Peppy: Proteogenomic Search Software

Proteogenomic searching is a useful method for identifying novel proteins, annotating genes and detecting peptides unique to an individual genome. The approach, however, can be laborious, as it often requires search segmentation and the use of several unintegrated tools. Furthermore, many proteogenomic efforts have been limited to small genomes, as large genomes can prove impractical due to the required amount of computer memory and computation time. We present Peppy, a software tool designed to perform every necessary task of proteogenomic searches quickly, accurately and automatically. The software generates a peptide database from a genome, tracks peptide loci, matches peptides to MS/MS spectra and assigns confidence values to those matches. Peppy automatically performs a decoy database generation, search and analysis to return identifications at the desired false discovery rate threshold. Written in Java for cross-platform execution, the software is fully multithreaded for enhanced speed. The program can run on regular desktop computers, opening the doors of proteogenomic searching to a wider audience of proteomics and genomics researchers. Peppy is available at http://geneffects.com/peppy

Mesothelin\u27s minimal MUC16 binding moiety converts TR3 into a potent cancer therapeutic via hierarchical binding events at the plasma membrane

TRAIL has been extensively explored as a cancer drug based on its tumor-selective activity profile but it is incapable per se of discriminating between death receptors expressed by normal host cells and transformed cancer cells. Furthermore, it is well documented that surface tethering substantially increases its biologic activity. We have previously reported on Meso-TR3, a constitutive TRAIL trimer targeted to the biomarker MUC16 (CA125), in which the entire ectodomain of human mesothelin was genetically fused to the TR3 platform, facilitating attachment to the cancer cells via the MUC16 receptor. Here, we designed a truncation variant, in which the minimal 64 amino acid MUC16 binding domain of mesothelin was incorporated into TR3. It turned out that the dual-domain biologic Meso64-TR3 retained its high MUC16 affinity and bound to the cancer cells quickly, independent of the TR3/death receptor interaction. Furthermore, it was substantially more potent than Meso-TR3 and TR3 in vitro and in a preclinical xenograft model of MUC16-dependent ovarian cancer. Phenotypically, Meso64-TR3 is more closely related to non-targeted TR3, evident by indistinguishable activity profiles on MUC16-deficient cancers and similar thermal stability characteristics. Overall, Meso64-TR3 represents a fully human, MUC16-targetd TRAIL-based biologic, ideally suited for exploring preclinical and clinical evaluation studies in MUC16-dependent malignancies

Novel treatment option for MUC16-positive malignancies with the targeted TRAIL-based fusion protein Meso-TR3

BACKGROUND: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. METHODS: Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. RESULTS: Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. CONCLUSIONS: Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies

The novel drug candidate S2/IAPinh improves survival in models of pancreatic and ovarian cancer

Cancer selective apoptosis remains a therapeutic challenge and off-target toxicity has limited enthusiasm for this target clinically. Sigma-2 ligands (S2) have been shown to enhance the cancer selectivity of small molecule drug candidates by improving internalization. Here, we report the synthesis of a novel drug conjugate, which was created by linking a clinically underperforming SMAC mimetic (second mitochondria-derived activator of caspases; LCL161), an inhibitor (antagonist) of inhibitor of apoptosis proteins (IAPinh) with the sigma-2 ligand SW43, resulting in the new chemical entity S2/IAPinh. Drug potency was assessed via cell viability assays across several pancreatic and ovarian cancer cell lines in comparison with the individual components (S2 and IAPinh) as well as their equimolar mixtures (S2 + IAPinh) both in vitro and in preclinical models of pancreatic and ovarian cancer. Mechanistic studies of S2/IAPinh-mediated cell death were investigated in vitro and in vivo using syngeneic and xenograft mouse models of murine pancreatic and human ovarian cancer, respectively. S2/IAPinh demonstrated markedly improved pharmacological activity in cancer cell lines and primary organoid cultures when compared to the controls. In vivo testing demonstrated a marked reduction in tumor growth rates and increased survival rates when compared to the respective control groups. The predicted mechanism of action of S2/IAPinh was confirmed through assessment of apoptosis pathways and demonstrated strong target degradation (cellular inhibitor of apoptosis proteins-1 [cIAP-1]) and activation of caspases 3 and 8. Taken together, S2/IAPinh demonstrated efficacy in models of pancreatic and ovarian cancer, two challenging malignancies in need of novel treatment concepts. Our data support an in-depth investigation into utilizing S2/IAPinh for the treatment of cancer

On the Ortho:Para Ratio of H3+ in Diffuse Molecular Clouds

The excitation temperature T_01 derived from the relative intensities of the J = 0 (para) and J = 1 (ortho) rotational levels of H2 has been assumed to be an accurate measure of the kinetic temperature in interstellar environments. In diffuse molecular clouds, the average value of T_01 is ~70 K. However, the excitation temperature T(H3+) derived from the (J,K) = (1,1) (para) and (1,0) (ortho) rotational levels of H3+ has been observed to be ~30 K in the same types of environments. In this work, we present observations of H3+ in three additional diffuse cloud sight lines for which H2 measurements are available, showing that in 4 of 5 cases T_01 and T(H3+) are discrepant. We then examine the thermalization mechanisms for the ortho:para ratios of H3+ and H2, concluding that indeed T_01 is an accurate measure of the cloud kinetic temperature, while the ortho:para ratio of H3+ need not be thermal. By constructing a steady-state chemical model taking into account the nuclear-spindependence of reactions involving H3+, we show that the ortho:para ratio of H3+ in diffuse molecular clouds is likely governed by a competition between dissociative recombination with electrons and thermalization via reactive collisions with H2.Comment: 13 pages, 8 figures, 5 tables, accepted for publication in Ap

