Long noncoding RNAs expression in gastric cancer

Long noncoding RNAs (lncRNAs) have been involved in the pathogenesis of several human cancers including gastric cancer. In the current study, we selected five lncRNAs namely NEAT1, TUG1, PANDA, UCA1, and GHET1 to assess their expressions in gastric cancer samples compared with adjacent noncancerous tissues (ANCTs) from the same patients. Some previous reports have shown contribution of these lncRNAs in gastric cancer. However, we aimed to explore their associations with patients’ clinicopathological data and their potential as diagnostic biomarkers. Significant associations were found between site of primary tumor and relative expression of all lncRNAs in cancer samples compared with ANCTs. Besides, GHET1 relative expression was associated with lymph node status. The diagnostic power of GHET1 was higher from other lncRNAs. Combination of GHET1, TUG1, UCA1, and PANDA increased the diagnostic power and significance (AUC = 0.8; P < 0.0001). The current study supports participation of lncRNAs in the pathogenesis of gastric cancer and highlights their potential as diagnostic biomarkers.     

Nucleic Acid Template and the Risk of a PCR-Induced HIV-1 Drug Resistance Mutation

Background

The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses.

Methods and Findings

We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04% vs. 0.25%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses.

Conclusions

This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.     

Contrast Agents for Quantitative MicroCT of Lung Tumors in Mice

The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (μCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available μCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. μCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4–ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative μCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced μCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.Abbreviations: μCT, microCT, HU, Hounsfield units, RECIST, response evaluation criteria in solid tumorsLung cancer is the leading cause of cancer death worldwide, and non-small–cell lung cancer is the most common form of lung cancer that is diagnosed.¹⁹ Various animal models have been used to mimic these cancers, understand their biology, and evaluate potential therapeutics.^14,20,27 Traditionally, the method to study the disease in vivo has been to inject human tumor derived cell lines subcutaneously in immunodeficient mice (xenografts) or to orthotopically implant tumors in tissues of interest. Xenografts, despite being of human origin, are not ideal models because tumor cell–stroma interactions cannot be reconstituted in the system. Moreover, the tumor microenvironment has been shown to be essential in predicting cancer cell survival, progression, metastasis, and response to therapy.^14,20,27 To overcome these issues, several investigators have propagated tumors orthotopically by either direct injection of tumor cells into the organ of choice, intravenous injection of tumor cell lines, or implantation of patient-derived tumor biopsy samples into immunodeficient mice. These models simulate the tumor microenvironment and bear a closer resemblance to clinical cancer than do xenografts.^11,27 In the past decade, tremendous progress has been made in development of genetically engineered mouse models that reconstitute facets of human disease in the organ of choice. In these models, the tumors are developed in immunocompetent hosts with intact tumor–stroma interactions, and the tumors can be controlled temporally and spatially.^7,14,25,26Despite the many advantages of genetically engineered and orthotopic models, their use has been limited, primarily due to the heterogeneity and technical difficulties associated with monitoring disease progression.¹⁷ Conventional optical imaging is not widely used with genetically engineered and orthotopic models, because these modalities provide low spatial resolution, limited tissue penetration,^1,17 and rarely accomdate reporter genes like luciferase or fluorescent proteins.³⁰ Nuclear imaging (for example, positron-emission tomography and MRI) has been used in evaluating lung tumor models,^7,13,33 but these modalities are not readily available in all facilities, require specialized laboratories (for example, radionucleotide synthesis), and do not achieve accurate quantification of nodular tumors.³³ X-ray CT has been used in human clinical practice to assess lung tumor nodules to predict likelihood of malignancy and to monitor the response of tumors to treatment.^18,23 Response Evaluation Criteria in Solid Tumors (RECIST) scores based on CT imaging data are used routinely in clinical trials and practice.³² Similarly, high-resolution microCT (μCT) scanners have been used successfully to image lung tumors^{6,15,17,24,25,34} in small animals such as rodents. The investigators in the aforementioned studies took advantage of the natural air–tissue contrast within the thorax to identify tumors. However, this method was unable to distinguish tumor and soft tissue from nearby vascular structures.^25,29 This limitation decreased the accuracy of tumor margin demarcation and tumor volumetric measurements.The goal of our study was to compare 3 commercially available μCT contrast agents (products containing iopamidol, iodinated lipid, and inorganic nanoparticulate) to evaluate and accurately quantify lung tumors in preclinical models. Our results showed that, among those we tested, the nanoparticulate product was the best contrast agent for tumor visualization and quantitation. In addition, we demonstrated the utility of contrast-enhanced μCT in a preclinical evaluation of the efficacy of crizotinib (PF-2341066), a small-molecule multikinase inhibitor,^9,10 in a genetically modified mouse model of lung cancer.     

Clinical relevance of SKP2 alterations in metastatic melanoma

In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2.     

The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization,degradation, and toxicity

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry–based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2_911–919, a nine amino acid segment within a flexible interdomain region (LRRK2_853–981), which we designate the “regulatory loop” (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2’s association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2’s interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase–dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.     

Regulation of nitrogen partitioning in field-grown almond trees: Effects of fruit load and foliar nitrogen applications

Two treatments were employed to influence the amount of amino nitrogen (N) transport in phloem. In walnut trees (Juglans regia L.), developing fruit significantly reduced the efflux of foliar-applied ¹⁵N-enriched urea from treated spurs over a 33-day period in comparison with similarly-treated defruited spurs. Those data suggest that local aboveground demand for N influences vascular transport of amino N. In another experiment, a 1% urea solution was applied foliarly to 5-year old `Mission' almond trees [Prunus dulcis (Mill.) D. A. Webb] to increase the concentration of amino N in the phloem. The effect of foliar N treatments on a) the transport and distribution of labelled urea N within the trees over the experimental period and b) the uptake of soil-applied labelled N were determined by replicated whole tree excavation, fractionation into various tree components and mass spectrometric analyses of the ¹⁴N/¹⁵N ratios. Concentrations and composition of amino acids in the phloem and xylem saps of control trees and trees receiving foliar-applied urea were also determined. In foliar urea-treated trees, the amino acid concentrations increased significantly in leaf and bark phloem exudate, within 24 and 96 h, respectively. Foliar-applied urea N was translocated to the roots of almond trees over the experimental period and decreased soil N uptake. The results of these experiments are consistent with the hypothesis that aboveground N demand affects the amount of amino N cycling between shoots and roots, and may be involved in the regulation of soil N uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.     

Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach

Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.     

Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells

Vascular endothelial growth factor (VEGF) is an essential regulator of normal and abnormal blood vessel growth. A monoclonal antibody (mAb) that targets VEGF suppresses tumor growth in murine cancer models and human patients. We investigated cellular and molecular events that mediate refractoriness of tumors to anti-angiogenic therapy. Inherent anti-VEGF refractoriness is associated with infiltration of the tumor tissue by CD11b+Gr1+ myeloid cells. Recruitment of these myeloid cells is also sufficient to confer refractoriness. Combining anti-VEGF treatment with a mAb that targets myeloid cells inhibits growth of refractory tumors more effectively than anti-VEGF alone. Gene expression analysis in CD11b+Gr1+ cells isolated from the bone marrow of mice bearing refractory tumors reveals higher expression of a distinct set of genes known to be implicated in active mobilization and recruitment of myeloid cells. These findings indicate that, in our models, refractoriness to anti-VEGF treatment is determined by the ability of tumors to prime and recruit CD11b+Gr1+ cells.