Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.

Pursuing Impactful Quantitative Proteomics Using QC-Channels in Every Spectrum and Trend-Design in Experiment.

False changes discovered by quantitative proteomics reduce the trust of biologists in proteomics and limit the applications of proteomics to unlock biological mechanisms, which suppresses the application of proteomics techniques in the pharmaceutical industry more than it does in academic research. To remove false changes that arise during LC-MS/MS data acquisition, we evaluated the contributions of peptide abundance and number of unique peptides on reproducibility. Lower abundance and only one unique peptide have a higher risk of generating a higher coefficient of variation (CV), resulting in less accurate quantification. However, the abundance of peptides in samples is not adjustable and discarding proteins quantified by only one unique peptide is not a choice either. Indeed, a large percentage of proteins are accurately quantified by only one unique peptide. Therefore, to improve the calculations of the CV, we leverage a new function in PEAKS called QC-channels which enables technical replicates of each spectrum to be evaluated prior to calculation of the CV. While the QC-channels function in PEAKS significantly reduced the false quantification, random false changes still exist due to known or unknown reasons. To address this challenge, we present the idea of Trend-design to track trend changes rather than changes from two points to remove false quantifications and reveal consequential changes responding to a treatment or condition. The idea was confirmed by molecules with different affinity and dose in the current study. The combination of QC-channels and Trend-design enables a more impactful quantitative proteomics to allow unlocking biological mechanisms using proteomics.

Multiomics analysis reveals metabolic subtypes and identifies diacylglycerol kinase α (DGKA) as a potential therapeutic target for intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and lethal hepatobiliary tumor with few therapeutic strategies. The metabolic reprogramming of tumor cells plays an essential role in the development of tumors, while the metabolic molecular classification of iCCA is largely unknown. Here, we performed an integrated multiomics analysis and metabolic classification to depict differences in metabolic characteristics of iCCA patients, hoping to provide a novel perspective to understand and treat iCCA. METHODS: We performed integrated multiomics analysis in 116 iCCA samples, including whole-exome sequencing, bulk RNA-sequencing and proteome analysis. Based on the non-negative matrix factorization method and the protein abundance of metabolic genes in human genome-scale metabolic models, the metabolic subtype of iCCA was determined. Survival and prognostic gene analyses were used to compare overall survival (OS) differences between metabolic subtypes. Cell proliferation analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, RNA-sequencing and Western blotting were performed to investigate the molecular mechanisms of diacylglycerol kinase α (DGKA) in iCCA cells. RESULTS: Three metabolic subtypes (S1-S3) with subtype-specific biomarkers of iCCA were identified. These metabolic subtypes presented with distinct prognoses, metabolic features, immune microenvironments, and genetic alterations. The S2 subtype with the worst survival showed the activation of some special metabolic processes, immune-suppressed microenvironment and Kirsten rat sarcoma viral oncogene homolog (KRAS)/AT-rich interactive domain 1A (ARID1A) mutations. Among the S2 subtype-specific upregulated proteins, DGKA was further identified as a potential drug target for iCCA, which promoted cell proliferation by enhancing phosphatidic acid (PA) metabolism and activating mitogen-activated protein kinase (MAPK) signaling. CONCLUSION: Via multiomics analyses, we identified three metabolic subtypes of iCCA, revealing that the S2 subtype exhibited the poorest survival outcomes. We further identified DGKA as a potential target for the S2 subtype.

Spectroscopic characterization of heteronuclear iron-chromium carbonyl cluster anions.