Malic enzyme 1 absence in synovial sarcoma shifts antioxidant system dependence and increases sensitivity to ferroptosis induction with ACXT-3102

PURPOSE: To investigate the metabolism of synovial sarcoma (SS) and elucidate the effect of malic enzyme 1 absence on SS redox homeostasis. EXPERIMENTAL DESIGN: ME1 expression was measured in SS clinical samples, SS cell lines, and tumors from an SS mouse model. The effect of ME1 absence on glucose metabolism was evaluated utilizing Seahorse assays, metabolomics, and C13 tracings. The impact of ME1 absence on SS redox homeostasis was evaluated by metabolomics, cell death assays with inhibitors of antioxidant systems, and measurements of intracellular reactive oxygen species (ROS). The susceptibility of ME1-null SS to ferroptosis induction was interrogated in vitro and in vivo. RESULTS: ME1 absence in SS was confirmed in clinical samples, SS cell lines, and an SS tumor model. Investigation of SS glucose metabolism revealed that ME1-null cells exhibit higher rates of glycolysis and higher flux of glucose into the pentose phosphate pathway (PPP), which is necessary to produce NADPH. Evaluation of cellular redox homeostasis demonstrated that ME1 absence shifts dependence from the glutathione system to the thioredoxin system. Concomitantly, ME1 absence drives the accumulation of ROS and labile iron. ROS and iron accumulation enhances the susceptibility of ME1-null cells to ferroptosis induction with inhibitors of xCT (erastin and ACXT-3102). In vivo xenograft models of ME1-null SS demonstrate significantly increased tumor response to ACXT-3102 compared with ME1-expressing controls. CONCLUSIONS: These findings demonstrate the translational potential of targeting redox homeostasis in ME1-null cancers and establish the preclinical rationale for a phase I trial of ACXT-3102 in SS patients. See related commentary by Subbiah and Gan, p. 3408

The Extended Environment of M17: A Star Formation History

M17 is one of the youngest and most massive nearby star-formation regions in the Galaxy. It features a bright H II region erupting as a blister from the side of a giant molecular cloud (GMC). Combining photometry from the Spitzer GLIMPSE survey with complementary infrared (IR) surveys, we identify candidate young stellar objects (YSOs) throughout a 1.5 deg x 1 deg field that includes the M17 complex. The long sightline through the Galaxy behind M17 creates significant contamination in our YSO sample from unassociated sources with similar IR colors. Removing contaminants, we produce a highly-reliable catalog of 96 candidate YSOs with a high probability of association with the M17 complex. We fit model spectral energy distributions to these sources and constrain their physical properties. Extrapolating the mass function of 62 intermediate-mass YSOs (M >3 Msun), we estimate that >1000 stars are in the process of forming in the extended outer regions of M17. From IR survey images from IRAS and GLIMPSE, we find that M17 lies on the rim of a large shell structure ~0.5 deg in diameter (~20 pc at 2.1 kpc). We present new maps of CO and 13CO (J=2-1) emission, which show that the shell is a coherent, kinematic structure associated with M17 at v = 19 km/s. The shell is an extended bubble outlining the photodissociation region of a faint, diffuse H II region several Myr old. We provide evidence that massive star formation has been triggered by the expansion of the bubble. The formation of the massive cluster ionizing the M17 H II region itself may have been similarly triggered. We conclude that the star formation history in the extended environment of M17 has been punctuated by successive waves of massive star formation propagating through a GMC complex.Comment: 31 pages, 15 figures, accepted for publication in ApJ. For a version with higher-quality figures, see http://www.astro.wisc.edu/glimpse/Povich2009_M17.pd

MetaCyto: A Tool for Automated Meta-analysis of Mass and Flow Cytometry Data

While meta-analysis has demonstrated increased statistical power and more robust estimations in studies, the application of this commonly accepted methodology to cytometry data has been challenging. Different cytometry studies often involve diverse sets of markers. Moreover, the detected values of the same marker are inconsistent between studies due to different experimental designs and cytometer configurations. As a result, the cell subsets identified by existing auto-gating methods cannot be directly compared across studies. We developed MetaCyto for automated meta-analysis of both flow and mass cytometry (CyTOF) data. By combining clustering methods with a silhouette scanning method, MetaCyto is able to identify commonly labeled cell subsets across studies, thus enabling meta-analysis. Applying MetaCyto across a set of ten heterogeneous cytometry studies totaling 2,926 samples enabled us to identify multiple cell populations exhibiting differences in abundance between demographic groups

Delayed Rectifier and A-Type Potassium Channels Associated with Kv 2.1 and Kv 4.3 Expression in Embryonic Rat Neural Progenitor Cells

BACKGROUND: Because of the importance of voltage-activated K(+) channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes. METHODOLOGY/PRINCIPAL FINDINGS: Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and betaIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current. CONCLUSIONS/SIGNIFICANCE: We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K(+) currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells