Analysis Of Target Proteins Research Articles

Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method.

Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine residues facing the hydrophilic environment, even within the transmembrane domain. Cysteine crosslinking experiments provide us with information about the distance between two cysteine residues. The combination of these methods enables us to obtain information about the structural changes of the target protein. Here, we describe the detailed protocol for structural analysis using SCAM.

Analyses of long non-coding RNA and mRNA profiles in the spinal cord of rats using RNA sequencing during the progression of neuropathic pain in an SNI model

ABSTRACTThe pathogenesis of neuropathic pain (NP) is characterized by an increased responsiveness of nociceptive neurons in the nervous system. However, the molecular mechanisms underpinning the NP still remain elusive. Recent data suggest that long non-coding RNAs (lncRNAs) regulate expression of NP-associated genes. Herein, we analyzed lncRNAs and mRNA profiles in the spinal cord of rats by RNA sequencing during the progression of NP in a spared nerve injury (SNI) model. Sprague-Dawley (SD) rats were employed for the establishment of the SNI models, and nociceptive responses to mechanical and thermal stimuli were measured 3 hours prior to surgery and on postoperative days 1, 3, 7 and 14, with L4-5 spinal cords extracted from three SD rats under deep anesthesia at each time point after behavioral test. SNI rats exhibited higher sensitivity to mechanical stimuli from days 1 to 14. Mechanical hyperalgesia reached a steady peak at day14 after surgery, whereas thermal allodynia did not develop. The results of second-generation sequencing suggested that the expression profiles of lncRNAs and mRNAs were significantly altered in spinal cords of SNI rats versus the control rats at different stages during NP. Differentially expressed (DE) lncRNAs and mRNAs were demonstrated at each stage during the NP course using Volcano Plot, Venn and Hcluster heatmap analyses. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses were performed to predict the functionalities of differentially expressed lncRNAs and target genes. Protein interaction networks were constructed based on the correlation analyses of DE lncRNA target proteins at 7 and 14 days after SNI, respectively. Taken together, our results revealed the profiles of lncRNAs and mRNAs in the rat spinal cord under an NP condition. These lncRNAs and mRNAs may represent new therapeutic targets for the treatment of NP.

Solvent-Responsive Molecularly Imprinted Nanogels for Targeted Protein Analysis in MALDI-TOF Mass Spectrometry.

Molecular imprinted poly(acrylamido)-derivative nanogels have shown their selectivity to bind the protein human serum transferrin (HTR) and also showed their capability for instantaneous solvent-induced modification upon the addition of acetonitrile. Integrated to matrix-assisted laser desorption/ionization time-of-flight mass analysis the HTR-imprinted solvent-responsive nanogels permitted the determination of HTR straight from serum and offered novel perspectives in targeted protein analysis.

Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins

Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation.

The pros and cons of increased trypsin-to-protein ratio in targeted protein analysis

The effect of increasing the trypsin amount in bottom-up based targeted protein analysis is evaluated. By applying an increased trypsin-to-protein ratio (1:1 (w/w)) after heat denaturation (60°C), reduction and alkylation, the digestion time could be reduced profoundly compared to conventional digestion conditions (ratio 1:40, overnight) without compromising method sensitivity or digestion repeatability. The procedure was obtained after a systematic evaluation of trypsin level and trypsin quality using a set of three model proteins: human chorionic gonadotropin (hCG), bovine serum albumin (BSA) and cytochrome C (CytC). All peptides monitored were produced at similar or higher levels after 45min at trypsin-to-protein ratio 1:1, compared to conventional overnight digestion (exception: CytC using modified trypsin, required up to 4h (at 1:1 ratio) in order achieve this). Peptide decay due to chymotryptic activity was observed at longer digestion times, but the effect was circumvented using digestion times <4h. The accelerated digestion protocol (1:1 (w/w), 45min) was applied to realistic human serum samples containing the biomarker protein hCG to demonstrate its applicability.

Trajectory analysis of drug-research trends in pancreatic cancer on PubMed and ClinicalTrials.gov

Increasing interest in developing treatments for pancreatic cancer has led to a surge in publications in the field. Analyses of drug-research trends are needed to minimize risk in anti-cancer drug development. Here, we analyzed publications on anti-cancer drugs extracted from PubMed records and ClinicalTrials datasets. We conducted a drug cluster analysis by proposing the entity Dirichlet Multinomial Regression (eDMR) technique and in-depth network analysis of drug cluster and target proteins. The results show two distinct research clusters in both the ClinicalTrials dataset and the PubMed records. Specifically, various targets associated with anti-cancer drugs are investigated in new drug testing while the diverse chemicals are studied together with a standard therapeutic agent in the academic literature. In addition, our study confirms that drug research published in PubMed is preceded by clinical trials. Although we only evaluate drugs for pancreatic cancer in the present study, our method can be applied to drug-research trends of other diseases.

Binding position analysis of target proteins with the use of amidopyrene probes as LA-LDI enhancing tags.

Amidopyrene-conjugated compounds can be detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. When actin, a cytoskeletal protein, was labeled with an excess amount of amidopyrene N-hydroxysuccinate (apy-OSu), eight apy-labeled actin peptides were predominantly detected by LA-LDI MS. Then actin was labeled with an amidopyrene NHS ester of the antitumor marine macrolide aplyronine A (ApA-apy-OSu) to form a 1 : 1 conjugate. The sequence of an apy-labeled peptide was established as A(108)PLNPKANR(116) by MS/MS analysis, in which the NHS ester moiety specifically reacted with the ε-amino group of K113. While the fragmentation at the linker part reduces the detection sensitivity of apy-labeled peptides on LA-LDI MS, our chemical probe method is useful for analyzing the binding modes of various ligands and target biomacromolecules that include multiple and weak interactions.

Dysregulated Serum MiRNA Profile and Promising Biomarkers in Dengue-infected Patients.

Objectives: Pathological biomarkers and mechanisms of dengue infection are poorly understood. We investigated a new serum biomarker using miRNAs and performed further correlation analysis in dengue-infected patients.Methods: Expression levels of broad-spectrum miRNAs in serum samples from three patients with dengue virus type 1 (DENV-1) and three healthy volunteers were separately analyzed using miRNA PCR arrays. The expressions of the five selected miRNAs were verified by qRT-PCR in the sera of 40 DENV-1 patients and compared with those from 32 healthy controls. Receiver operating characteristic (ROC) curve and correlation analyses were performed to evaluate the potential of these miRNAs for the diagnosis of dengue infection.Results: MiRNA PCR arrays revealed that 41 miRNAs were upregulated, whereas 12 miRNAs were down-regulated in the sera of DENV-1 patients compared with those in healthy controls. Among these miRNAs, qRT-PCR validation showed that serum hsa-miR-21-5p, hsa-miR-590-5p, hsa-miR-188-5p, and hsa-miR-152-3p were upregulated, whereas hsa-miR-146a-5p was down-regulated in dengue-infected patients compared with healthy controls. ROC curves showed serum hsa-miR-21-5p and hsa-miR-146a-5p could distinguish dengue-infected patients with preferable sensitivity and specificity. Correlation analysis indicated that expression levels of serum hsa-miR-21-5p and hsa-miR-146a-5p were negative and positively correlated with the number of white blood cells and neutrophils, respectively. Functional analysis of target proteins of these miRNAs in silico indicated their involvement in inflammation and cell proliferation.Conclusion: Dengue-infected patients have a broad “fingerprint” profile with dysregulated serum miRNAs. Among these miRNAs, serum hsa-miR-21-5p, hsa-miR-146a-5p, hsa-miR-590-5p, hsa-miR-188-5p, and hsa-miR-152-3p were identified as promising serum indicators for dengue infection.

MicroRNA Profiling Implies New Markers of Gemcitabine Chemoresistance in Mutant p53 Pancreatic Ductal Adenocarcinoma.

BackgroundNo reliable predictors of susceptibility to gemcitabine chemotherapy exist in pancreatic ductal adenocarcinoma (PDAC). MicroRNAs (miR) are epigenetic gene regulators with tumorsuppressive or oncogenic roles in various carcinomas. This study assesses chemoresistant PDAC for its specific miR expression pattern.MethodsGemcitabine-resistant variants of two mutant p53 human PDAC cell lines were established. Survival rates were analyzed by cytotoxicity and apoptosis assays. Expression of 1733 human miRs was investigated by microarray and validated by qRT-PCR. After in-silico analysis of specific target genes and proteins of dysregulated miRs, expression of MRP-1, Bcl-2, mutant p53, and CDK1 was quantified by Western blot.ResultsBoth established PDAC clones showed a significant resistance to gemcitabine (p<0.02) with low apoptosis rate (p<0.001) vs. parental cells. MiR-screening revealed significantly upregulated (miR-21, miR-99a, miR-100, miR-125b, miR-138, miR-210) and downregulated miRs (miR-31*, miR-330, miR-378) in chemoresistant PDAC (p<0.05). Bioinformatic analysis suggested involvement of these miRs in pathways controlling cell death and cycle. MRP-1 (p<0.02) and Bcl-2 (p<0.003) were significantly overexpressed in both resistant cell clones and mutant p53 (p = 0.023) in one clone.ConclusionConsistent miR expression profiles, in part regulated by mutant TP53 gene, were identified in gemcitabine-resistant PDAC with significant MRP-1 and Bcl-2 overexpression. These results provide a basis for further elucidation of chemoresistance mechanisms and therapeutic approaches to overcome chemoresistance in PDAC.

Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe.

Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S)-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014) [1] have been deposited to the ProteomeXchange with identifier PXD001200.

Aptamer Recognition Induced Target-Bridged Strategy for Proteins Detection Based on Magnetic Chitosan and Silver/Chitosan Nanoparticles Using Surface-Enhanced Raman Spectroscopy.

Poor selectivity and biocompability remain problems in applying surface-enhanced Raman spectroscopy (SERS) for direct detection of proteins due to similar spectra of most proteins and overlapping Raman bands in complex mixtures. To solve these problems, an aptamer recognition induced target-bridged strategy based on magnetic chitosan (MCS) and silver/chitosan nanoparticles (Ag@CS NPs) using SERS was developed for detection of protein benefiting from specific affinity of aptamers and biocompatibility of chitosan (CS). In this process, one aptamer (or antibody) modified MCS worked as capture probes through the affinity binding site of protein. The other aptamer modified Raman report molecules encapsulated Ag@CS NPs were used as SERS sensing probes based on the other binding site of protein. The sandwich complexes of aptamer (antibody)/protein/aptamer were separated easily with a magnet from biological samples, and the concentration of protein was indirectly reflected by the intensity variation of SERS signal of Raman report molecules. To explore the universality of the strategy, three different kinds of proteins including thrombin, platelet derived growth factor BB (PDGF BB) and immunoglobulin E (lgE) were investigated. The major advantages of this aptamer recognition induced target-bridged strategy are convenient operation with a magnet, stable signal expressing resulting from preventing loss of report molecules with the help of CS shell, and the avoidance of slow diffusion-limited kinetics problems occurring on a solid substrate. To demonstrate the feasibility of the proposed strategy, the method was applied to detection of PDGF BB in clinical samples. The limit of detection (LOD) of PDGF BB was estimated to be 3.2 pg/mL. The results obtained from human serum of healthy persons and cancer patients using the proposed strategy showed good agreement with that of the ELISA method but with wider linear range, more convenient operation, and lower cost. The proposed strategy holds great potential in highly sensitive and selective analysis of target proteins in complex biological samples.

Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737.