Infrared photodissociation spectroscopy has been used to investigate CrFe(CO)n- (n = 4-9) clusters in the gas phase. Comparison of the observed spectra in the carbonyl stretching frequency region with those predicted for low-lying isomers by DFT calculations showed that the observed CrFe(CO)n- (n = 4-8) clusters could be characterized to have Cr-Fe bonded (OC)4Fe-Cr(CO)n-4 structures. The coexistence of isomers with the (OC)Fe-Cr(CO)5 and (OC)3Fe-Cr(CO)4 structures was also observed for CrFe(CO)6- and CrFe(CO)7- anions, respectively. The CrFe(CO)n- (n = 4-8) complexes were strongly bonded systems. The CrFe(CO)8- complex was a coordination-saturated cluster, and the CrFe(CO)9- anion was characterized to contain a CrFe(CO)8- core tagged by one CO molecule. Bonding analysis revealed that the Cr-Fe bonds in the CrFe(CO)n- (n = 4-8) clusters were predominantly σ-type single bonds. The iron center in the Fe(CO)4 moiety and the chromium center in the Cr(CO)5 moiety fulfilled the 18-electron configuration for the CrFe(CO)n- (n = 4-6) clusters. As in the CrFe(CO)n- (n = 7, 8) complexes, the iron center in the Fe(CO)4 moiety exhibited a 17-electron configuration, while the chromium center in the Cr(CO)4 moiety exhibited a 16-electron configuration. These findings provide valuable insights into the structure and bonding mechanism of heterometallic carbonyl clusters.

New Insights into Radio-Resistance Mechanism Revealed by (Phospho)Proteome Analysis of Deinococcus Radiodurans after Heavy Ion Irradiation.

Deinococcus radiodurans (D. radiodurans) can tolerate various extreme environments including radiation. Protein phosphorylation plays an important role in radiation resistance mechanisms; however, there is currently a lack of systematic research on this topic in D. radiodurans. Based on label-free (phospho)proteomics, we explored the dynamic changes of D. radiodurans under various doses of heavy ion irradiation and at different time points. In total, 2359 proteins and 1110 high-confidence phosphosites were identified, of which 66% and 23% showed significant changes, respectively, with the majority being upregulated. The upregulated proteins at different states (different doses or time points) were distinct, indicating that the radio-resistance mechanism is dose- and stage-dependent. The protein phosphorylation level has a much higher upregulation than protein abundance, suggesting phosphorylation is more sensitive to irradiation. There were four distinct dynamic changing patterns of phosphorylation, most of which were inconsistent with protein levels. Further analysis revealed that pathways related to RNA metabolism and antioxidation were activated after irradiation, indicating their importance in radiation response. We also screened some key hub phosphoproteins and radiation-responsive kinases for further study. Overall, this study provides a landscape of the radiation-induced dynamic change of protein expression and phosphorylation, which provides a basis for subsequent functional and applied studies.

Overexpression of vascular endothelial growth factor enhances the neuroprotective effects of bone marrow mesenchymal stem cell transplantation in ischemic stroke.

Although bone marrow mesenchymal stem cells (BMSCs) might have therapeutic potency in ischemic stroke, the benefits are limited. The current study investigated the effects of BMSCs engineered to overexpress vascular endothelial growth factor (VEGF) on behavioral defects in a rat model of transient cerebral ischemia, which was induced by middle cerebral artery occlusion. VEGF-BMSCs or control grafts were injected into the left striatum of the infarcted hemisphere 24 hours after stroke. We found that compared with the stroke-only group and the vehicle- and BMSCs-control groups, the VEGF-BMSCs treated animals displayed the largest benefits, as evidenced by attenuated behavioral defects and smaller infarct volume 7 days after stroke. Additionally, VEGF-BMSCs greatly inhibited destruction of the blood-brain barrier, increased the regeneration of blood vessels in the region of ischemic penumbra, and reducedneuronal degeneration surrounding the infarct core. Further mechanistic studies showed that among all transplant groups, VEGF-BMSCs transplantation induced the highest level of brain-derived neurotrophic factor. These results suggest that BMSCs transplantation with vascular endothelial growth factor has the potential to treat ischemic stroke with better results than are currently available.

[Effects of vaccination status on the disease severity of patients with coronavirus disease 2019].

OBJECTIVE: To evaluate the effect of 2019 novel coronavirus inactivated vaccine on the disease severity of patients with Delta variant of coronavirus disease 2019. METHODS: A retrospective analysis was performed on 704 patients with coronavirus disease 2019 infected with Delta variant who were older than 18 years old and admitted in the coronavirus disease 2019 designated hospital of Yangzhou (Subei Hospital New Area Branch) from July 2021 to September 2021. They were divided into severe (severe, critical) group and non-severe (light, ordinary) group according to the clinical characteristics of patients. According to the vaccination status, they were divided into 0-dose group, 1-dose group and 2-dose group. We evaluated the effects of vaccination on the severity of the disease and the production of antibodies, and analyzed the influencing factors leading to the severe group of coronavirus disease 2019. RESULTS: The proportion of severe group in the 2-dose vaccinated group was significantly lower than that in the 1-dose vaccinated group and 0-dose vaccinated group [3.02% (7/232) vs. 9.48% (22/232), 15.83% (38/240), P < 0.05]. The time from onset to admission (day: 1.97±1.66 vs. 2.66±2.70), age (years: 45.3±12.2 vs. 63.6±17.0), direct bilirubin [DBil (µmol/L): 3.70±1.83 vs. 5.30±5.13], lactate dehydrogenase [LDH (U/L): 240.69±74.29 vs. 256.30±85.18], creatinine [SCr (µmol/L): 63.38±19.86 vs. 70.23±25.43], interleukin-6 [IL-6 (ng/L): 7.32 (1.54, 17.40) vs. 18.38 (8.83, 33.43)], creatine kinase [CK (U/L): 66.00 (43.00, 99.75) vs. 78.00 (54.50, 144.00)] and D-dimer [mg/L: 0.30 (0.08, 0.49) vs. 0.41 (0.23, 0.69)] of patients in the 2-dose group were significantly lower than those in the 0-dose group (all P < 0.05), while platelet [PLT (×109/L): 176.69±60.25 vs. 149.25±59.07], white blood cell count [WBC (×109/L): 5.43±1.77 vs. 5.03±1.88] and lymphocyte [LYM (×109/L): 1.34±0.88 vs. 1.17±0.50] were significantly higher than those in the 0-dose group (all P < 0.05). The titer of immunoglobulin G (IgG) in the 2-dose group was significantly higher than those in the 1-dose group and 0-dose group on the 10th day after admission [U/L: 130.94 (92.23, 326.31), 113.18 (17.62, 136.20), 117.85 (33.52, 156.73), both P < 0.05], and higher than 0-dose group on the 16th day [U/L: 156.12 (120.32, 167.76) vs. 126.52 (61.34, 149.57), P < 0.05]. The proportion of complete 2-dose vaccination [10.45% (7/67) vs. 35.32% (225/637)], LYM (×109/L: 1.09±0.32 vs. 1.25±0.56) and PLT (×109/L: 138.55±68.03 vs. 166.93±59.70) in the severe group were significantly lower than those in the non-severe group (P < 0.05), while the time from onset to admission (day: 3.01±2.99 vs. 2.25±2.09), the length of hospital stay (day: 28±18 vs. 16±6), male proportion [77.61% (52/67) vs. 34.54% (220/637)], age (years: 69.13±12.63 vs. 52.28±16.53), DBil [µmol/L: 4.20 (3.18, 6.65) vs. 3.60 (2.80, 4.90], LDH (U/L: 310.61±98.33 vs. 238.19±72.14), SCr (µmol/L: 85.67±38.25 vs. 65.98±18.57), C-reactive protein [CRP (µmol/L): 28.12 (11.32, 42.23) vs. 8.49 (2.61, 17.58)], IL-6 [ng/L: 38.38 (24.67, 81.50) vs. 11.40 (4.60, 22.07)], CK [U/L: 140.00 (66.00, 274.00) vs. 72.80 (53.00, 11.00)] and the D-dimer [mg/L: 0.46 (0.29, 0.67) vs. 0.35 (0.19, 0.57)] in the severe group were significantly higher than those in the non-severe group (all P < 0.05). Multivariate regression analysis showed that the odds ratio (OR) of severe group was 0.430 (P = 0.010) in the 1-dose group and the 2-dose group compared with the 0-dose group. However, the risk of severe group was 0.381-fold in the 2-dose group compared with the 0-dose group [OR = 0.381, 95% confidence interval (95%CI) was 0.121-1.199] which was not statistically significant, when the age was included in the regression analysis (P > 0.05). PLT (OR = 0.992, 95%CI was 0.986-0.998) were protective factors, but older than 60 years old (OR = 3.681, 95%CI was 1.637-8.278), CK (OR = 1.001, 95%CI was 1.000-1.001), IL-6 (OR = 1.006, 95%CI was 1.002-1.010), SCr (OR = 1.020, 95%CI was 1.007-1.033) were risk factors for severe group (all P < 0.05). CONCLUSIONS: Compared with the 0-dose vaccinated patients, the coronavirus disease 2019 patients infected with delta variant and fully vaccinated with 2-dose 2019 novel coronavirus inactivated vaccine had lower level of IL-6, SCr, CK and D-dimer, and higher PLT, LYM and IgG titer, who were not easy to develop into the severe condition.

