Conditional Deletion of the Pten Gene in the Mouse Prostate Induces Prostatic Intraepithelial Neoplasms at Early Ages but a Slow Progression to Prostate Tumors

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, Pten^LoxP:Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of Pten^LoxP/LoxP:Osr1-Cre mice as early as 2 weeks of age. Intriguingly, Pten^LoxP/LoxP:Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. Pten^LoxP/+:Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of Pten^LoxP/LoxP:Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated Pten^LoxP/LoxP:Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.     

β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar¹,Ile⁴,Ile⁸]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.     

Immunization with a Thermostable Newcastle Disease Virus K148/08 Strain Originated from Wild Mallard Duck Confers Protection against Lethal Viscerotropic Velogenic Newcastle Disease Virus Infection in Chickens

Newcastle disease (ND) is one of the most devastating poultry infections because of its worldwide distribution and accompanying economical threat. In the present study, we characterized the ND virus (NDV) K148/08 strain from wild mallard duck, with regard to safety, thermostability, immunogenicity, and protective efficacy against velogenic ND viral infection. The NDV K148/08 strain offered enhanced immunogenicity and safety relative to commercially available vaccine strains. The NDV K148/08 strain was safe in 1-day-old SPF chicks after vaccination using a coarse or cabinet-type fine sprayer. We demonstrated that the NDV K148/08 strain elicited high levels of antibody responses and provided protective efficacy against lethal NDV challenge. In addition, the thermostability of the NDV K148/08 strain was as high as that of the thermostable V4 strain. Therefore, the NDV K148/08 strain may be useful to ensure NDV vaccine performance and effectiveness in developing countries, especially in remote areas without cold chains.     

The Smartphone Addiction Scale: Development and Validation of a Short Version for Adolescents

Objective

This study was designed to investigate the revised and short version of the smartphone addiction scale and the proof of its validity in adolescents. In addition, it suggested cutting off the values by gender in order to determine smartphone addiction and elaborate the characteristics of smartphone usage in adolescents.

Method

A set of questionnaires were provided to a total of 540 selected participants from April to May of 2013. The participants consisted of 343 boys and 197 girls, and their average age was 14.5 years old. The content validity was performed on a selection of shortened items, while an internal-consistency test was conducted for the verification of its reliability. The concurrent validity was confirmed using SAS, SAPS and KS-scale. Receiver operating characteristics analysis was conducted to suggest cut-off.

Results

The 10 final questions were selected using content validity. The internal consistency and concurrent validity of SAS were verified with a Cronbach''s alpha of 0.911. The SAS-SV was significantly correlated with the SAS, SAPS and KS-scale. The SAS-SV scores of gender (p<.001) and self-evaluation of smartphone addiction (p<.001) showed significant difference. The ROC analysis results showed an area under a curve (AUC) value of 0.963(0.888–1.000), a cut-off value of 31, sensitivity value of 0.867 and specificity value of 0.893 in boys while an AUC value of 0.947(0.887–1.000), a cut-off value of 33, sensitivity value of 0.875, and a specificity value of 0.886 in girls.

Conclusions

The SAS-SV showed good reliability and validity for the assessment of smartphone addiction. The smartphone addiction scale short version, which was developed and validated in this study, could be used efficiently for the evaluation of smartphone addiction in community and research areas.     

Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing

Background

Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.

Methods and Findings

We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.

Conclusions

Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.     

Drought Responses of Foliar Metabolites in Three Maize Hybrids Differing in Water Stress Tolerance

Maize (Zea mays L.) hybrids varying in drought tolerance were treated with water stress in controlled environments. Experiments were performed during vegetative growth and water was withheld for 19 days beginning 17 days after sowing. Genotypic comparisons used measured changes of leaf water potential or results were expressed by time of treatment. Total dry matter of the drought tolerant hybrid on the final harvest was 53% less than that of the intermediate and susceptible maize hybrids when plants were water sufficient. This showed that maize hybrids selected for extreme drought tolerance possessed a dwarf phenotype that affected soil water contents and leaf water potentials. Changes of shoot and root growth, leaf water potential, net photosynthesis and stomatal conductance in response to the time of water stress treatment were diminished when comparing the drought tolerant to the intermediate or susceptible maize hybrids. Genotypic differences were observed in 26 of 40 total foliar metabolites during water stress treatments. Hierarchical clustering revealed that the tolerant maize hybrid initiated the accumulation of stress related metabolites at higher leaf water potentials than either the susceptible or intermediate hybrids. Opposite results occurred when changes of metabolites in maize leaves were expressed temporally. The above results demonstrated that genotypic differences were readily observed by comparing maize hybrids differing in drought tolerance based on either time of treatment or measured leaf water potential. Current findings provided new and potentially important insights into the mechanisms of drought tolerance in maize.     

Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains

Background

Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes.

Methods/Principal Findings

Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported.

Conclusions/Significance

The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.     

Tumor-infiltrating PD1-Positive Lymphocytes and FoxP3-Positive Regulatory T Cells Predict Distant Metastatic Relapse and Survival of Clear Cell Renal Cell Carcinoma

BACKGROUND: Clear cell renal cell carcinoma (CRCC) is the most common malignant tumor of the kidney, and the clinical outcome of CRCC is related with the metastatic potential of CRCC. A significant proportion of metastatic CRCC remains incurable. Recently, immunotherapy against specific targets such as programmed death 1 (PD1) has been adapted for fatal cases of CRCC. MATERIALS AND METHODS: In this study, we aimed to evaluate the potential of tumor-infiltrating PD1-positive lymphocytes or FoxP3-positive regulatory T cells (Tregs) as predictors of the metastatic potential or prognosis of CRCC and investigate possible correlations with Epstein-Barr virus (EBV) infection in 199 cases of CRCC. RESULTS: PD1 positivity, high Treg number, and EBV infection all predicted poor overall survival (OS) by univariate analysis. PD1 positivity and high Treg numbers were also significantly correlated with more distant metastatic relapse (DMR) and poor relapse-free survival (RFS) by univariate analysis. PD1 positivity and high Treg number were independent prognostic indicators for OS. In addition, PD1 positivity was an independent predictor of RFS and DMR. EBV infection was an independent predictor of OS of CRCC. CONCLUSION: This study demonstrates that intratumoral infiltration of PD1-positive or FoxP3-positive lymphocytes can be used as significant prognostic indicators of CRCC and PD1 positivity could be very helpful in the prediction of latent distant metastasis of CRCCs. Therefore, evaluation of the infiltration of PD-positive cells or Tregs in CRCC may be useful diagnostic tools for the selection of patients who could benefit from PD1- or Treg-based immunotherapy.     

Expression of DBC1 and Androgen Receptor Predict Poor Prognosis in Diffuse Large B Cell Lymphoma

BACKGROUND: Recently, deleted in breast cancer 1 (DBC1) has been suggested as a poor prognostic indicator of various human cancers and may possibly have a role as a coactivator of androgen receptor (AR). However, their roles in lymphoma are still unknown. MATERIALS AND METHODS: We investigated the effect of the expression of DBC1 and AR in diffuse large B cell lymphoma (DLBCL). Immunohistochemical expression of DBC1 and AR were evaluated in 101 DLBCL samples by tissue microarray. RESULTS: Positive expression of DBC1 and AR was seen in 73% and 70% of DLBCL, respectively. In total DLBCL patients, DBC1 and AR expression were significantly associated with high clinical stage, elevated serum lactate dehydrogenase levels, and high international prognostic index scores, and they predicted shorter overall survival (OS) and relapse-free survival (RFS) by univariate analysis. DBC1 expression was also an independent prognostic indicator by multivariate analysis (OS, P = .017; RFS, P = .004). Especially, both DBC1 and AR expression significantly correlated with shorter OS and RFS in non-germinal center B cell (non-GCB)-type DLBCL by univariate analysis. In multivariate analysis, DBC1 expression was an independent prognostic predictor for OS (P = .035) and AR expression significantly correlated with RFS (P = .005). CONCLUSION: We demonstrate that the expression of DBC1 and AR are significant prognostic indicators for DLBCL patients, especially for unfavorable non-GCB-type DLBCL.     

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells

Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Identifying agents that target these cells will advance the development of more effective therapies. Recent converging evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti- hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib). c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls. These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.