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Supplementary Data 1-9 from Overexpression of Glycosylphosphatidylinositol (GPI) Transamidase Subunits Phosphatidylinositol Glycan Class T and/or GPI Anchor Attachment 1 Induces Tumorigenesis and Contributes to Invasion in Human Breast Cancer

Supplementary Data 1 from LOXL1 and LOXL4 Are Epigenetically Silenced and Can Inhibit Ras/Extracellular Signal-Regulated Kinase Signaling Pathway in Human Bladder Cancer

Promoter hypermethylation is one of the common mechanisms leading to gene silencing in various human cancers. Using a combination of pharmacologic unmasking and microarray techniques, we identified 59 candidate hypermethylated genes, including LOXL1, a lysyl oxidase-like gene, in human bladder cancer cells. We further showed that LOXL1 and LOXL4 are commonly silenced genes in human bladder cancer cells, and this silence is predominantly related to promoter methylation. We also found LOXL1 and LOXL4 gene methylation and loss of expression in primary bladder tumors. In addition, somatic mutations were identified in LOXL4, but not in LOXL1 in bladder cancer. Moreover, reintroduction of LOXL1 and LOXL4 genes into human bladder cancer cells leads to a decrease of colony formation ability. Further studies indicated that the overexpression of LOXL1 and LOXL4 could antagonize Ras in activating the extracellular signal-regulated kinase (ERK) signaling pathway. Thus, our current study suggests for the first time that lysyl oxidase-like genes can act as tumor suppressor genes and exert their functions through the inhibition of the Ras/ERK signaling pathway in human bladder cancer. [Cancer Res 2007;67(9):4123–9]

Supplementary Figure 1 Legend from ΔNp63α Up-Regulates the Hsp70 Gene in Human Cancer

Based on the oncogenic role of phosphatidylinositol glycan (PIG) class U in human tumors, we explored the role of two additional subunits of the glycosylphosphatidylinositol (GPI) transamidase complex in human breast cancer. We found that PIG class T (PIG-T) and GPI anchor attachment 1 (GPAA1) were overexpressed in breast cancer cell lines and primary tumors. Forced expression of PIG-T and GPAA1 transformed NIH3T3 cells in vitro and increased tumorigenicity and invasion of these cells in vivo. Suppression of PIG-T expression in breast cancer cell lines led to inhibition of anchorage-independent growth. Moreover, we found that PIG-T and GPAA1 expression levels positively correlated with paxillin phosphorylation in invasive breast cancer cell lines. Furthermore, suppression of PIG-T and GPAA1 expression led to a decrease in paxillin phosphorylation with a concomitant decrease in invasion ability. These results suggest that the GPI transamidase complex is composed of a group of proto-oncogenes that individually or as a group contribute to breast cancer growth. This aberrant growth is mediated, at least partially, by phosphorylation of paxillin, contributing to invasion and progression of breast cancer. (Cancer Res 2006; 66(22): 9829-36) (Cancer Res 2006; 66(20): 9829-36)

HSP70, a stress response protein, is known to be a determinant of cell death and cell transformation. We show that different isoforms of p63 have different transcriptional activities on hsp70 genes. ΔNp63α, an abundantly expressed isoform of p63, activates (in vitro and in vivo), whereas TAp63γ down-regulates the expression of hsp70. We further show that the transactivation domain at the NH2 terminus of p63 represses, whereas the COOH terminus activates hsp70 transcription. In addition, ΔNp63α regulates transcription of the hsp70 gene through its interaction with the CCAAT binding factor and NF-Y transcription factors which are known to form a complex with the CCAAT box located in the hsp70 promoter. Moreover, ΔNp63α expression correlates with HSP70 expression in all head and neck cancer cell lines. Finally, we show colocalization of ΔNp63α and HSP70 in the epithelium and coexpression of both proteins in 41 primary head and neck cancers. Our study provides strong evidence for the physiologic association between ΔNp63α and hsp70 in human cancer, thus further supporting the oncogenic potential of ΔNp63α.