Activation of the ß­TrCP/IκBα/inflammation axis limits the sensitivity of liver cancer cells to neddylation inhibition.

Upregulation of protein neddylation occurs in numerous types of human cancer, including liver cancer. MLN4924, a potent neddylation­inhibiting pharmacological agent, demonstrates anticancer ability in numerous cancers. However, the sensitivity of MLN4924 in liver cancer remains unsatisfactory due to factors causing resistance. RT­qPCR and western blotting were utilized to assess the mRNA and protein levels of genes, respectively. Cell Counting Kit­8 assay and colony formation assays were employed to assess cell viability and proliferation. The pathway of protein degradation and stability were determined by western blotting after treatment with MG132 and cycloheximide. An immunoprecipitation assay was utilized to detect the ubiquitination of protein. An in vitro ubiquitination assay was used to determine the ubiquitin linkage. To the best of our knowledge, the present study was the first to demonstrate that NF­κB inhibitor α (IκBα) downregulation and subsequent inflammation in response to MLN4924 limited the antitumor potential of MLN4924. Ectopic expression of IκBα enhanced the antitumor potential of MLN4924 in liver cancer cells. Moreover, the results of the present study demonstrated that MLN4924 decreased IκBα via promoting the K48 linkage of ubiquitin to IκBα. Mechanistic studies demonstrated that MLN4924 enhanced the protein stability of ß­transducin repeat­containing protein (ß­TrCP), promoting the ubiquitination of IκBα, which led to the ubiquitin­mediated degradation of IκBα. In addition, the results of the present study also demonstrated that ß­TrCP knockdown markedly inhibited MLN4924 from suppressing the growth of liver cancer cells, via attenuating MLN4924­mediated IκBα downregulation and inflammation. Collectively, these results indicated that the ß­TrCP/IκBα/inflammation pathway may act as a novel resistance factor of MLN4924, and targeting ß­TrCP may be beneficial for the treatment of liver cancer.

A Practical Method for Determination of Nine Nucleosides in Tricholoma matsutake by UPLC/MS and Quantitative Analysis of Multicomponents Using Single Marker Method.

