To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; growth & development ; physiology ; ultrastructure ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Humans ; Virus Release ; Virus Replication

The present study was designed to observe the influence of cerebral ischemia/reperfusion injury on learning and memory in hyperlipidemic rats and estimate the changes of activity of autonomic nervous system. Twenty-three male Wistar rats were randomly divided into three groups, named control group (C group, n=10), hyperlipidemia group (H group, n=6) and hyperlipidemia-ischemia group (HI group, n=7), respectively. The rats in H and HI group were fed a high-fat diet for 2 weeks and the rats in all groups were examined through Morris water maze (MWM) task. The rats in HI group underwent ischemia/reperfusion by 2-vessel occlusion (2-VO) method, and had electrocardiogram (ECG) recording simultaneously. The MWM task and ECG recording were taken again after 7 d of recuperation. The following results were obtained: (1) In the second place navigation performance and probe trial performance, the frequency of memory in quadrant of hidden-platform and memory score decreased significantly in HI group compared to that in C and H groups. (2) The heart rate in HI group decreased slowly after ischemia; the power at high frequency band (HF) reduced gradually, meanwhile the power at middle frequency band (MF) and the ratio of power at MF and HF decreased clearly compared to baseline value. (3) After 7 d of ischemia/reperfusion, the heart rate in HI group was significantly higher than that in H group (P<0.05). While there was no statistical change in the power at MF, the power at HF decreased and the ratio of MF/HF increased significantly (P<0.05). The data demonstrated that ischemia/reperfusion decreased the activity of autonomic nervous system, and the reduction of sympathetic nerve activity was much more than that of vagus nerve activity. The results suggest that the hippocampus neuron injury caused by ischemia induces cognitive disorder and imbalance of vago-sympathetic nerve activity accompanied by vagus nerve suppression.
Animals ; Autonomic Nervous System ; physiopathology ; Brain Ischemia ; etiology ; physiopathology ; Heart Rate ; physiology ; Hippocampus ; physiopathology ; Hyperlipidemias ; complications ; Learning Disorders ; etiology ; physiopathology ; Male ; Memory Disorders ; etiology ; physiopathology ; Random Allocation ; Rats ; Rats, Wistar ; Reperfusion Injury ; pathology ; physiopathology

OBJECTIVETo establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum.

METHODSTwo DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA.

RESULTSThe standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively.

CONCLUSIONThe Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.

DNA, Viral ; blood ; Humans ; Parvovirus B19, Human ; genetics ; Polymerase Chain Reaction ; methods

OBJECTIVEThis study explored the dynamic expression of the E3 ubiquitin-protein ligase gene, Arkadia, in response to carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model and investigated the differential expression that occurs following treatment with the anti-fibrotic bone morphogenetic protein-7 (BMP-7).

METHODSThirty healthy male imprinting control region (ICR) mice were randomly assigned to three groups: normal (control; n = 6), CCl4-induced model group (model; n = 18), and CCl4-induced model with BMP-7 treatment group (treatment; n = 6). The model group was further divided into three subgroups (n = 6 each) for analysis at 4, 8 and 12 weeks after fibrosis induction. Liver fibrosis was induced by hypodermic injections of 60% CCl4 /peanut oil (5 mL/kg) to the hind legs of mice two-times per week in alternating legs for a period of 12 weeks. At week 9, the treatment group of CCl4-induced mice were given an intraperitoneal injection of BMP-7 (300 pg/g) simultaneously with that day's hypodermic injection of 60% CCl4 /peanut oil, and then every other day for a period of four weeks. The pathological changes in liver tissues were observed after staining with hematoxylin-eosin (HE) and Masson's trichrome. Messenger RNA (mRNA) and protein expression of Arkadia in liver were evaluated using reverse transcription-polymerase chain reaction and immunohistochemistry and Western blotting, respectively.