Supplementary Figure 1 from ΔNp63α Up-Regulates the Hsp70 Gene in Human Cancer

Brain tissue of Magnetic Resonance Imaging is precisely segmented and quantified, which aids in the diagnosis of neurological diseases such as epilepsy, Alzheimer's, and multiple sclerosis. Recently, UNet-like architectures are widely used for medical image segmentation, which achieved promising performance by using the skip connection to fuse the low-level and high-level information. However, In the process of integrating the low-level and high-level information, the non-object information (noise) will be added, which reduces the accuracy of medical image segmentation. Likewise, the same problem also exists in the residual unit. Since the output and input of the residual unit are fused, the non-object information (noise) of the input of the residual unit will be in the integration. To address this challenging problem, in this paper we propose a Purified Residual U-net for the segmentation of brain tissue. This model encodes the image to obtain deep semantic information and purifies the information of low-level features and the residual unit from the image, and acquires the result through a decoder at last. We use the Dilated Pyramid Separate Block (DPSB) as the first block to purify the features for each layer in the encoder without the first layer, which expands the receptive field of the convolution kernel with only a few parameters added. In the first layer, we have explored the best performance achieved with DPB. We find the most non-object information (noise) in the initial image, so it is good for the accuracy to exchange the information to the max degree. We have conducted experiments with the widely used IBSR-18 dataset composed of T-1 weighted MRI volumes from 18 subjects. The results show that compared with some of the cutting-edge methods, our method enhances segmentation performance with the mean dice score reaching 91.093% and the mean Hausdorff distance decreasing to 3.2606.

Chikungunya fever, caused by Chikungunya virus (CHIKV), is an Aedes mosquito-borne disease present worldwide, and millions of CHIKV infections have been reported. Treatment for CHIKV includes supportive care and anti-inflammatory medications, but there are currently no antiviral treatments or vaccines. Nonstructural protein 2 (nsP2) of CHIKV is the most important functional protein mediating virus replication and amplification, making it an ideal antiviral target for CHIKV. In this study, we determined the CHIKV nsP2 Epitope Rich Region, expressed recombinant nsP2 protein, and isolated 5 nsP2-specific nanobodies (Nb-A2, Nb-A9, Nb-D7, Nb-D12 and Nb-E12) from a phage display library comprising variable domains of Camellidae heavy chain-only antibodies (VHH). We subsequently established a stable Nbs-expressing HEK293T cell line to explore antiviral function. The results showed that Nb-A9 inhibited CHIKV replication at the early stage of CHIKV infection in HEK293T cells, and protected cells against CHIKV-induced cytopathic effect (CPE). This is possibly the first report of an Nbs-based strategy against CHIKV nsP2, Nb-A9 has great potential for developing a novel antiviral drug to treat CHIKV infection. The acquisition of antibodies has laid a foundation for further research on the function of CHIKV nsP2 and the development of therapeutic drugs.

Lead telluride nanowires deposited by electrochemical atomic layers have broad application prospects in the field of photodetectors. In this work, using the method of electrochemical atomic layer deposition, we obtained different morphologies of lead telluride materials by controlling the deposition parameters, such as deposition time, temperature, and potential, and characterized them using SEM, TEM, XPS, and other techniques. A lead telluride nanowire detector with good performance was prepared. The photoresponsivity of the detector is 102 mA W−1, the detectivity is 2.1 × 108 Jones, and the response time and recovery time are 0.52 s and 0.54 s respectively at 2.7 μm wavelength laser irradiation.

Wenzhou mammarenavirus (WENV) is a zoonotic pathogen newly discovered in east and southeast Asia. WENV has been found in wild rodent animals around the world while its standing is barely understood in Guangzhou city, where is known as a region of outbreak hotspot for zoonotic emerging infectious diseases. To investigate the prevalence and genomic characteristics of mammarenavirus in Guangzhou City, lung tissue samples from wild rodent species were collected from five districts of Guangzhou City in the year 2015 and 2016. The viral RNA was extracted and then subjected to mammarenavirus-specific PCR. The result revealed approximately 1.0% (3/306) nucleic acid positivity for lung tissue samples obtained from three rodent species: Mus musculus, Rattus flavipectus, and Rattus norvegicus. Viral metagenomic sequencing of three samples was then carried out and two full segment L and three full segment S sequences were obtained. Phylogenetics analysis indicated the sequences of the new mammarenavirus strain have 76.2% - 94.4% similarity to known WENV encoded genes, with the highest similarity to the WENV 9–24 strain. Population structure analysis grouped all known WENV into seven lineages, and this WENV Guangzhou strain was grouped with WENV 9–24 as well. Though the seroprevalence result was not available, our data provides the first nucleic acid evidence of circulating WENV in Guangzhou city, and it suggested WENV had a broader host tropism than previously known.