Nucleosides can be used as quality evaluation indicators of Tricholoma matsutake. In this work, a new ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS) strategy for quantitative analysis of multiple components using a single marker (QAMS) was proposed to determine nine nucleosides (adenosine, cytidine, guanosine, inosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyuridine) in T. matsutake. Guanosine was set as the internal reference substance, whose content in T. matsutake was determined using the conventional external standard method. Relative correction factors (RCFs) between guanosine and eight other nucleosides were measured. The concentrations of the eight components were calculated with the obtained RCFs by QAMS. An ultrasonic extraction method is used for sample preparation. This method was validated to be sensitive, precise, and accurate with the LODs of 0.31-1.9 ng, the overall intraday and interday variations less than 4.08%, and the overall recovery over 89.0%. The correlation coefficients (r 2) of the calibration curves were higher than 0.9918. The values of vector angle analysis were above 0.99845, which indicates no significant differences between the conventional external standard method and the present QAMS method. As far as we know, this is also the first report of UPLC/MS analysis of nucleoside compounds by QAMS, providing an efficient and feasible quality assessment method for other natural products containing nucleosides.

Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach.

Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C-like proteases (3CLpros) from CoVs (also called main proteases) are essential for viral replication and are also involved in polyprotein cleavage and immune regulation, making them attractive and effective targets for the development of antiviral drugs. Herein, the 3CLpro from the porcine epidemic diarrhea virus, an enteropathogenic CoV, was used as a model to identify novel crucial residues for enzyme activity. First, we established a rapid, sensitive, and efficient luciferase-based biosensor to monitor the activity of PDEV 3CLproin vivo. Using this luciferase biosensor, along with confirming the well-known catalytic residues (His41 and Cys144), we identified 4 novel proteolytically inactivated mutants of PDEV 3CLpro, which was also confirmed in mammalian cells by biochemical experiments. Our molecular dynamics (MD) simulations showed that the hydrogen bonding interactions occurring within and outside of the protease's active site and the dynamic fluctuations of the substrate, especially the van der Waals contacts, were drastically altered, a situation related to the loss of 3CLpro activity. These data suggest that changing the intermolecular dynamics in protein-substrate complexes eliminates the mechanism underlying the protease activity. The discovery of novel crucial residues for enzyme activity in the binding pocket could potentially provide more druggable sites for the design of protease inhibitors. In addition, our in-depth study of the dynamic substrate's envelope model using MD simulations is an approach that could augment the discovery of new inhibitors against 3CLpro in CoVs and other viral 3C proteases.-Zhou, J., Fang, L., Yang, Z., Xu, S., Lv, M., Sun, Z., Chen, J., Wang, D., Gao, J., Xiao, S. Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach.

INPP4B promotes colorectal cancer cell proliferation by activating mTORC1 signaling and cap-dependent translation.

BACKGROUND AND OBJECTIVE: Inositol polyphosphate 4-phosphatase type II (INPP4B) is over-expressed in CRC tissues, and emerges as an oncogene. However, the mechanism by which INPP4B regulates CRC cell proliferation remains largely unclear. In this study, we aimed to investigate the regulatory mechanisms of INPP4B in CRC. MATERIALS AND METHODS: The expression levels of mRNA were detected by qRT-PCR. The expression levels of protein were determined by Western blot. Cell Counting Kit-8 (CCK-8) assays and BrdU incorporation assays were performed to evaluate cell proliferation abilities. Bicistronic luciferase assays and the m7GTP pull down assay were performed to measure the cap-dependent translation in cells. RESULTS: INPP4B promotes CRC cell proliferation by increasing mTORC1 activity. Furthermore, it was shown that the activation of mTORC1 signaling by INPP4B led to increased cap-dependent translation, which is essential for INPP4B-mediated CRC cell proliferation. Finally, it was demonstrated that increased AKT and serum and glucocorticoid-inducible kinase 1 activity contributed to the activation of cap-dependent translation induced by INPP4B. CONCLUSION: Collectively, the present study reveals INPP4B promotes colorectal cancer cell proliferation by activating mTORC1 signaling and cap-dependent translation.

Gut microbial dysbiosis may predict diarrhea and fatigue in patients undergoing pelvic cancer radiotherapy: a pilot study.

