Aberrant epigenetic and genetic marks are seen in myelodysplastic leukocytes and reveal Dock4 as a candidate pathogenic gene on chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.     

Mutant profilin suppresses mutant actin-dependent mitochondrial phenotype in Saccharomyces cerevisiae

In the Saccharomyces cerevisiae actin-profilin interface, Ala(167) of the actin barbed end W-loop and His(372) near the C terminus form a clamp around a profilin segment containing residue Arg(81) and Tyr(79). Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function.     

Molecular modeling of Mycobacterium tuberculosis dUTpase: docking and catalytic mechanism studies

Mycobacterium tuberculosis is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of M. tuberculosis infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-TB agents are necessary. Therefore, dUTpase was selected as a target enzyme to combat M. tuberculosis. In this work, molecular modeling methods involving docking and QM/MM calculations were carried out to investigate the binding orientation and predict binding affinities of some potential dUTpase inhibitors. Our results suggest that the best potential inhibitor investigated, among the compounds studied in this work, is the compound dUPNPP. Regarding the reaction mechanism, we concluded that the decisive stage for the reaction is the stage 1. Furthermore, it was also observed that the compounds with a -1 electrostatic charge presented lower activation energy in relation to the compounds with a -2 charge.     

Neurofibromatosis Type 1 Alternative Splicing Is a Key Regulator of Ras Signaling in Neurons

Neurofibromatosis type I (Nf1) is a GTPase-activating protein (GAP) that inactivates the oncoprotein Ras and plays important roles in nervous system development and learning. Alternative exon 23a falls within the Nf1 GAP domain coding sequence and is tightly regulated in favor of skipping in neurons; however, its biological function is not fully understood. Here we generated mouse embryonic stem (ES) cells with a constitutive endogenous Nf1 exon 23a inclusion, termed Nf1 23aIN/23aIN cells, by mutating the splicing signals surrounding the exon to better match consensus sequences. We also made Nf1 23aΔ/23aΔ cells lacking the exon. Active Ras levels are high in wild-type (WT) and Nf1 23aIN/23aIN ES cells, where the Nf1 exon 23a inclusion level is high, and low in Nf1 23aΔ/23aΔ cells. Upon neuronal differentiation, active Ras levels are high in Nf1 23aIN/23aIN cells, where the exon inclusion level remains high, but Ras activation is low in the other two genotypes, where the exon is skipped. Signaling downstream of Ras is significantly elevated in Nf1 23aIN/23aIN neurons. These results suggest that exon 23a suppresses the Ras-GAP activity of Nf1. Therefore, regulation of Nf1 exon 23a inclusion serves as a mechanism for providing appropriate levels of Ras signaling and may be important in modulating Ras-related neuronal functions.     

Stochastic multi-attribute analysis (SMAA) as an interpretation method for comparative life-cycle assessment (LCA)

Purpose

Comparative life-cycle assessments (LCAs) today lack robust methods of interpretation that help decision makers understand and identify tradeoffs in the selection process. Truncating the analysis at characterization is misleading and existing practices for normalization and weighting may unwittingly oversimplify important aspects of a comparison. This paper introduces a novel approach based on a multi-criteria decision analytic method known as stochastic multi-attribute analysis for life-cycle impact assessment (SMAA-LCIA) that uses internal normalization by means of outranking and exploration of feasible weight spaces.

Methods

To contrast different valuation methods, this study performs a comparative LCA of liquid and powder laundry detergents using three approaches to normalization and weighting: (1) characterization with internal normalization and equal weighting, (2) typical valuation consisting of external normalization and weights, and (3) SMAA-LCIA using outranking normalization and stochastic weighting. Characterized results are often represented by LCA software with respect to their relative impacts normalized to 100 %. Typical valuation approaches rely on normalization references, single value weights, and utilizes discrete numbers throughout the calculation process to generate single scores. Alternatively, SMAA-LCIA is capable of exploring high uncertainty in the input parameters, normalizes internally by pair-wise comparisons (outranking) and allows for the stochastic exploration of weights. SMAA-LCIA yields probabilistic, rather than discrete comparisons that reflect uncertainty in the relative performance of alternatives.