BAG3 is overexpressed in several malignancies and mediates a non-canonical, selective form of (macro)autophagy. By stabilizing pro-survival Bcl-2 proteins in complex with HSP70, BAG3 can also exert an apoptosis-antagonizing function. ABT-737 is a high affinity Bcl-2 inhibitor that fails to target Mcl-1. This failure may confer resistance in various cancers. Urothelial cancer cells were treated with the BH3 mimetics ABT-737 and (-)-gossypol, a pan-Bcl-2 inhibitor which inhibits also Mcl-1. To clarify the importance of the core autophagy regulator ATG5 and BAG3 in ABT-737 treatment, cell lines carrying a stable lentiviral knockdown of ATG5 and BAG3 were created. The synergistic effect of ABT-737 and pharmaceutical inhibition of BAG3 with the HSF1 inhibitor KRIBB11 or sorafenib was also evaluated. Total cell death and apoptosis were quantified by FACS analysis of propidium iodide, annexin. Target protein analysis was conducted by Western blotting. Knockdown of BAG3 significantly downregulated Mcl-1 protein levels and sensitized urothelial cancer cells to apoptotic cell death induced by ABT-737, while inhibition of bulk autophagy through depletion of ATG5 had no discernible effect on cell death. Similar to knockdown of BAG3, pharmacological targeting of the BAG3/Mcl-1 pathway with KRIBB11 was capable to sensitize both cell lines to treatment with ABT-737. Our results show that BAG3, but not bulk autophagy has a major role in the response of bladder cancer cells to BH3 mimetics. They also suggest that BAG3 is a suitable target for combined therapies aimed at synergistically inducing apoptosis in bladder cancer.

Genetically encoded sensors of protein hydrodynamics and molecular proximity

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry

Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.

Myeloperoxidase-dependent Lipid Peroxidation Promotes the Oxidative Modification of Cytosolic Proteins in Phagocytic Neutrophils

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.

Abscission research: what we know and what we still need to study

Purpose of review: Abscission is a programmed developmental process initiated by auxin depletion. This review summarizes the mechanisms leading to auxin depletion in the abscission zone (AZ), evaluates the methods for estimation of the spatio-temporal auxin levels, demonstrates how auxin depletion occurs during natural, stress-induced, and artificially-induced organ abscission, and presents new evidence for early and late events resulting from auxin depletion which lead to organ abscission.Findings: Auxin depletion occurs during natural developmental processes which end in organ abscission (leaf and flower senescence, fruit ripening, and self-pruning) and stress-induced abscission, and following artificial organ removal in the tomato model system. Stress-induced auxin depletion is mediated by increased ethylene and reactive oxygen species (ROS) production and carbohydrate starvation. Similar changes in auxin-related genes occurred in both flower AZ (FAZ) and leaf AZ (LAZ) following flower or leaf removal, respectively, suggesting a similar regulation of the abscission process of these organs. Auxin depletion resulted from decreased indole-3-acetic acid (IAA) biosynthesis and transport, as well as from enhanced IAA transport autoinhibition (ATA), conjugation and oxidative IAA catabolism. Functional analyses of several target genes delaying abscission, such as Knotted-Like Homeobox Protein1 (KD1), Tomato Proline Rich Protein (TPRP), Ethylene Responsive Factor52 (ERF52), and Ribonuclease LX (LX), shed light on various events operating in response to auxin depletion in tomato FAZ and/or LAZ. The information gained allows a better understanding of the abscission process driven by auxin depletion, and might lead to development of improved methods for abscission control in horticultural crops.Direction for future research: A better understanding of abscission regulation as it pertains to auxin depletion will require advanced molecular tools such as microarrays, new generation sequencing (NGS), transcriptomic, functional, and proteomic analyses of target genes and proteins found to operate in the abscission process.

Dysregulated Serum MicroRNA Expression Profile and Potential Biomarkers in Hepatitis C Virus-infected Patients.

Objectives: Circulating microRNAs (miRNAs) play critical roles in pathogen-host interactions. Aberrant miRNA expression profiles might have specific characteristics for virus strains, and could serve as noninvasive biomarkers for screening and diagnosing infectious diseases. In this study, we aimed to find new potential miRNA biomarkers of hepatitis C virus (HCV) infection.Methods: Expression levels of broad-spectrum miRNAs in serum samples from 10 patients with HCV viremia and 10 healthy volunteers were analyzed using miRNA PCR arrays. Subsequently, the differential expression of four selected miRNAs (miR-122, miR-134, miR-424-3p, and miR-629-5p) was verified by qRT-PCR in the serum of 39 patients compared with that in 29 healthy controls. Receiver operating characteristic (ROC) curve analysis was performed to evaluate their potential for the diagnosis of HCV infection.Results: miRNA PCR array assays revealed differential expression of 106 miRNAs in sera of HCV patients compared with that in healthy controls. Serum hsa-miR-122, miR-134, miR-424-3p, and miR-629-5p were well identified. The ROC curves showed that miR-122, miR-134, miR-424-3p, and miR-629-5p could distinguish HCV patients with preferable sensitivity and specificity. In addition, Correlation analysis indicated serum miR-122 expression was positive correlation with ALT/AST levels. Functional analysis of target proteins of these miRNAs indicated the involvement of viral replication, inflammation, and cell proliferation.Conclusion: HCV patients have a broad 'fingerprint' profile with dysregulated serum miRNAs compared with that in healthy controls. Among these, serum hsa-miR-122, miR-134, miR-424-3p, and miR-629-5p are identified as promising indication factors of the serum miRNA profile of HCV infection. Particularly, miR-122 could be one of serum biomarkers for early pathological process of HCV. However, more miRNA biomarkers and biological functions of these miRNAs require further investigation.

Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response.

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

Abstract 1422: Validation of primary non-small cell lung cancer model: Stable isotope-resolved metabolomics (SIRM) reveals functional biochemistry

Abstract Stable isotope-resolved metabolomics (SIRM) provides a comprehensive and functional readout of cellular activity and is therefore a useful tool both for biomarker discovery and fundamental cancer biology. We have established a primary lung squamous cell carcinoma line (PSLC1), isolated from the lung tumor tissue of a non-small cell lung cancer patient, for which the metabolic profile has been established. We used SIRM to characterize the metabolic activity of the cells in mouse xenografts and in cell culture. Here we report the metabolic activities of the cell line in culture using two different tracers to evaluate central metabolism. PSLC1 cells were grown in either [U-13C]-glucose or [U-13C,15N]-glutamine labeled medium. Cell extracts and medium were prepared and analyzed by 1H-NMR, HSQC, TOCSY, GC-MS, and FTICR-MS. Cells exhibited linear glucose consumption that persisted even as proliferation plateaued. The conversion of glucose to lactate occurred at a strikingly high 89%. Anaplerotic pathways involving pyruvate carboxylation and glutaminolysis were active and appeared to be dependent on cellular proliferation. Glutamine represented an important contributor for biosynthetic and bioenergetic cellular needs, including de novo pyrimidine biosynthesis and glucose-independent TCA cycling. In addition, glutamine served as the major precursor in proline and glutathione biosynthesis. Distinct glutamine utilization and labeling patterns indicated that this cancer might be driven by C-MYC overexpression. C-MYC overexpression was observed by targeted protein analysis guided by metabolomics-edited transcriptomics analysis (Pro-META). The ability of SIRM to generate hypotheses about protein expression demonstrates its potential use as a first line biomarker detection method. Comparison with upstream in situ and xenograft data will allow us to assess model validity and the effects of tumor microenvironment. In turn, the in vitro data will help us interpret human in situ data more robustly. Supported by grant R25-CA-134283 and 1P01CA163223-01A1 from the National Cancer Institute Citation Format: Connor J. Kinslow, Teresa W-M Fan, Jin L. Tan, Pawel K. Lorkiewicz, Ramya Balasubramaniam, Yihua Cai, Jun Yan, Andrew N. Lane. Validation of primary non-small cell lung cancer model: Stable isotope-resolved metabolomics (SIRM) reveals functional biochemistry. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1422. doi:10.1158/1538-7445.AM2014-1422

Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions: a study on postmenopausal monozygotic twin pairs.

MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen’s causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes’ IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.