Fatigue and diarrhea are the most frequent adverse effects of pelvic radiotherapy, while their etiologies are largely unknown. The aim of this study is to investigate the correlations between fatigue, diarrhea, and alterations in gut microbiota induced by pelvic radiotherapy. During the 5-week treatment of pelvic radiotherapy in 11 cancer patients, the general fatigue score significantly increased and was more prominent in the patients with diarrhea. The fatigue score was closely correlated with the decrease of serum citrulline (an indicator of the functional enterocyte mass) and the increases of systemic inflammatory proteins, including haptoglobin, orosomuoid, α1-antitrypsin and TNF-α. Serum level of lipopolysaccharide (LPS) was also elevated, especially in the patients with diarrhea indicating epithelial barrier breach and endotoxemia. Pyrosequencing analysis of 16S rRNA gene revealed that microbial diversity, richness, and the Firmicutes/Bacteroidetes ratio were significantly altered prior to radiotherapy in patients who later developed diarrhea. Pelvic radiotherapy induced further changes in fecal microbial ecology, some of which were specific to the patients with or without diarrhea. Our results indicate that gut microbial dysbiosis prior to radiation therapy may be exploited to predict development of diarrhea and to guide preventive treatment options. Radiation-induced dysbiosis may contribute to pelvic radiation disease, including mucositis, diarrhea, systemic inflammatory response, and pelvic radiotherapy-associated fatigue in cancer patients.

Nm23-H1 protein binds to APE1 at AP sites and stimulates AP endonuclease activity following ionizing radiation of the human lung cancer A549 cells.

Non-metastatic protein-23 homolog-1 (Nm23-H1) is a multifunctional protein with DNase and histidine protein kinase activities. Human apurinic endonuclease-1 (APE1) is the AP endonuclease DNA base excision repair (BER) enzyme involved in several important cellular functions. Since the relationship between Nm23-H1 and APE1 proteins is unclear, we evaluated their interaction at different time points after irradiating human lung cancer A549 cells with X-rays. We found that Nm23-H1 and APE1 overexpression was induced by irradiation in a dose- and time-dependent manner. Subcellular distribution pattern of both proteins was reversed after irradiation. After irradiation, APE1 that initially showed nuclear localization was gradually increased in the cytoplasm, whereas Nm23-H1 that mainly showed cytoplasmic localization was gradually increased in the nuclei of A549 cells. Nm23-H1 and APE1 interaction was demonstrated by His-pull-down and co-immunoprecipitation assays. The presence of Nm23-H1/APE1 complex in X-ray-irradiated A549 cells was also detected by DNA affinity precipitation analysis of a DNA fragment containing an AP site. Although the AP endonuclease activity of Nm23-H1 was too weak to be detected, the AP endonuclease activity of APE1 was increased with the enhanced Nm23-H1 expression. In conclusion, our data point to a mechanism by which Nm23-H1 protects cells against oxidative stress through the engagement of DNA BER enzyme APE1.

Aberrant expression of the p53-inducible antiproliferative gene BTG2 in hepatocellular carcinoma is associated with overexpression of the cell cycle-related proteins.

We previously reported that the abnormal BTG2 expression was related to genesis/development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate the BTG2 expression in HCC compared with p53, cyclin D1, and cyclin E. For this purpose, modified diethylnitrosamine (DEN)-induced primary HCC rat model was established. Target proteins and mRNAs were measured by western blot and RT-PCR/northern blot, respectively. In rat liver, expression of BTG2 and other proteins was determined by western blot, and BTG2 mRNA in HCC/normal tissues was detected by high-flux tissue microarray (TMA) and in situ hybridization (ISH). BTG2 mRNA/protein expression was increased in fetal liver, 7701, and LO2 cell lines but decreased in HepG2 cells. BTG2/p53 were expressed early after DEN treatment, peaked at 5 weeks and decreased gradually thereafter. Cyclin-D1/Cyclin-E expression increased significantly with the tumor progression. BTG2 mRNA was expressed in 71.19% HCC by ISH and correlated with differentiation. Expression of p53/cyclin D1/cyclin E was positive in 82.35/94.12/76.47% BTG2 mRNA-negative tissues, respectively. BTG2 protein expression was lost in 32.2% (19/59) HCC tissues, and the mRNA/protein expression correlated significantly with the increasing tumor grade (P < 0.05). In conclusion, BTG2 expression is commonly impaired in HCC which may be a factor involved in deregulation of cyclin-D1/cyclin-E expression during hepatocarcinogenesis.

