Role and mechanism of protein kinase D in the replication of human coronavirus 229E / 中国人兽共患病学报

This study investigated the role of protein kinase D(PKD)in the replication of human coronavirus 229E(HCoV-229E)and its potential molecular mechanisms.Using MRC-5 cells as a model,we assessed the expression levels of PKD family genes and proteins through qPCR and western blotting.We knocked down Prkd3 with siRNA and inhibited PKD activity with CRT0066101 to examine the replication lev-els of HCoV-229E-infected cells.Cell viability was assessed with CCK8,viral titration was determined with the TCID50,and immunofluorescence staining was used to assess HCoV-229E expression and the structure of the trans-Golgi network(TGN).The level of phosphatidylinositol 4,5-bisphosphate(PI(4,5)P2)was measured to reflect phosphatidylinositol 4-kinaseⅢβ(PI4KⅢβ)activity.PI4KⅢβ activity was inhibited with a PI4KⅢβ inhibitor(BQR695)to observe changes in TGN struc-ture and HCoV-229E replication after cell infection.CRT0066101 was used to inhibit PKD activity in Vero-E6 cells,and HCoV-229E replication levels were examined post-infection.The expression levels of PKD family genes and proteins,including Prkd1,Prkd2 and Prkd3,significantly increased during HCoV-229E infection.Knockdown of PKD3 significantly inhibited HCoV-229E mRNA levels and titers with respect to those in the control group(t=8.999,P＜0.001;t=6.920,P＜0.001),whereas overexpression of PKD3 increased HCoV-229E mRNA levels and titers(t=6.630,P＜0.001;t=5.794,P＜0.001).CRT0066101 also significantly inhibited HCoV-229E mRNA levels and titers(t=6.931,P＜0.001;t=4.055,P＜0.01).Im-munofluorescence staining indicated that CRT0066101 inhibited TGN fission caused by HCoV-229E infection.Inhibition of PI4KⅢβ activity significantly decreased PI(4,5)P2 levels,and BQR695 rescued the elevated PI(4,5)P2 levels caused by PKD overexpression(t=6.671,P＜0.01).BQR695 decreased TGN fission in HCoV-229E-infected cells,and HCoV-229E mRNA levels and titers(t=5.151,P＜0.001;t=7.744,P＜0.001).CRT0066101 inhibited HCoV-229E mRNA levels and titers in Vero-E6 cells(t=7.480,P＜0.001;t=7.228,P＜0.01).This study revealed that PKD regulates TGN fission through modu-latingPI4KⅢβ and is involved in HCoV-229E replication.PKD inhibitors and PI4KⅢβ inhibitors significantly decreased HCoV-229E replication,thus providing important insights for future research onthe treatment of coronavirus infections.

A descriptive analysis of injury triage, surge of medical demand, and resource use in an university hospital after 8.12 Tianjin Port Explosion, China / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>The 8.12 Tianjin Port Explosion in 2015 caused heavy casualties. Pingjin Hospital, an affiliated college hospital in Tianjin, China participated in the rescue activities. This study aims to analyze the emergency medical response to this event and share experience with trauma physicians to optimize the use of medical resource and reduce mortality of critical patients.</p><p><b>METHODS</b>As a trauma centre at the accident city, our hospital treated 298 patients. We retrospectively analyzed the data of emergency medical response, including injury triage, injury type, ICU patient flow, and medical resource use.</p><p><b>RESULTS</b>There were totally 165 deaths, 8 missing, and 797 non-fatal injuries in this explosion. Our hospital treated 298 casualties in two surges of medical demand. The first one appeared at 1 h after explosion when 147 wounded were received and the second one at 4 h when 31 seriously injured patients were received, among whom 29 were transferred from Tianjin Emergency Center which was responsible for the scene injury triage. After reexamination and triage, only 11 cases were defined as critical ill patients. The over-triage rate reached as high as 62.07%. Seventeen patients underwent surgery and 17 patients were admitted to the intensive care unit.</p><p><b>CONCLUSIONS</b>The present pre-hospital system is incomplete and may induce two surges of medical demand. The first one has a much larger number of casualties than predicted but the injury level is mild; while the second one has less wounded but almost all of them are critical patients. The over-triage rate is high. The hospital emergency response can be improved by an effective re-triage and implementation of a hospital-wide damage control.</p>

NVP-BEZ235 inhibits proliferation and colony-forming capability of CD34(+)CD38(-) human acute myeloid leukemia stem cells / 中国实验血液学杂志

This study was aimed to explore the effect of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle and colony forming capability of CD34(+)CD38(-) human acute myeloid leukemia (AML) KG1a cells. Flow cytometry was used to detect expression of CD34 and CD38 on the surface of human AML KG1a cells; Trypan blue assay was used to analyze the effect of NVP-BEZ235 at various concentrations on proliferation of KG1a cells; flow cytometry was performed to examine the cell cycle of KG1a cells after NVP-BEZ235 treatment; Soft agar colony-forming experiment was used to detect the colony forming ability of KG1a cells treated with NVP-BEZ235 at various concentrations. The results indicated that the percentage of CD34(+)CD38(-) AML KG1a cells was (98.02 ± 0.72)%. NVP-BEZ235 (0.125 - 1 µmol/L) inhibited the proliferation of KG1a cells in a time-and dose-dependent manner (P < 0.05) and the 50% inhibition concentrations (IC50) at 24 h and 48 h were 0.597 µmol/L and 0.102 µmol/L, respectively. KG1a cells were arrested at G0/G1 phase after treating with 0.5 µmol/L NVP-BEZ235 for 24 h, it was significantly higher than that of control group (83.2 ± 3.80)% vs (43.47 ± 9.60)% (P < 0.05). KG1a cells treated with NVP-BEZ235 (0 - 1 µmol/L) for 14 d and 21 d, the number of colony decreased respectively from (375.67 ± 21.46) per 2500 KG1a cells and (706.33 ± 87.31) per 2500 KG1a cells to 0, with statistical significance (P < 0.05). It is concluded that NVP-BEZ235 can inhibit proliferation and colony-forming capability of CD34(+)CD38(-) human AML KG1a cells.