BACKGROUNDIschemia-reperfusion injury is considered an inevitable event that compromises posttransplant outcomes. Numerous treatments have been proposed to reduce its impact. However, most of them have had limited success, as none of them can completely avoid graft ischemia.METHODSIschemia-free liver transplantation (IFLT) comprises surgical techniques to enable continuous oxygenated blood supply to brain-dead donor livers during procurement, preservation and implantation using normothermic machine perfusion technology. In this nonrandomized study, 38 donor livers were transplanted using IFLT and were compared to 130 livers procured and transplanted using a conventional procedure (CLT).RESULTSOne patient (2.6%) suffered early allograft dysfunction in the IFLT group, compared with 43.8% of patients in the CLT group (absolute risk difference, 41.2 percentage points; 95% confidence interval, −31.3, −51.1). The median (range) peak aspartate aminotransferase levels within the first week (336, 149-4112 vs. 1445, 149-25083 U/L, PCONCLUSIONSIFLT provides a new approach to minimize ischemia-reperfusion injury and improve post-transplant outcomes.Clinical trial registryThis trial is registered with http://www.chictr.org.cn, number ChiCTR-OPN-17012090.

The goal of this study was to compare the microbiota in different pig-present settings in China. Bioaerosol samples from pig farms and slaughterhouses and nasal samples from pig farmers and slaughterhouse workers were collected in Guangdong, southern China. The bacterial genomic DNA was isolated and subjected to 16S sequencing. The data were analyzed using QIIME2 with the DADA2 pipeline. A total of 14,923,551 clean reads and 2785 operational taxonomic units (OTUs) were obtained, which were mostly grouped into 4 phyla (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) and 220 families. The microbiota richness of nasal samples in pig-present workers was higher than that of bioaerosols collected in the vicinity of the pig enclosures. There were 31.7% (620/1954) shared OTUs between pig farm bioaerosols and pig farmers which was higher than that between pig slaughterhouses and slaughterhouse workers (23.4%, 364/1553) (p

BACKGROUNDThe ongoing worldwide outbreak of the 2019-nCoV is markedly similar to the severe acute respiratory syndrome (SARS) outbreak 17 years ago. During the 2002-2003 SARS outbreak, healthcare workers formed a special population of patients. Although virus-specific IgG play important roles in virus neutralization and prevention against future infection, limited information is available regarding the long term persistence of IgG after infection with SARS-like coronavirus.METHODSA long-term prospective cohort study followed 34 SARS-CoV-infected healthcare workers from a hospital with clustered infected cases during the 2002-2003 SARS outbreak in Guangzhou, China, with a 13-year follow-up. Serum samples were collected annually from 2003-2015. Twenty SARS-CoV-infected and 40 non-infected healthcare workers were enrolled in 2015, and their serum samples were collected. All sera were tested for IgG antibodies with ELISA using whole virus and a recombinant nucleocapsid protein of SARS-CoV, as a diagnostic antigen.RESULTSAnti SARS-CoV IgG was found to persist for up to 12 years. IgG titers typically peaked in 2004, declining rapidly from 2004-2006, and then continued to decline at a slower rate. IgG titers in SARS-CoV-infected healthcare workers remained at a significantly high level until 2015. Patients treated with corticosteroids at the time of infection were found to have lower IgG titers than those without.CONCLUSIONSIgG antibodies against SARS-CoV can persist for at least 12 years. The presence of SARS-CoV IgG might provide protection against SARS-CoV and other betacoronavirus. This study provides valuable information regarding humoral immune responses against SARS-CoV and the 2019-nCoV.

