Objective To investigate the calcium transient of CA1 pyramidal neurons induced by potassium blocker 4-aminopyridine (4-AP) in acute hippocampal slices to explore the relation between potassium channel function and calcium transient, and their mechanism. Methods Fluorescent probe was employed to mark the hippocampai neurons in acute brain slices of rats; confocal microscopy was used to perform calcium imaging to observe the influences of different concentrations of 4-AP and perfusate with/without calcium on calcium transient of CA1 pyramidal neurons. Results The response of [Ca2+]I to lower concentration of 4-AP (<15 mmol/L) was in a dose-dependent manner (r2=0.910, P=0.000); the higher the concentration of 4-AP (20-80 mmol/L), the lower the peak level of calcium transient. The latency and amplitude of calcium transient induced by 4-AP were obviously reduced when the extracellular condition was switched to an absence of calcium, which was significantly different as compared with that with calcium (P<0.05). Conclusion Blockade of potassium channels with 4-AP can increase [Ca2+]I in the hippocampal pyramidal neurons of acute slices. The increase of [Ca2+]1 to 4-AP could be ascribe to calcium release from intracellular stores and calcium influx from extracellular matrix.

OBJECTIVETo construct a cell model of 4.1R gene knockout in murine macrophage cell line RAW264.7 using CRISPR/Cas9 technique.

METHODSThree high?grade small?guide RNAs (sgRNAs) that could specifically identify 4.1R gene were synthesized and inserted into lentiCRISPRv2 plasmid. RAW264.7 cells were infected with sgRNA?Cas9 lentivirus from 293T cells transfected with the recombinant sgRNA?lentiCRISPRv2 plasmid, and the positive cells were screened using puromycin and the monoclonal cells were obtained. The expression of 4.1R protein in the monoclonal cells was measured by Western blotting, and the mutation site was confirmed by sequence analysis. Result A 4.1R gene knockout RAW264.7 cell line was obtained, which showed a 19?bp deletion mutation in the 4.1R gene sequence and obviously enhanced proliferation.

CONCLUSIONWe successfully constructed a 4.1R gene knockout macrophage cell line using CRISPR/Cas9 technique, which may facilitate further investigation of the function of 4.1R in macrophages.

Paracetamol was used as a model drug in this study to investigate the synergetic effects of lipid coating and beta-cyclodextrin (beta-CD) inclusion for masking the bitter taste of poorly soluble drugs. To control the concentration as low as possible of the free drug which produced a bitter taste, a kinetic model was established to calculate the drug distribution theoretically among the free drug in medium, lipid coated particles and molecular inclusion on the basis of the preparation and characterization of the lipid microspheres, so as to select the proper amount of beta-CD. Finally, the synergetic drug delivery systems were prepared and characterized by 1H nuclear magnetic resonance (1H NMR), molecular simulation and the electronic tongue. As a result, the drug release rate constant (k) of the lipid microspheres coated with octadecanol was determined as 0.001 270 s(-1). Then, the synergetic drug delivery systems were prepared with the ratio of 6.74 : 1 (w/w) for beta-CD and paracetamol. The chemical shift values for the fingerprint peaks of paracetamol all increased and hydrogen bonds were formed between the oxygen on the phenolic hydroxyl group, the nitrogen on the imino in paracetamol and the hydrogens on the hydroxyl groups in beta-CD. The results tested by the electronic tongue indicated that the paracetamol, lipid microspheres, beta-CD inclusion and their mixture showed different taste characteristics, with the bitterness order of the synergetic drug delivery systems approximately lipid microspheres < beta-CD inclusion < paracetamol, which confirmed the synergetic taste masking effects of lipid coating and beta-CD molecular inclusion. In summary, the synergetic taste masking was jointly achieved through the retard of the drug release by the lipid coating and the inclusion of the free paracetamol by beta-CD through hydrogen bonds.
Acetaminophen ; administration & dosage ; chemistry ; Administration, Oral ; Drug Delivery Systems ; Electrical Equipment and Supplies ; Electrochemical Techniques ; instrumentation ; methods ; Hydrogen Bonding ; Kinetics ; Lipids ; chemistry ; Microspheres ; Solubility ; Taste ; drug effects ; beta-Cyclodextrins ; chemistry

OBJECTIVETo investigate the plasma level of extracellular histones in patients with silicosis, and to explore the role of extracellular histones in the pathogenesis of pulmonary fibrosis in silicosis.

METHODSSixty-two patients with silicosis were enrolled as the silicosis group, consisting of 23 patients with stage I silicosis, 25 with stage II silicosis, and 14 with stage III silicosis; sixty workers who had a history of occupational exposure to silica dust for more than 2 years and had not been diagnosed with silicosis were enrolled as the silica dust exposure group; sixty-five healthy workers without a history of occupational exposure to dust were enrolled as healthy controls. Enzyme-linked immunosorbent assay was applied to measure the plasma levels of plasma extracellular histone (H4) and transforming growth factor-β(TGF-β).

