lymph node dissection on the prognosis in advanced gastric carcinoma.

METHODSClinicopathological data of 395 advanced gastric cancer patients undergoing radical operation in Harbin Medical University Cancer Hospital in 2011 were retrospectively analyzed. Logistic regression was used to investigate the influencing factors. Cox model was used to evaluate the prognostic factors. Association of survival with the number of lymph node dissection in different N stage was further examined.

RESULTSLymph node metastasis was found in 307 patients(77.7%), including 88 of N0, 86 of N1, 111 of N2 and 110 of N3. Overall 3-year survival rate was 65.3%. Borrmann classification (χ(2)=32.045, P=0.000), histopathological type (χ(2)=5.595, P=0.018), depth of invasion (χ(2)=27.227, P=0.000) and the number of lymph node dissection (χ(2)=12.337, P=0.000) were influencing factors of metastatic lymph node. Tumor location(OR=2.86, 95% CI: 1.80~4.53, P=0.000), depth of invasion (OR=1.44, 95% CI: 1.12~1.85, P=0.004) and the number of metastatic lymph node (OR=1.58, 95% CI: 1.33~1.87, P=0.000) were independent prognostic factors(all P < 0.05), and number of lymph node dissection was not associated with prognosis (P > 0.05). Subgroup analysis revealed overall survival rate of stage N3 patients with ≥ 40 of lymph node dissection was obviously higher as compared to those with < 40, while such difference was not found in N0, N1, N2 groups (all P > 0.05).

CONCLUSIONSLymph node staging of advanced gastric cancer is associated with Borrmann classification, histopathological type, depth of invasion and number of lymph node dissection. Dissection of 20 lymph nodes is suitable for stage N0, N1 and N2, while ≥ 40 lymph nodes should be dissected in stage N3.

OBJECTIVETo investigate the differences of clinicopathological features and prognosis in patients with advanced gastric cancer in different locations after radical gastrectomy, and to provide optimized therapies.

METHODSClinicopathological data of 421 patients with advanced gastric cancer undergoing radical gastrectomy in our department from January to December 2011 were analyzed retrospectively. Patients were divided into 3 groups according to cancer locations, including 48 cases of upper gastric cancer, 74 cases of middle gastric cancer and 299 cases of lower gastric cancer. Clinicopathological features and prognosis were compared among groups.

RESULTSThere were significant differences among 3 groups in lesion size(P=0.001), subtypes(P=0.033), pT classification(P=0.010), TNM staging(P=0.019) and lymph node metastasis (P=0.000). Ratio of lesion size >5 cm, and T4, N3, stage III( patients was significantly higher in upper gastric cancer group as compared to middle and lower group (all P<0.05). The survival curves showed that the general prognosis of upper gastric cancer group was worse than that of middle and lower gastric cancer group (P<0.05), while no significant difference was found between middle and lower gastric cancer group (P=0.027). Multivariate analysis revealed that depth of invasion (P=0.034, HR=1.918) was independent prognostic factor of advanced upper gastric cancer, lymph node metastasis (P=0.022, HR=1.406) was independent prognostic factor of advanced middle gastric cancer, and depth of invasion (P=0.022, HR=1.359) and lymph node metastasis (P=0.000, HR=1.519) were independent prognostic factors of advanced lower gastric cancer.

CONCLUSIONAs compared to advanced middle and lower gastric cancer, advanced upper gastric cancer possesses bigger cancer lesion, deeper depth of invasion, easier metastasis of lymph nodes, later TNM staging, and worse prognosis.

KDM5B is a histone H3K4me2/3 demethylase. The PHD1 domain of KDM5B is critical for demethylation, but the mechanism underlying the action of this domain is unclear. In this paper, we observed that PHD1KDM5B interacts with unmethylated H3K4me0. Our NMR structure of PHD1KDM5B in complex with H3K4me0 revealed that the binding mode is slightly different from that of other reported PHD fingers. The disruption of this interaction by double mutations on the residues in the interface (L325A/D328A) decreases the H3K4me2/3 demethylation activity of KDM5B in cells by approximately 50% and increases the transcriptional repression of tumor suppressor genes by approximately twofold. These findings imply that PHD1KDM5B may help maintain KDM5B at target genes to mediate the demethylation activities of KDM5B.
Binding Sites ; genetics ; Crystallography, X-Ray ; Gene Expression Regulation ; HEK293 Cells ; Histones ; chemistry ; metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases ; chemistry ; genetics ; metabolism ; Lysine ; chemistry ; metabolism ; Magnetic Resonance Spectroscopy ; Methylation ; Microscopy, Fluorescence ; Models, Molecular ; Mutation ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Repressor Proteins ; chemistry ; genetics ; metabolism