A discrete dynamic model for human epidemics was developed in present study. The model included major parameters as transmission strength and its decline parameters, mean incubation period, hospitalization time, non-hospitalization daily mortality, non-hospitalization daily recovery rate, and hospitalization proportion, etc. Sensitivity analysis of the model indicated the total cumulative cases significantly increased with initial transmission strength, hospitalization time. The total cumulative cases significantly decreased with transmission strength’s decline and hospitalization proportion, and linearly decreased with non-hospitalization daily mortality and non-hospitalization daily recovery rate. In a certain range, the total cumulative cases significantly increased with mean incubation period. Sensitivity analysis demonstrated that dynamic change of transmission strength is one of the most important and controllable factors. In addition, reducing the delay for hospitalization is much effective in weakening disease epidemic. Non-hospitalization recovery rate is of importance for enhancing immunity to recover from the disease.

BACKGROUNDSThe ongoing new coronavirus (2019-nCoV) pneumonia outbreak is spreading in China and has not reached its peak. Five millions of people had emigrated from Wuhan before the city lockdown, which potentially represent a source of virus spreaders. Case distribution and its correlation with population emigration from Wuhan in early epidemic are of great importance for early warning and prevention of future outbreak.METHODSThe officially reported cases of 2019-nCoV pneumonia were collected as of January 30, 2020. Time and location information of these cases were extracted analyzed with ArcGIS and WinBUGS. Population migration data of Wuhan City and Hubei province were extracted from Baidu Qianxi and analyzed for their correlation with case number.FINDINGSThe 2019-nCoV pneumonia cases were predominantly distributed in Hubei and other provinces of South China. Hot spot provinces included Sichuan and Yunnan provinces that are adjacent to Hubei. While Wuhan city has the highest number of cases, the time risk is relatively stable. Numbers of cases in some cities are relatively low, but the time risks are continuously rising. The case numbers of different provinces and cities of Hubei province were highly correlated with the emigrated populations from Wuhan. Lockdown of 19 cities of Hubei province, and implementation of nationwide control measures efficiently prevented the exponential growth of case number.INTERPRETATIONPopulation emigrated from Wuhan was the main infection source for other cities and provinces. Some cities with low case number but were in rapid increase. Due to the upcoming Spring Festival return transport wave, understanding of the trends of risks in different regions is of great significance for preparedness for both individuals and institutions.FUNDINGSNational Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, State Key Program of National Natural Science of China.

The ongoing large-scale pneumonia outbreak in China is caused by the 2019-nCoV, a new coronavirus highly similar to SARS-CoV in the SARS outbreak. The cause and consequence of the outbreak remain largely unknown as it is still in its early stage. As many aspects of the new virus are similar to SARS in 2003, knowledge, patterns and lessons of the SARS-CoV outbreak are valuable resources for responding to the Wuhan 2019-nCoV outbreak. Using epidemiological surveys and analyses from the early stage of the SARS outbreak, we assessed and compared the characteristics of those two outbreaks and predicted the possible outcome for the current 2019-nCoV outbreak. Like the SARS-CoV, the 2019-nCoV has a high human-to-human transmission capability and healthcare workers and family members are high risk populations. Because the early outbreak stage coincides with the Chinese spring festival travel rush, it is a challenge to prevent and control the spread of the virus. In this situation, the emergence and movement of a 2019-nCoV super-spreader is difficult to identify. Using the reported case data so far (as of Jan 23, 2019), a logistic model was built and the cumulative and daily counts of the 2019-nCoV cases were predicted. The cumulative counts of 2019-nCoV cases was estimated about 2-3 times the total number of SARS, and the peak incidence is predicted to be in early or middle February. Regional migration should be limited or prohibited to prevent emergence and movement of a super-spreader. There is an urgent need to establish enhanced surveillance and implement efficient measures nationwide to control this epidemic.

Additional file 8. Meta-analysis for the potential influencing factor: occupations.

Additional file 3. Funnel plots of (a) overall studies (b) studies on male (c) studies on female.