RESULTSCompared with healthy controls [(0.82±0.67) μg/ml], the silica dust exposure group[(4.14±2.85) μg/ml] and silicosis group[(9.50±5.04) μg/ml] had significant increases in plasma level of H4 (P<0.01). The plasma level of H4 was significantly correlated with the stage of silicosis(r=0.8955, P=0.0388). The silicosis group had a significantly higher plasma level of TGF-β than the silica dust exposure group and healthy controls(P <0.05). In the patients with silicosis, the plasma level of H4 was significantly correlated with that of TGF-β(r=0.5375, P<0.01).

CONCLUSIONThe plasma level of extracellular histones increases significantly in the pathogenesis of silicosis, and extracellular histones may play an important role in the progression of fibrosis in silicosis.

Case-Control Studies ; Disease Progression ; Dust ; Histones ; blood ; Humans ; Occupational Exposure ; Silicon Dioxide ; Silicosis ; blood ; pathology ; Transforming Growth Factor beta ; blood

The complete nucleotide sequence of a cDNA clone encoding weever ribosomal protein L8, WeevL8, is described here. The WeevL8 cDNA has 848 nucleotides in full length and encodes a 257 amino-acid protein with a calculated molecular mass of 28.02 kDa. Two conserved domains have been identified in WEEVL8. It was concluded fromsequence alignment that WeevL8 gene was quite conservative, consistent with its role as a house-keeping gene. A phylogenetic analysis made by L8 protein showed the similar phylogenetic relationship to that with 18s rDNA. The high similarity supports the notion that ribosomal protein L8 can also be used as phylogenetic criterion.

Aim To screen the differential microRNA (miRNA) expression profiles of different metastatic po-tential liver cancer cell lines,and predict miRNAs-reg-ulated target genes and their functions. Methods To-tal RNA was extracted and the miRNA expression pro-files were obtained by miRNA microarray chip hybrid-ization. The miRNAs whose expression had significant difference were selected by analyzing the miRNA difference expression profiles of the two different meta-static potential liver cancer cell lines, namely MHCC-97H(high-metastasis) and Hep3B(non-metastasis), which were compared with normal hepatocytes L02 re-spectively. Moreover, we analyzed the miRNA differ-ential expression profile between liver cancer cell lines MHCC-97H and Hep3B. The miRNAs were verified by qPCR and target genes were predicted by four softwares (TargetScan, miRanda, miRWalk, miRDB). To un-derstand the biological functions of predicted target genes, bioinformatics analysis was performed. Results The miRNA microarray results showed that the ex-pression of miR-192-5p and miR-215-5p significantly increased in liver cancer cell lines (MHCC-97H, Hep3B) when compared with normal hepatocytes L02, while miR-130a-3p and miR-196a-5p were significantly reduced; compared with Hep3B, the expression of miR-224-5p markedly increased in liver cancer cell line MHCC-97H, while miR-146a-5p, miR-483-3p and miR-200b-3p were significantly reduced. The re-sults of qRT-PCR were consistent with chip results. Conclusion There are differences of miRNA expres-sion profiles in different metastatic potential liver canc-er cell lines MHCC-97H, Hep3B, and they may par-ticipate in regulating the development and invasion of hepatocellular carcinoma.

Background:BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) is an important cause of dysfunction and failure of renal transplants.This study aimed to assess the diagnostic performance of morning urine specific gravity (MUSG) in diagnosing BKPyVAN in kidney transplant recipients.Methods:A total of 87 patients,including 27 with BKPyVAN,22 with isolated BKPyV viruria,18 with T cell-mediated rejection (TCMR),and 20 with stable graft function,were enrolled in the First Affiliated Hospital of Sun Yat-Sen University from March 2015 to February 2017.MUSG at biopsy and during a follow-up period of 24 months after biopsy was collected and analyzed.Receiver operating characteristic (ROC) curve analysis was used to determine the ability of MUSG to discriminate BKPyVAN.Results:At biopsy,the MUSG of BKPyVAN group (1.008 ± 0.003) was significantly lower than that of isolated BK viruria group (1.013 ± 0.004,P ＜ 0.001),TCMR group (1.011 ± 0.003,P =0.027),and control group (1.014 ± 0.006,P ＜ 0.001).There was no significant difference in MUSG among the isolated BK viruria group,TCMR group,and control group (P =0.253).In BKPyVAN group,the timing and trend of MUSG elevate were consistent with the timing and trend of the decline of viral load in urine and plasma,reaching a statistical difference at 3 months after treatment (1.012 ± 0.003,P ＜ 0.001) compared with values at diagnosis.ROC analysis indicated that the optimal cut-off value of MUSG for diagnosis of BKPyVAN was 1.009,with an area under the ROC curve (AUC) of 0.803 (95％ confidence interval [CI]:0.721-0.937).For differentiating BKPyVAN and TCMR,the optimal MUSG cut-off value was 1.010,with an AUC of 0.811 (95％ CI:0.687-0.934).Condusion:Combined detection of MUSG and BKPyV viruria is valuable for predicting BKPyVAN and distinguishing BKPyVAN from TCMR in renal transplant recipients.

