Novel Posttranslational Modification in LKB1 Activation and Function

Cancer cells display dramatic alterations in cellular metabolism to meet their needs of increased growth and proliferation. In the last decade, cancer research has brought these pathways into focus, and one emerging issue that has come to attention is that many oncogenes and tumor-suppressors are intimately linked to metabolic regulation (Jones and Thompson, 2009). One of the key tumor-suppressors involved in metabolism is Liver Kinase B1 (LKB1). LKB1 is the major upstream kinase of the evolutionarily conserved metabolic sensor—AMP-activated protein kinase (AMPK). Activation of the LKB1/AMPK pathway provides a survival advantage for cells under energy stress. LKB1 forms a heterotrimeric complex and is activated through binding of the two regulatory proteins, STRAD and MO25. LKB1 has been shown to be a tumor-suppressor in various mouse models; however, recent studies suggest that LKB1 has pro-oncogenic functions. How the LKB1 activity and the LKB1-STRAD-MO25 complex are maintained and regulated and how LKB1 regulates cancer development are largely unclear. Here we show that K63-linked LKB1 polyubiquitination by the Skp1-Cul1-F-box-protein/Skp2 (Skp2-SCF) ubiquitin ligase complex is critical for LKB1 activation by a mechanism of maintaining the LKB1-STRAD-MO25 complex integrity. We further demonstrate that oncogenic Ras acts upstream of Skp2 to promote LKB1 polyubiquitination by activating the Skp2-SCF ubiquitin ligase complex. Moreover, Skp2-mediated LKB1 polyubiquitination is required for energy stress-induced cell survival. We also detected upregulation and positive correlation of Skp2 and LKB1 expression in late-stage hepatocellular carcinoma (HCC), and their overexpression predicts poor survival outcome of HCC patients. Finally, we show that Skp2-mediated LKB1 polyubiquitination is important for HCC tumor growth in a mouse subcutaneous xenograft tumor model. Our study provides new insights into the upstream regulation of LKB1 activation and suggests a potential target, the Ras/Skp2/LKB1 axis, for cancer therapy

Regulation of Skp2 Expression and Activity and Its Role in Cancer Progression

The regulation of cell cycle entry is critical for cell proliferation and tumorigenesis. One of the key players regulating cell cycle progression is the F-box protein Skp2. Skp2 forms a SCF complex with Skp1, Cul-1, and Rbx1 to constitute E3 ligase through its F-box domain. Skp2 protein levels are regulated during the cell cycle, and recent studies reveal that Skp2 stability, subcellular localization, and activity are regulated by its phosphorylation. Overexpression of Skp2 is associated with a variety of human cancers, indicating that Skp2 may contribute to the development of human cancers. The notion is supported by various genetic mouse models that demonstrate an oncogenic activity of Skp2 and its requirement in cancer progression, suggesting that Skp2 may be a novel and attractive therapeutic target for cancers

Metabolic classification of microbial genomes using functional probes

<p>Abstract</p> <p>Background</p> <p>Microorganisms able to grow under artificial culture conditions comprise only a small proportion of the biosphere's total microbial community. Until recently, scientists have been unable to perform thorough analyses of difficult-to-culture microorganisms due to limitations in sequencing technology. As modern techniques have dramatically increased sequencing rates and rapidly expanded the number of sequenced genomes, in addition to traditional taxonomic classifications which focus on the evolutionary relationships of organisms, classifications of the genomes based on alternative points of view may help advance our understanding of the delicate relationships of organisms.</p> <p>Results</p> <p>We have developed a proteome-based method for classifying microbial species. This classification method uses a set of probes comprising short, highly conserved amino acid sequences. For each genome, <it>in silico </it>translation is performed to obtained its proteome, based on which a probe-set frequency pattern is generated. Then, the probe-set frequency patterns are used to cluster the proteomes/genomes.</p> <p>Conclusions</p> <p>Features of the proposed method include a high running speed in challenge of a large number of genomes, and high applicability for classifying organisms with incomplete genome sequences. Moreover, the probe-set clustering method is sensitive to the metabolic phenotypic similarities/differences among species and is thus supposed potential for the classification or differentiation of closely-related organisms.</p

Fatigue and sleep disturbance related to perceived stress in Chinese HIV-positive individuals: A mixed methods study

Background Few studies of HIV+ individuals in China have examined the associations between HIV-related stress with sleep disturbance and fatigue, which are common complaints among people living with HIV/AIDS (PLWHA). We carried out this study to examine the relationships among perceived stress, sleep disturbance, and fatigue in PLWHA in China. Methods A mixed methods study design was used during data collection in Shanghai, China, from December 2009 to March 2010. Qualitative in-depth interviews were conducted with 19 HIV+ females. Additionally, cross-sectional audio computer-assisted self-interviews (ACASI) were conducted to collect quantitative data from a convenience sample of 107 HIV+ patients (84% were male) including the following scales: 1) Perceived Stress Scale for PLWHA, 2) General Sleep Disturbance Scale, and 3) Fatigue Scale. Results The major themes that emerged from the in-depth interviews were around life stress with HIV, sleep disturbance, and fatigue. Participants presented varying amounts of stress around worrying about whether to disclose their diagnosis and whether they might transmit the disease to their family. In addition, in the cross-sectional data, 40% of the participants reported clinically significant sleep disturbances (GSDS \u3e 3) with an average of 3 nights of disturbed sleep in the past week (M=2.87, SD=1.21) and moderate fatigue severity (M=5.24, SD=2.27). In mediation analyses, the data suggests that the relationship between perceived stress and fatigue was largely (53%) mediated through sleep disturbance. Conclusions Chinese PLWHA described how stress had caused them to become sleepless and fatigued. The quantitative data also demonstrated significant levels of sleep disturbance and fatigue, where were due to perceived stress with HIV disease. A systematic self-management intervention to decrease perceived stress should be designed and implemented in mental health resource-limited settings such as China in order to reduce sleep disturbance and fatigue

Antiretroviral Therapy (ART) Side Effect Impacted on Quality of Life, and Depressive Symptomatology: A Mixed-Method Study

Antiretroviral therapy (ART) is known for its side effects. In this paper, we describe ART side effects as experienced by Chinese HIV+ individuals. This study presents two stages of a research project, combining qualitative in-depth interviews (29 HIV+ participants) with quantitative statistical data analysis (N = 120). All data was collected between July 2005 to March 2008 at Beijing\u27s Ditan Hospital. Consent was obtained from each participant for the qualitative interview and again for the quantitative survey. During in-depth interviews, Chinese HIV+ patients reported experiencing digestive discomfort, skin rashes, numbness, memory loss, nightmares, and dizziness, which not only brought them physical discomfort, but also interrupted different dimensions of their social lives. Furthermore, multiple regression analyses revealed that those who reported more severe side effects also experienced greater depressive mood after controlling for other clinical and psychosocial factors. ART side effects are one of the primary reasons causing HIV+ individuals to delay or stop taking life-saving medication; therefore, clinical interventions are critically needed to assist HIV+ individuals in managing ART side effects. ART side effects reinforced existing negative attitudes toward ART and lead to lower ART adherence. Future research should focus on developing culturally sensitive interventions to enhance HIV+ selfmanagement, to alleviate physical and psychological burden from ART and HIV

Trypsin-induced proteome alteration during cell subculture in mammalian cells

<p>Abstract</p> <p>Background</p> <p>It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.</p> <p>Methods</p> <p>In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.</p> <p>Results</p> <p>36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.</p> <p>Conclusions</p> <p>In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.</p

Patients Infected with CRF07_BC Have Significantly Lower Viral Loads than Patients with HIV-1 Subtype B: Mechanism and Impact on Disease Progression

The circulating recombinant form (CRF) 07_BC is the most prevalent HIV-1 strain among injection drug users (IDUs) in Taiwan. It contains a 7 amino-acid deletion in its p6gag. We conducted a cohort study to compare viral loads and CD4 cell count changes between patients infected with subtype B and CRF07_BC and to elucidate its mechanism. Twenty-one patients infected with CRF07_BC and 59 patients with subtype B were selected from a cohort of 667 HIV-1/AIDS patients whom have been followed up for 3 years. Generalized estimated equation was used to analyze their clinical data and the results showed that patients infected with CRF07_BC had significantly lower viral loads (about 58,000 copies per ml less) than patients with subtype B infection (p = 0.002). The replicative capacity of nine CRF07_BC and four subtype B isolates were compared and the results showed that the former had significantly lower replicative capacity than the latter although all of them were CCR5- tropic and non-syncytium inducing viruses. An HIV-1-NL4-3 mutant virus which contains a 7 amino-acid deletion in p6gag (designated as 7d virus) was generated and its live cycle was investigated. The results showed that 7d virus had significantly lower replication capacity, poorer protease-mediated processing and viral proteins production. Electron microscopic examination of cells infected with wild-type or 7d virus demonstrated that the 7d virus had poorer and slower viral maturation processes: more viruses attached to the cell membrane and higher proportion of immature virions outside the cells. The interaction between p6gag and Alix protein was less efficient in cells infected with 7d virus. In conclusion, patients infected with CRF07_BC had significantly lower viral loads than patients infected with subtype B and it may due to the deletion of 7 amino acids which overlaps with Alix protein-binding domain of the p6gag

Clinical characteristics and risk behavior as a function of HIV status among heroin users enrolled in methadone treatment in northern Taiwan

<p>Abstract</p> <p>Background</p> <p>Methadone treatment was introduced in Taiwan in 2006 as a harm-reduction program in response to the human immunodeficiency virus (HIV), which is endemic among Taiwanese heroin users. The present study was aimed at examining the clinical and behavioral characteristics of methadone patients in northern Taiwan according to their HIV status.</p> <p>Methods</p> <p>The study was conducted at four methadone clinics. Participants were patients who had undergone methadone treatment at the clinics and who voluntarily signed a consent form. Between August and November 2008, each participant completed a face-to-face interview that included questions on demographics, risk behavior, quality of life, and psychiatric symptoms. Data on HIV and hepatitis C virus (HCV) infections, methadone dosage, and morphine in the urine were retrieved from patient files on the clinical premises, with permission of the participants.</p> <p>Results</p> <p>Of 576 participants, 71 were HIV positive, and 514 had hepatitis C. There were significant differences between the HIV-positive and HIV-negative groups on source of treatment payment, HCV infection, urine test results, methadone dosage, and treatment duration. The results indicate that HIV-negative heroin users were more likely to have sexual intercourse and not use condoms during the 6 months prior to the study. A substantial percent of the sample reported anxiety (21.0%), depression (27.2%), memory loss (32.7%), attempted suicide (32.7%), and administration of psychiatric medications (16.1%). There were no significant differences between the HIV-positive and HIV-negative patients on psychiatric symptoms or quality of life.</p> <p>Conclusions</p> <p>HIV-positive IDUs were comorbid with HCV, indicating the need to refer both HIV- and HCV-infected individuals for treatment in methadone clinics. Currently, there is a gap between psychiatric/psychosocial services and patient symptoms, and more integrated medical services should be provided to heroin-using populations.</p