[Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells].

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Classification and prediction of outcome in traumatic brain injury based on computed tomographic imaging.

Traumatic brain injury (TBI) is a common and potentially devastating problem. The classification of TBI is necessary for accurate diagnosis and the prediction of outcomes. The increased use of early sedation, intubation and ventilation in more severely injured patients has decreased the value of the Glasgow Coma Scale for the purposes of classification. An alternative is the classification of TBI according to morphological criteria based on computed tomography (CT) investigations. This article reviews the current classification and prediction of outcomes in TBI based on CT imaging. Classifications based on the presence or absence of intracranial local lesions, diffuse injury, signs of subarachnoid or intra-ventricular haemorrhage and fractures or foreign bodies are considered, and their predictive value is discussed. Future studies should address the complicated issue of how optimally to combine CT characteristics for prognostic purposes and how to improve on currently used CT classifications to predict outcomes more accurately.

[Effects of infant feeding practice on eczema during early childhood in Shanghai, Hohhot, and Fuzhou].

Objective: To estimate the prevalence of eczema in early childhood and effect of infant feeding practice on eczema by different regions of China with diverse climate and dietary patterns. Method: A questionnaire survey was conducted from June 2012 to October 2012 in Shanghai, Hohhot, and Fuzhou. The parent or guardian of the children aged between 2.5 to 3.5 years attending routine health visit in the chosen communities were invited to complete a modified questionnaire of the International Study of Asthma and Allergy in Childhood (ISAAC). Logistic regression model was used to analyze of the family history of allergy, duration of breastfeeding, timing of introduction of complementary foods and other potential confounders. Result: A total of 2 242 children were interviewed, 750 from Shanghai, 716 from Hohhot, and 776 from Fuzhou. The prevalence of eczema in early childhood was significantly different among Shanghai (16.9%, 95%CI 16.87-16.93), Hohhot (34.5%, 95%CI 34.46-34.54)and Fuzhou (44.3%, 95%CI 44.26-44.34). The difference was statistically significant between 3 groups (χ2=72.05, P<0.05). Introducing complementary food after the age of 6 months was associated with a decreased risk for eczema when compared to introduction between 4 to 6 months(odds ratio (OR) 0.58, 95%CI 0.41-0.81) in Fuzhou, while there was no significant association between timing of introduction of complementary foods and eczema in Shanghai and Hohhot. Conclusion: The prevalence of eczema during early childhood is various among three cities. The relationship between timing of introduction of complementary foods and eczema in Fuzhou is different from that in Shanghai and Hohhot. The role of climate and dietary patterns on prevalence of eczema needs further studies.

Phytotoxicity of dredged sediment from urban canal as land application.

Phytotoxicity of dredged sediment from Hangzhou section of the Grand Canal as land application was evaluated by pakchoi (Brassica chinensis L.) germination tests and pot experiments. Germination rates of pakchoi in the dredged sediment and in sediment-applied soils were both significantly higher than that in the soil controls, while the germination rate between the sediment-applied soils was no significant difference. In pot experiments, plant height and biomass were increased by the dredged sediment application rate in the rate of lower than 540 t ha(-1), but decreased when the application rate was over this rate. Concentrations of Zn and Cu in pakchoi were linearly increased with the increasing of the application rate of the dredged sediment. Both plant height and biomass of pakchoi in sediment-treated red soil were higher than that in sediment-treated paddy soil, regardless the application rate. The results suggest that plant biomass of pakchoi may be used as an indicator of the phytotoxicity of the dredged sediment. It also showed that red soil is more suitable to accept the dredged sediment than paddy soil, and 270 t ha(-1) is a safe application rate both in red soil and paddy soil.

Up-to-date observations of space energetic particles made by the Chinese satellite Fengyun-1 (B).

This paper presents the observational results of space energetic particles obtained by the Cosmic Ray Composition Monitor (CRCM) onboard the Chinese satellite, Fengyun-1(B). These results, including those of a few solar proton events, the geomagnetically trapped particles and the anomalous cosmic ray components, were obtained from 3 September 1990 to 15 February 1991. The observed elements include H, He, C, N, O and Fe of energies from 4-23 MeV/u. It was found that the proton fluxes of the inner Radiation Belt (IRB) increased obviously during the period of solar proton event (SPE). A few kinds of heavy ions (Z > or = 6) were also detected in the IRB. As to the anomalous cosmic ray component (ACRC), in addition to C, N and O, anomalous iron particles were also recorded.

Prolonged infection in rhesus macaques with simian immunodeficiency virus (SIVmac239) results in animal-specific and rarely tissue-specific selection of nef variants.

We analyzed the sequence of nef genes from different tissues of three rhesus macaques that had been infected with molecularly cloned SIVmac239 for 88 to 92 weeks. Comparison of the predicted amino acid sequences revealed that each macaque had selected out specific amino acid substitutions and that most of this variation (70%) was confined to four regions, amino acids 39 to 75, 90 to 105, 153 to 167, and 191 to 217, comprising 36% of the protein. The nef genes in these animals underwent extensive genetic variation with average nucleotide and amino acid substitution rates varying from 0.86 to 2.84% and 2.47 to 6.27%, respectively, although tissue-specific selection of nef variants occurred in only 1 of 14 tissues examined in this study. Comparison of the rate of nucleotide and amino acid substitutions in the nef genes to those previously reported in the env in the central nervous system (CNS) and lymph node (LN) revealed that the predicted amino acid substitution rates for Nef were much higher than for the gp120 region of env in the CNS and LN tissues for one macaque. In the two other macaques, the predicted amino acid substitution rates were similar between these two proteins in LN tissues, but the amino acid substitution rates in Nef were significantly higher than in the gp120 from the CNS. Comparison of the nucleotide substitutions in the region of overlap between the env and the nef revealed that approximately 83% of the nucleotide substitutions in this area resulted in a Nef amino acid sequence change, 26% of the nucleotide substitutions resulted in a gp41 amino acid change, and 9.5% of nucleotide substitutions resulted in amino acid sequence changes in both proteins, suggesting a preference for the selection of amino acid substitutions in the Nef in these animals. Our results indicate that in animals infected with SIVmac239 for prolonged periods, variation in the nef occurs at rates similar to or exceeding that observed for the env gene.