CEA is an independent prognostic indicator that is associated with reduced survival and liver metastases in SCLC.

Small-cell lung cancer (SCLC) is classified into two stages, limited-stage small-cell lung cancer (LS-SCLC) and extensive-stage small-cell lung cancer (ES-SCLC), with ES-SCLC being associated with reduced survival. However, disease stage alone is not a very precise predictor of survival time. The aim of this study is to search for a prognostic indicator that can be used in conjunction with the staging system in SCLC. We performed a prospective analysis of 116 patients with SCLC undergoing standard chemotherapy with or without radiotherapy. Expression levels of 12 tumor markers were measured at the time of diagnosis by a commercial protein chip system, and patients were followed up for a maximum of 54 weeks. CEA was the most frequently detected tumor marker in SCLC, with 32.8% of samples scoring positive, and was also the only marker that correlated with overall survival. The average survival time was 16.78 months for patients with CEA below 5 ng/ml and 11.4 months for patients with CEA above 5 ng/ml (P < 0.001). In contrast, no association was found between tumor stage and overall survival. CEA expression was independent of tumor stage (P = 0.930). CEA is an independent prognostic factor that can be used in conjunction with disease stage in SCLC.

Clinical study of recombinant adenovirus-p53 combined with fractionated stereotactic radiotherapy for hepatocellular carcinoma.

OBJECTIVE: The purpose of our study was to evaluate the feasibility and treatment outcomes of recombinant adenovirus-p53 (rAd-p53, trademarked as Gendicine) combined with fractionated stereotactic radiotherapy (fSRT) in treatment of primary hepatocellular carcinoma (HCC). METHODS: We randomly enrolled 40 patients with HCC treated by fSRT alone (fSRT group) or rAd-p53 combined with fSRT (combined group). Tumor size was 2-5.2 cm (average 3.2 cm). We prescribed 50 Gy in 10 fractions at the 50%-80% isodose line of the planning target volume for 2 weeks in two groups. The combined group was treated with two intratumoral injections of rAd-p53 on day 1 and 8 while fSRT started on day 3. Tumor response was assessed after treatment using modified WHO criteria. The follow-up period was 11-44 months (median 35 months). RESULTS: The overall response rate of fSRT group was 70%, with 4 patients showing complete response (20%), 10 partial response (50%) and 6 stable disease (30%). Correspondingly the overall response rate of combined group was 85%, with 7 patients showing complete response (35%), 10 partial response (50%) and 3 stable disease (15%). The 1-year survival rates of fSRT group and combined group were 70.0% and 90.0%, respectively. The 1-year disease-free survival rates of fSRT group and combined group were 65% and 85%, respectively. These treatments were well tolerated, because grade 3 or 4 toxicity was not observed. CONCLUSION: These results suggest that rAd-p53 combined with fSRT is a relatively safe and effective method for treating primary hepatocellular carcinoma compared with only fSRT. Thus, rAd-p53 combined with fractionated SRT may be preferred as a choice of local treatment for primary HCC when the patients are inoperable or when the patients refuse operation.

MicroRNA-224 is upregulated in HepG2 cells and involved in cellular migration and invasion.

BACKGROUND AND AIM: MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics. METHODS: We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 (miR-224) can influence the biological behaviors of HepG2 cells. RESULTS: MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK4 and MMP9, which were involved in the invasion of tumor, was found. CONCLUSIONS: Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.