Objective To construct the pEGFP-C1-CXCL1 eukaryotic expression vector and to investigate the effect of CXCL1 on the proliferation of HepG2 cells under endoplasmic reticulum stress ( ERS).Methods Fragments of CXCL1 were obtained from the cDNA library of HepG2 cells before CXCL1 was cloned into a pEGFP-C1 vector for a recombinant plasmid pEGFP-C1-CXCL1 which was screened and identified by PCR and sequence alignment .Then,the recombinant plas-mid of pEGFP-C1-CXCL1 was transfected into human 293 T cell line and the expression of CXCL 1 was detected by fluores-cence microscopy and Western blotting.pEGFP-C1-CXCL1was furhter transfected into HepG2 cells, and CCK8 was used to detect the inhibitory effect of CXCL1 on tumor proliferation induced by TM in hepatocellular carcinoma .Results pEGFP-C1-CXCL1 was vertified by sequencing analysis .Fluorescence microscopy showed that pEGFP-C1-CXCL1 was transfected into 293T.CXCL1 expression was detected by Western blotting .CCK8 showed that TM inhibited tumor proliferation , while overexpression of CXCL1 decreased the inhabitory rate on cell proliferation of HepG 2 cells under ER stress compared to pEGFP-C1 group and the control group .Conclusion A recombinant pEGFP-C1-CXCL1 plasmid is successfully constructed that can be expressed stably in human 293T cells.Overexpression of CXCL1 can effectively reduce the inhabitory rate of HCC cells induced by the ER stress.

Objective To explore the possible role of voltage-gated potassium channel-interacting protein 1 (KChIP1) in the pathogenesis of epilepsy. Methods Sprague Dawley female adult rats were treated with pentylenettrazole (PTZ) to develop acute and chronic epilepsy models. The approximate coronal sections of normal and epilepsy rat brain were processed for immunohistochemistry. Double-labeling confocal microscopy was used to determine the coexistence of KChIP1 and gamma-aminobutyric acid (GABA). Results KChIP1 was expressed abundantly throughout adult rat brain. KChIP1 is highly co-localize with GABA transmitter in hippocampus and cerebral cortex. In the acute PTZ-induced convulsive rats, the number of KChIP1-postive cells was significantly increased especially in the regions of CA1 and CA3 (P < 0.05); whereas the chronic PTZ-induced convulsive rats were found no changes. The number of GABA-labeled and co-labeled neurons in the hippocampus appeared to have no significant alteration responding to the epilepsy-genesis treatments. Conclusion KChIP1 might be involved in the PTZ-induced epileptogenesis process as a regulator to neuronal excitability through influencing the properties of potassium channels. KChIP1 is preferentially expressed in GABAergic neurons, but its changes did not couple with GABA in the epileptic models.

OBJECTIVETo explore the effects of zinc supplementation on zinc and calcium levels in serum and tissue in burned rats.

METHODSEighty SD rats were randomly divided into C group (control group without scald, n = 8), and N, W, H groups (each consisting of 24 rats), in which the rats were exposed to scalding resulting in partial thickness burns covering 15% of the total body surface area on the back, and then they were fed with diets containing zinc 40 microg/g in N and W groups, and 80 microg/g in H group. A cream containing zinc 761.1 microg/g was applied on the wound in W group at the same time. Eight rats of each group were sacrificed on day 1, 3 and 7 after scald respectively. Venous blood and samples of liver, femur and scald skin were harvested. Zinc and calcium contents in serum and tissues were determined with atomic absorption spectrophotometer.

RESULTSThe serum Zn(2+) levels in N, W groups were lower than C group, however, it was obviously higher in H group (up to 16.2 micromol/L) on day 1 after scald. The liver Zn(2+) showed an increasing tendency in all groups, while Ca(2+) level declined in H group, but increased in N, W group. The bone Zn(2+) and Ca(2+) levels showed a progressive declination in all groups from day 1 to 7 after scald. The changes were more obviously in N group than H group (P < 0.05). The Zn(2+) content of the scalded skin increased obviously in H group on first day after scald and in W group on 7th day after scald. The Ca(2+) contents of scalded skin showed marked increases in all groups, especially in N group, but least in W group.

CONCLUSIONThere are obvious changes in Zn(2+) and Ca(2+) contents of serum and tissues after scald injury and zinc supplementation. The effects of zinc supplementation on calcium level in the tissue need to be further studied.

Animals ; Burns ; drug therapy ; metabolism ; Calcium ; blood ; metabolism ; Dietary Supplements ; Disease Models, Animal ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Zinc ; administration & dosage ; metabolism ; pharmacology