RESULTSMouse models of liver fibrosis were successfully established by CCl4 exposure. Arkadia, Smad7 and TGF-beta1 mRNA levels were up-regulated in the model group in a time-dependent manner (vs. control group), and BMP-7 treatment led to significant down-regulation of the CCl4-induced expression of the three genes (vs. control group: F = 812.80, 451.46, and 998.96, respectively; P less than 0.01). At week 12, the mRNA levels of Arkadia, Smad7, and TGF-b1 were significantly lower in the BMP-7 treatment group than in the model group (t = 12.108, 18.737, and 16.364, respectively; P less than 0.01). Arkadia, Smad7, and TGF-b1 protein staining was weak in the portal area of control liver tissue. In contrast, the model group showed significantly stronger staining for all three proteins in the portal area and in the cytoplasm of liver cells. The staining of Arkadia, Smad7, and TGF-b1 proteins was significantly lower in the treatment group (vs. control group: F = 8.399, 609.690, and 900.561, respectively; P < 0.01). At week 12, the protein levels of Arkadia, Smad7, and TGF-b1 were significantly lower in the treatment group than in the model group (t = 23.438, 11.667, and 42.889, respectively; P < 0.01).

CONCLUSIONArkadia expression gradually increased along with the development of liver fibrosis but was suppressed by treatment with the anti-fibrotic factor, BMP-7.

Animals ; Bone Morphogenetic Protein 7 ; pharmacology ; Liver ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred ICR ; Ubiquitin-Protein Ligases ; metabolism ; Up-Regulation

Purpose To observe the effect of Necrostatin-1 (Nec-1),a necroptosis inhibitor,on the inflammation in unilateral ureter obstruction mice.Methods Male C57BL/6J mice were randomly divided into sham operation group,unilateral ureter obstruction operation group and UUO + Nec-1 treatment group,and the mice were sacrificed at 7th day after operated.Scr and BUN were measured.The pathological changes in the kidney were observed by HE staining.Immunohistochemistry and Western blot were used to detect the protein expression of necroptosis-related indicators RIP1,RIP3,MLKL and inflammatory cytokines TNF-α,IL-1β and MCP-1.Results Compared with sham operation group,the expression of RIP1,RIP3 and MLKL protein increased in the renal tissue of UUO mice,accompanied with increased expression of TNF-α,IL-1β and MCP-1.Nec-1 treatment significantly decreased above-mentioned protein expression in UUO mice,and also reduced renal interstitial inflammation and renal tubal injury according to HE staining.Scr and BUN levels suggested improved renal function.Conclusion Nec-1 could relieve the inflammatory reaction in renal tissue of the UUO mice by inhibiting necroptosis,which may be a new target for the treatment of secondary inflammation.

Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25-30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P<0.05). And there was no significant difference between DBD group and DBCD group (P>0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent.

Objective To compare the differences between Hertel exophthalmometry and CT on exophthalmos measurement,and explore its practical application value in forensic medicine.Methods Fifty-six normal individuals without eye injuries or diseases were selected as the control group.The absolute values of exophthalmos were measured in the standardized CT image workstation.The difference of binocular exophthalmos was compared in normal group.Forty-seven cases with unilateral orbital fracture were divided into injured eye group and normal eye group according to the presence of binocular orbital fractures.The differences of the absolute values of eophthalmos from the same eye and the relative values of eophthalmos between two eyes for same person measured by Hertel exophthalmometry and CT were analyzed.Results There was no statistical difference of exophthalmos between the normal eyes measured by CT method.In the normal eye group,the absolute value of exophthalmos measured by CT method was (16.66±5.41) rm,which was (16.16±4.45)mm when measured by the Hertel exophthalmometry method.There was no statistical difference between two groups (P＞0.05).In the injured eye group,there was statistical difference between the absolute values of exophthalmos measured by two measurement methods (P＜0.05).There was no statistical difference of the relative values of exophthalmos between the injured eyes and the normal eyes by two measurement methods (P＞0.05).Conclusion CT method has a good consistency with Hertel exophthalmometry,which can be applied into the practice of medicolegal expertise.