Simian immunodeficiency virus SIVmac chimeric virus whose env gene was derived from SIV-encephalitic brain is macrophage-tropic but not neurovirulent.

We inoculated four rhesus macaques with molecularly cloned simian immunodeficiency virus SIVmac239/17E env, a chimeric virus whose env gene was derived from the brain of an SIV-encephalitic macaque. Blood and lymphoid tissues had high frequencies of infected cells. The virus was neuroinvasive, but productive virus replication did not occur in the brain, and animals did not develop encephalitis.

Pathogenesis of lymphocyte-tropic and macrophage-tropic SIVmac infection in the brain.

SIVmac239 replicates productivity in activated CD4+ T lymphocytes, but inefficiently in macrophages from rhesus macrophages. Inoculation of the virus into animals results in an acute, highly productive burst of virus replication in activated T lymphocytes in lymphoid tissues and infected cells invade the central nervous system (CNS). This phase lasts a few weeks and is eventually followed by development of immunosuppression of different degrees of severity, opportunistic infections, and tumors related to the loss of T lymphocytes. On rare occasions, infected immunosuppressed animals develop encephalitis and/or interstitial pneumonia, syndromes that are associated with selection of mutant viruses that replicate efficiently in macrophages of these tissues. Usually, however, brains of animals dying with AIDS caused by SIVmac239 appear histologically normal. Is the brain infected with virus? We report here on a macaque dying with AIDS, a neuroinvasive tumor and interstitial pneumonia associated with macrophage-tropic virus. Except for focal infiltration of tumor cells, the brain was normal histologically. We examined the virus and viral DNA from different tissues and found that lymphocytes but not macrophages from lymph nodes and spleen yielded virus, whereas macrophages but not lymphocytes from the lung produced virus. No virus was recovered from the brain but small amounts of viral p27 were present in the brain homogenate. Viral sequences were present in the brain as determined by PCR from tissue DNA. Comparison showed that the viral sequences in the brain closely resembled those from the spleen. Presumably, the virus caused a minimally productive infection detectable by production of small amounts of p27, but was not accompanied by any histopathological changes. It is unclear why the macrophage-tropic virus in the lung failed to 'take-off' in the brain of this animal. To determine whether this virus had encephalitic potential, we inoculated the lung homogenate containing cell-free, macrophage tropic virus into a young pigtail macaque, a species known to be sensitive to primate lentiviral infections. This animal developed severe encephalitis 10 weeks later. Virus from the brain was very similar to the inoculum virus, proving its encephalitic potential. Possible reasons for the differences in neurovirulence of this virus between the two animals remain speculative.

Lymphocyte-tropic simian immunodeficiency virus causes persistent infection in the brains of rhesus monkeys.

Molecularly cloned SIVmac239 is the prototypical SIVmac lymphocyte-tropic virus that replicates productively in lymphocytes but poorly in macrophages. In macaques, the virus causes activation and productive infection of T lymphocytes which invade the central nervous system (CNS) early after infection in the animal. However, infected animals develop immunosuppression and AIDS but rarely overt neurological disease. In this study, we examined multiple regions of the brain and spinal cord for the presence of SIV env sequences and histological lesions in five macaques that had been infected with SIVmac239 for 1.7 to 2.25 years. Histopathological examination of the brain revealed no lesions consistent with encephalitis; however, viral DNA was found in all five brains. In one animal the virus caused infection in a widely disseminated pattern from the frontal cortex to the distal end of the spinal cord, whereas in the other four animals infection in the CNS occurred in a nonspecific, focal pattern. Sequence analyses were performed on gp120 sequences isolated from selected regions of the CNS and compared to gp120 sequences isolated from corresponding lymph nodes, a tissue known to support productive replication of SIVmac239. Examination of the viral sequences from the CNS tissue from two animals (macaques 10F and 14F) revealed a low mutation rate when compared to the sequences isolated from the lymph node tissues. The percentage change in the amino acid sequence was approximately 1% for CNS clones versus > or = 3% for clones isolated from the lymph node. The majority of the CNS viral sequences of macaques 10F and 14F had none of the genetic markers shown in a previous study to be associated with macrophage-tropic variants and indeed retained a nucleotide sequence of similar to the original lymphocyte-tropic virus used for inoculation despite almost 2 years of persistent infection in the animals. Construction of chimeric viruses with V1-V5 regions of selected macaque 10F and macaque 14F CNS-gp120 clones confirmed the predicted lymphocyte-tropic nature of these env genes. In contrast, the gp120 sequences isolated from the CNS tissue of one of the other three animals (macaque 13F) had a mutation rate comparable to that observed for the lymph node clones. The CNS clones from this animal had amino acid substitutions that were previously shown to be associated with macrophage tropism. Compared to the chimeric viruses constructed with V1-V5 sequences from macaques 10F and 14F, viruses constructed with the V1-V5 sequences of several macaque 13F brain clones did not yield infectious virus.(ABSTRACT TRUNCATED AT 400 WORDS)