Results and discussion

All methods favored liquid over powder detergent. However, each method results in different conclusions regarding the environmental tradeoffs. Graphical outputs at characterization of comparative assessments portray results in a way that is insensitive to magnitude and thus can be easily misinterpreted. Typical valuation generates results that are oversimplified and unintentionally biased towards a few impact categories due to the use of normalization references. Alternatively, SMAA-LCIA avoids the bias introduced by external normalization references, includes uncertainty in the performance of alternatives and weights, and focuses the analysis on identifying the mutual differences most important to the eventual rank ordering.

Conclusions

SMAA-LCIA is particularly appropriate for comparative LCAs because it evaluates mutual differences and weights stochastically. This allows for tradeoff identification and the ability to sample multiple perspectives simultaneously. SMAA-LCIA is a robust tool that can improve understanding of comparative LCA by decision or policy makers.     

The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells

As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.     

Heterogeneity of Pancreatic Cancer Metastases in a Single Patient Revealed by Quantitative Proteomics

Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the administration of a combination of agents, with each agent targeted to the features of different subclones.Approximately half of the patients with pancreatic cancer are initially diagnosed with metastases to distal sites, with the commonest sites being the liver, lung, and peritoneum (1). Therapeutic strategies against metastases could help reduce the high mortality rates associated with this cancer (2). Understanding the nature of metastatic pancreatic cancer at a systems level can enable the discovery of potential targets for the development of targeted therapies.Pancreatic cancer has been shown to be a genetically evolving and heterogeneous disease (3–5). Clonal diversity and evolution of cancer genomes have also been demonstrated based on the isolation of distinct clonal populations purified directly from patient biopsies by means of flow cytometry followed by genomic characterization (6). A number of reports have documented the adoption of a proteomic approach for the discovery of potential biomarkers in pancreatic cancer (7, 8). However, these studies generally assume pancreatic cancers to be homogeneous, and the emphasis is placed on identifying molecules that are common across a broad array of tumors. There is a lack of studies systematically examining the proteomic changes or signaling pathways across pancreatic cancers to dissect the nature of the heterogeneity of each clone. An excellent setting in which the heterogeneity of tumors can be studied systematically is in a patient harboring metastases to several distant sites. To this end, we chose cells isolated from three metastatic pancreatic lesions of a single patient. The exomes of each tumor site were previously sequenced to study the progression of pancreatic cancer, and the results showed that all cell lines were identical for the genetic status of driver mutations (e.g. KRAS, TP53, and SMAD4) (9). Our hypothesis was that a better understanding of the proteomic consequences of the heterogeneity derived from genetic changes, and possibly other types of alterations, might provide additional opportunities to identify therapeutic targets.In order to precisely quantify differences across the proteomes of multiple metastatic pancreatic cancer lesions, we employed a SILAC-based¹ quantitative proteomics strategy combined with high-resolution mass spectrometry (10, 11). Based on changes observed at the whole-proteome level, we found that a class of cell surface receptors showed significant enrichment with the highest alteration of their expression among the three metastatic pancreatic cancer cell lines examined (i.e. peritoneum, lung, and liver). Because the total protein levels provide information about the static levels of proteins and not their activity per se, we decided to examine the activation of phosphorylation-driven pathways, many of which are activated by cell surface receptors. To globally examine tyrosine phosphorylation-based signaling pathways, we carried out mass spectrometric analysis of purified tyrosine phosphorylated peptides enriched using anti-phosphotyrosine antibodies. As a result, we observed differential activation of tyrosine kinases in the three different sites of metastases. For example, Axl receptor tyrosine kinase was found to be hyperphosphorylated in lung and liver metastases relative to peritoneal metastasis. Expression of Axl receptor tyrosine kinase in primary and matched pancreatic cancers on tissue microarrays was validated by immunohistochemistry. Given such unique patterns of activation of pathways, it was possible that tumors derived from different sites could show differences in their sensitivity to pathway inhibitors. To test this, we performed experiments in which we screened cell lines derived from each metastatic site against a panel of small molecule inhibitors. We observed that the three metastatic pancreatic cancers had differential sensitivities to different inhibitors. For example, cells derived from the peritoneal metastasis were highly sensitive to lapatinib, whereas greater sensitivity to the Axl inhibitor R428 was observed in the lung metastasis cell line. Finally, we showed that treatment of mice bearing xenografts from these different pancreatic cancer cell lines with R428, an inhibitor of Axl receptor tyrosine kinase, led to reduction of tumors with evidence of activation of Axl.