Additional file 4. Sensitivity analysis of included articles.

Additional file 1. The 9 items in adjusted AHRQ scale.

Additional file 7. Meta-analysis for the potential influencing factor: age.

Additional file 6. Meta-analysis for the potential influencing factor: sex.

Additional file 5. Forest plots of subgroup analyses.

The ongoing large-scale pneumonia outbreak in China is caused by the 2019-nCoV. As many aspects of the new virus are similar to SARS in 2003, knowledge and lessons of the SARS-CoV outbreak are valuable for responding to the 2019-nCoV outbreak. Using epidemiological surveys and analyses from the early stage of the SARS outbreak, we assessed and compared the characteristics of those two outbreaks. Like the SARS-CoV, the 2019-nCoV has a high human-to-human transmission capability and healthcare workers and family members are high risk populations. Using the reported case data so far, the cumulative counts of 2019-nCoV cases was estimated about 2-3 times the total number of SARS, and the peak incidence is predicted to be in early or middle February. Regional migration should be limited or prohibited to prevent emergence and movement of a super-spreader. Nationwide surveillance and efficient measures are needed to control this epidemic.

Additional file 2. Table of literature quality evaluation.

Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and have pleiotropic effects. It has been suggested that statins may be a potential treatment during the next influenza pandemic. In a previous study we found that a statin/caffeine combination protects BALB/c mice against Influenza A, subtypes haemagglutinin type 5 and neuraminidase type 1 (H5N1), H3N2 and H1N1 infection. The effect of statins alone on influenza virus infection, however, is not known. In this study, it was investigated whether fluvastatin is capable of inhibiting influenza A virus replication in vitro. The results demonstrated that the synthesis of viral RNA and protein was affected by fluvastatin treatment. Virus production was markedly reduced when fluvastatin was administered simultaneously with the virus; however, a greater inhibition was observed when fluvastatin was added following viral adsorption. The selectivity index [SI; 50% cytotoxic concentration (CC50)/50% inhibition concentration (IC50)], however, was only 21. It was further demonstrated that fluvastatin protects host cells against influenza-induced inflammation by reducing the production of tumour necrosis factor-α, interleukin 8 and interferon γ. In conclusion, the results demonstrated that fluvastatin exerted a minor inhibitory effect on influenza virus infection, which involved anti-inflammatory activities.

Background Little comprehensive analysis combining epidemiological and clinical data has been done with mosquito-borne diseases imported into Guangzhou by air travelers. Methods We screened international travelers (body temperature >36.5 °C) arriving at Guangzhou Baiyun International Airport, and recorded their epidemiological and clinical information. Whole-blood samples were collected for laboratory diagnosis of dengue virus (DENV), chikungunya virus (CHIKV), zika virus (ZIKV) infections and malaria. Results Between March 1, 2016 and December 31, 2017, 155 (6.6%) cases (100 of DENV, 21 of CHIKV, 1 of ZIKV, 34 of malaria, including one co-infection of DENV and CHIKV) were identified among 2350 febrile travelers. DENV (90.0%) and CHIKV (100.0%) cases mainly came from Southern and Southeast Asia. Malaria cases (91.2%) mainly came from sub-Saharan Africa. Traveling abroad (28/74, 37.8%) and living/working abroad (11/22, 50.0%) were the most common causes of DENV infection and malaria for Chinese, respectively. Cases with these four mosquito-borne diseases were more likely to have nervous, musculoskeletal and skin symptoms and signs than other febrile diseases (P Conclusions It is important to strengthen the surveillance of mosquito-borne diseases among tourists and workers returning from Southeast Asia, Southern Asia and sub-Saharan Africa, especially those with nervous, musculoskeletal and skin symptoms and signs.