BACKGROUND: Bone morphogenetic protein 2 (BMP-2) has a strong ability to induce and promote the osteogenic differentiation of mesenchymal stem cells. OBJECTIVE: To evaluate the BMP-2 effect on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on an injectable nano-hydroxyapatite/chitosan (nHA/CS) composite scaffold. METHODS: (1) Experiment 1: Passage 3 BMSCs were divided into two groups and cultured with the nHA/CS scaffold or cultured alone. Cell counting kit-8 was used to detect cell proliferation at 1, 3, 5, 7, 14 days of culture. (2) Experiment 2: Passage 3 BMSCs were seeded onto the nHA/CS scaffold and cultured in culture medium containing BMP-2 or not. Alkaline phosphatase activity in cells was detected at 3, 6, 9, 12, 15 days of culture. Cell counting kit-8 was used to detect cell proliferation at 1, 3, 5, 7, 14 days of culture. Alizarin red staining was used to observe the osteogenic differentiation of cells at 1 and 2 weeks of culture. RESULTS AND CONCLUSION: (1) Experiment 1: With the prolongation of culture time, the absorbance values in the two groups were gradually increased, but there was no significant difference between the two groups. At 7 days of culture, the BMSCs adhered tightly to the scaffold surface. (2) Experiment 2: With the prolongation of culture time, the alkaline phosphatase activities in the two groups were gradually increased, and moreover, the alkaline phosphatase activity in the experimental group was higher than that in the control group at different culture time (P < 0.05). The absorbance values in the two groups were also gradually increased, and the value in the experimental group was higher than that in the control group at different culture time (P < 0.05). At 1 and 2 weeks of culture, the number of calcified nodules was higher in the experimental group than the control group. To conclude, BMP-2 has a promotion role in the proliferation and differentiation of BMSCs cultured on the injectable nHA/CS scaffold.

Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25-30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P<0.05). And there was no significant difference between DBD group and DBCD group (P>0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent.
Allografts ; Animals ; Apoptosis ; Brain Death ; China ; Death ; Heart Arrest ; Hepatocytes ; pathology ; ultrastructure ; Humans ; In Situ Nick-End Labeling ; Liver ; pathology ; ultrastructure ; Liver Transplantation ; methods ; Microscopy, Electron ; Organ Preservation ; methods ; Swine ; Tissue Donors ; Tissue and Organ Procurement ; methods

Objective: To evaluate the short-term clinical efficacy and adverse events of volumetric modulated arc therapy (VMAT) in the treatment of locally advanced non-small cell lung cancer (NSCLC). Methods: From January to December 2016, 58 patients (47 male and 11 female) with unresectable locally advanced NSCLC received concurrent or sequential chemoradiotherapy. The radiation dose was ranged from 38 Gy to 66 Gy. The radiation dose was equal or higher than 56 Gy in 53 patients (92%). The median radiotherapy fraction was 30, 1.8 Gy to 3.0 Gy for each fraction. Twenty-eight patients (48%) received concurrent chemoradiotherapy. Results: The median follow-up time was 9 months. The 1-year overall survival (OS) rate was 84% and the 1-year progression-free survival (PFS) rate was 48%.Eleven patients (19%) suffered from symptomatic radiation pneumonitis and one of them died of radiation pneumonitis. Within 6 months after radiotherapy, 31 patients (53%) developed asymptomatic local pulmonary fibrosis on CT images. Seventeen patients (29%) suffered from grade Ⅱ esophagitis. Ten cases (17%) had ≥ grade Ⅲ adverse events and 9 of them presented with leucopenia. Conclusions VMAT yields high short-term clinical efficacy and tolerable adverse events in the treatment of locally advanced NSCLC, which does not increase the risk of pneumonitis.