Rapid induction of PC3/BTG2 gene by hepatopoietin or partial hepatectomy and its mRNA expression in hepatocellular carcinoma.

BACKGROUND: The anti-proliferative gene, PC3 (pheoch-romocytoma cell 3)/BTG2 (B-cell translocation gene 2), is one of the early growth response genes and belongs to the BTG/Tob protein family. This study aimed to assess the effects of recombinant human hepatopoietin (HPO) and partial hepatectomy on rapidly induced expression of immediate-early genes and to investigate the expression of PC3/BTG2 mRNA in hepatocellular carcinoma (HCC) at different stages of progression. METHODS: After a rat model of partial hepatectomy was established, we investigated gene expression within 1 hour after 2/3 partial hepatectomy by representational difference analysis and in a primary cultured hepatocyte system. The expression levels of PC3/BTG2 from liver tissues of the rat model were assessed by RT-PCR and Northern blotting. Meanwhile, the expression of BTG2 mRNA in a tissue microarray of HCC was determined by in situ hybridization. RESULTS: The PC3/BTG2 gene was rapidly induced after 2/3 partial hepatectomy and its expression peaked within 1-2 hours after operation. HPO rapidly induced the expression of the genes c-fos, LRF-1, and PC3 in primary cultured rat hepatocytes, which might be one of the molecular mechanisms by which HPO stimulates hepatocyte proliferation. Positive BTG2 mRNA expression was detected in 71.19% (42/59) of the HCC samples and in 75% (3/4) of the normal liver tissue samples obtained from the region around the HCC tissues. PC3/BTG2 mRNA was located mainly in the cytoplasm of HCC cells and its expression was related to the degree of differentiation. CONCLUSIONS: Recombinant human HPO and partial hepatectomy rapidly induce the expression of the PC3/BTG2 gene. PC3/BTG2 mRNA is highly expressed in HCC cells and its expression is related to the degree of cell differentiation. The abnormal expression of PC3/BTG2 is closely related to the genesis and development of HCC, so PC3/BTG2 may play an important role in these processes.

[The expression of B-cell translocation gene 2 in diethylnitrosamine-induced primary hepatocellular carcinoma rat model.].

OBJECTIVE: To investigate the expression and role of B-cell translocation gene 2(BTG2) in the carcinogenesis of hepatocellular carcinoma (HCC). METHODS: Modified Diethylnitrosamine (DEN)-induced primary hepatocellular carcinoma rat model was established. The expression of BTG2, p53 and cyclinD1 was detected by RT-PCR, western blot and immunohistochemistry. RESULTS: The BTG2 protein was predominantly localized in the nucleus, with faint cytoplasmic staining in normal liver cells; however, it is mainly a cytoplasmic protein in HCC cells. BTG2 was over-expressed during the early stage after DEN treatment, the expression level peaked at 5 weeks and then it gradually decreased to the normal level after 16 weeks. The expression of cyclin D1 and cyclin E was increased gradually after DEN treatment, and peaked at 16 weeks and 5 weeks respectively. A significant increase in p53 was not observed until 5 weeks after DEN treatment, and it gradually decreased after 16 weeks. CONCLUSIONS: Decreased expression of BTG2 may be an important step in carcinogenesis of HCC. BTG2 may positively regulate p53 expression and negatively regulate cyclin D1 expression in the carcinogenesis of HCC.

gamma-Secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer's disease.

The gamma-secretase complex plays a role in Alzheimer's disease and cancer progression. The development of clinically useful inhibitors, however, is complicated by the role of the gamma-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different gamma-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B gamma-secretase in a mouse Alzheimer's disease model led to improvements of Alzheimer's disease-relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total gamma-secretase activity in the human brain, and thus specific targeting of Aph1B-containing gamma-secretase complexes may help generate less toxic therapies for Alzheimer's disease.