Resistance to cisplatin-based chemotherapy is responsible for the majority of deaths from head and neck squamous cell carcinoma (HNSCC). In this study, using genome-wide gene expression analysis to investigate potential molecular mediators of HNSCC chemoresistance, we identified SERPINB2, a known inhibitor of extracellular serine proteinase urokinase-type plasminogen activator (uPA), as an important candidate. Whereas SERPINB2 is known to function as a suppressor of uPA molecular cascades, many of which play important roles in tumor invasion and metastasis, a role for SERPINB2 in cancer drug resistance has not been examined. By using quantitative real-time PCR and Western blot analysis, we determined that SERPINB2 mRNA and protein levels correlated with chemoresistance in HNSCC cell lines, and significantly lower SERPINB2 expression levels were observed in two cisplatin resistant HNSCC subclones compared to their isogenic drug-sensitive parental lines. Immunohistochemical analysis of HNSCC tumor tissues from patients treated with neoadjuvant cisplatin-based chemotherapy (n = 67 cases) revealed a significant association between SERPINB2 protein levels, tumor differentiation and patient relapse. Moreover, SERPINB2 down-regulation was a strong predictor of reduced overall survival in patients with HNSCC who received cisplatin-based chemotherapy (P = 0.001, log rank test). Studies using either siRNA-mediated down-regulation or forced over-expression of SERPINB2 in HNSCC cell lines confirmed a functional role for SERPINB2 in drug resistance. The findings were further supported using chemical inhibitors of STAT3 activity (a downstream effecter of uPAR signaling pathway), showing that STAT3 suppression altered HNSCC cell line cisplatin sensitivity. This is the first report on a role for SERPINB2 in acquired resistance to cisplatin in patients with HNSCC. © 2013 Wiley Periodicals, Inc.

We determined the efficacy, biological activity, pharmacokinetics and safety of the hypomethylating agent 5-azacitidine (Celgene Corp., Summit, New Jersey) in dogs with naturally occurring invasive urothelial carcinoma.We performed a preclinical phase I trial in dogs with naturally occurring invasive urothelial carcinoma to examine once daily subcutaneous administration of 5-azacitidine in 28-day cycles at doses of 0.10 to 0.30 mg/kg per day according to 2 dose schedules, including days 1 to 5 (28-day cohort) or days 1 to 5 and 15 to 19 (14-day cohort). Clinical efficacy was assessed by serial cystosonography, radiography and cystoscopy. Urinary 5-azacitidine pharmacokinetic analysis was also done. Pretreatment and posttreatment peripheral blood mononuclear cell and invasive urothelial carcinoma DNA, respectively, was analyzed for global and gene specific [CDKN2A (p14ARF)] methylation changes.Enrolled in the study were 19 dogs with naturally occurring invasive urothelial carcinoma. In the 28-day cohort the maximum tolerated dose was 0.20 mg/kg per day with higher doses resulting in grade 3 or 4 neutropenia in 4 of 6 dogs. In the 14-day cohort the maximum tolerated dose was 0.10 mg/kg per day with grade 3 or 4 neutropenia seen in 2 of 3 dogs treated at higher doses. No grade 3 or 4 nonhematological toxicity was observed during either dosing schedule. Of 18 dogs evaluable for tumor response partial remission, stable disease and progressive disease were observed in 4 (22.2%), 9 (50.0%) and 4 (22.2%), respectively. Consistent 5-azacitidine levels (205 to 857 ng/ml) were detected in urine. Pretreatment and posttreatment methylation analysis revealed no significant correlation with clinical response.Subcutaneous 5-azacitidine showed promising clinical activity in a canine invasive urothelial carcinoma model, thus meriting further development in humans with urothelial carcinoma.

Alpha-actinins are critical components of the actin cytoskeleton. Here we show that alpha-actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). Alpha-actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express alpha-actinin-1 and -4, alpha-actinin-4 interacted and colocalized with eNOS. Addition of alpha-actinin-4 directly inhibited eNOS recombinant protein, and overexpression of alpha-actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of alpha-actinin-4 by siRNA increased eNOS activity in BAECs. The alpha-actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on alpha-actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased alpha-actinin-4-eNOS interaction, leading to translocation of alpha-actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced alpha-actinin-4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with alpha-actinin-4 and calmodulin.

BACKGROUND Although severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA. METHODS SARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens. RESULTS The sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001). CONCLUSION N199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.

Promoter hypermethylation is one of the common mechanisms leading to gene silencing in various human cancers. Using a combination of pharmacologic unmasking and microarray techniques, we identified 59 candidate hypermethylated genes, including LOXL1, a lysyl oxidase-like gene, in human bladder cancer cells. We further showed that LOXL1 and LOXL4 are commonly silenced genes in human bladder cancer cells, and this silence is predominantly related to promoter methylation. We also found LOXL1 and LOXL4 gene methylation and loss of expression in primary bladder tumors. In addition, somatic mutations were identified in LOXL4, but not in LOXL1 in bladder cancer. Moreover, reintroduction of LOXL1 and LOXL4 genes into human bladder cancer cells leads to a decrease of colony formation ability. Further studies indicated that the overexpression of LOXL1 and LOXL4 could antagonize Ras in activating the extracellular signal-regulated kinase (ERK) signaling pathway. Thus, our current study suggests for the first time that lysyl oxidase-like genes can act as tumor suppressor genes and exert their functions through the inhibition of the Ras/ERK signaling pathway in human bladder cancer. [Cancer Res 2007;67(9):4123–9]

Aim: The resurgence of severe acute respiratory syndrome (SARS) is still a threat because the causative agent remaining in animal reservoirs is not fully understood, and sporadic cases continue to be reported. Developing high titers of anti-SARS hyperimmune globulin to provide an alternative pathway for emergent future prevention and treatment of SARS. Methods: SARS coronavirus (CoV)F69 (AY313906) and Z2-Y3 (AY394989) were isolated and identified from 2 different Cantonese onset SARS patients. Immunogen was prepared from SARS-CoV F69 strain. Six health horses were immunized 4 times and serum was collected periodically to measure the profile of specific IgG and neutralizing antibodies using indirect enzyme-linked immunosorbent assay and a microneutralization test. Sera were collected in large amounts at the peak, where IgG was precipitated using ammonium sulphate and subsequently digested with pepsin. The product was then purified using anion-exchange chromatography to obtain F(ab') 2 fragments. Results: The specific IgG and neutralizing antibody titers peaked at approximately week 7 after the first immunization, with a maximum value of 1:14210. The sera collected at the peak were then purified. Fragment of approximately 15 g F(ab') 2 was obtained from 1 litre antiserum and the purity was above 90% with the titer of 1:5120, which could neutralize the other strain (SARS-CoV Z2-Y3) as well. Conclusion: This research provides a viable strategy for the prevention and treatment of SARS coronavirus infection with equine hyperimmune globulin, with the purpose of combating any resurgence of SARS.

As in many other human cancers, overactivation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway occurs frequently in thyroid cancer, but the mechanism is not completely clear.Because activating mutations and genomic amplification of the PIK3CA gene, which encodes the p110a catalytic subunit of PI3K, are common in many cancers, we sought to investigate this phenomenon in thyroid tumors.To search for PIK3CA mutations, we isolated genomic DNA from primary thyroid tumors of various types and performed direct sequencing of the exons of PIK3CA gene that carry the most common mutations in other cancers. We used real-time quantitative PCR to investigate genomic amplification of the PIK3CA gene.We found no PIK3CA gene mutations in 37 benign thyroid adenomas, 52 papillary thyroid cancers, 25 follicular thyroid cancers, 13 anaplastic thyroid cancers, 13 medullary thyroid cancers, and seven thyroid tumor cell lines. We found a C3075T single-nucleotide polymorphism in exon 20 of this gene in two cases. With a copy number of 4 or more defined as amplification, we found PIK3CA gene amplification in four of 34 (12%) benign thyroid adenomas, three of 59 (5%) papillary thyroid cancer, five of 21 (24%) follicular thyroid cancer, none of 14 (0%) medullary thyroid cancer, and five of seven (71%) thyroid tumor cell lines. The PIK3CA gene amplification and consequent Akt activation were confirmed by fluorescence in situ hybridization and Western blotting studies using cell lines, respectively.These data suggest that mutation of the PIK3CA gene is not common, but its amplification is relatively common and may be a novel mechanism in activating the PI3K/Akt pathway in some thyroid tumors.

Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTA-tetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur. Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.