Single cell analysis: the new frontier in 'Omics'

Cellular heterogeneity arising from stochastic expression of genes, proteins, and metabolites is a fundamental principle of cell biology, but single cell analysis has been beyond the capabilities of 'Omics' technologies. This is rapidly changing with the recent examples of single cell genomics, transcriptomics, proteomics, and metabolomics. The rate of change is expected to accelerate owing to emerging technologies that range from micro/nanofluidics to microfabricated interfaces for mass spectrometry to third- and fourth-generation automated DNA sequencers. As described in this review, single cell analysis is the new frontier in Omics, and single cell Omics has the potential to transform systems biology through new discoveries derived from cellular heterogeneity

Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B, plays a critical role in modulating the phosphorylation-dependent CaM binding for IP{sub 3}R1. If combined with other phosphoprotein and phosphopeptide enrichment techniques such as IMAC, our method may serve as a general strategy to identify and characterize phosphorylation-dependent and functionally important protein complexes in mammalian cells

RNA Helicase DDX5 Regulates MicroRNA Expression and Contributes to Cytoskeletal Reorganization in Basal Breast Cancer Cells

RNA helicase DDX5 (also p68) is involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator, but its functional role in breast cancer remains elusive. Here, we report an integrative biology study of DDX5 in breast cancer, encompassing quantitative proteomics, global MicroRNA profiling, and detailed biochemical characterization of cell lines and human tissues. We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was upregulated in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by upregulation of tumor suppressor PDCD4 (a known miR-21 target); as well as upregulation of cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 downregulation. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182. Our results reveal a new functional role of DDX5 in breast cancer via the DDX5→miR-182→actin cytoskeleton pathway, and suggest the potential clinical utility of DDX5 and its downstream MicroRNAs in the theranostics of breast cancer

RETRACTED: A study on expression level and nutritional status of IGFBP-2 after left neck anastomosis combined with placement of feeding nutritional applicators carrying ^(125)I particles in the treatment of esophageal cancer

BackgroundTo explore the changes and significance of the expression level and nutritional status of human insulin-like growth factor binding protein-2 (IGFBP2) after the treatment of esophageal cancer with left neck anastomosis combined with placement of feeding nutritional applicators carrying ^(125)I particles.MethodsA total of 110 patients with esophageal cancer (observation group: left neck anastomosis combined with placement of feeding nutritional applicators carrying ^(125)I particles) and 100 healthy people (control group) were enrolled at the same period. Then enzyme-linked immunosorbent assay (ELISA) was carried out to detect level of IGFBP-2. Lymphocyte count and serum albumin were measured by immune analyzer and automatic protein analyzer to evaluate nutritional status. Logistic regression analysis was used to analyze the relationship between serum IGFBP-2, nutritional status and prognosis of esophageal cancer after combined treatment.ResultsThe albumin, lymphocyte absolute value and PNI detection value of the control group were lower than those of the observation group 1 month after treatment, and the difference was statistically significant compared with the control group. The detection value of IGFBP-2 in early patients before and after treatment was lower than that in middle and late patients, and the detection values of albumin, lymphocyte absolute value and PNI were higher than those in middle and late patients, the differences were statistically significant. Serum IGFBP-2 level was negatively correlated with PNI, and albumin and lymphocyte absolute value were positively correlated with PNI. The detection value of IGFBP-2 in patients with good prognosis was significantly lower than that in patients with poor prognosis, and the detection values of albumin, lymphocyte absolute value and PNI were significantly higher than those in patients with poor prognosis. The AUC (0.887,95% CI: 0.799-0.975) of IGFBP-2, albumin, lymphocyte absolute value and PNI in predicting poor prognosis of esophageal cancer was the largest, and the sensitivity and specificity were 94.12% and 92.47%, respectively.ConclusionsLeft neck anastomosis combined with ^(125)I particle application nutritional tube is helpful for the decrease of serum IGFBP-2 and the increase of various nutritional status indicators, which is beneficial for the improvement of the patient’s condition

Induction of Resistance Mediated by an Attenuated Strain of Valsa mali

To study the induced resistance in apple against Valsa mali var. mali (Vmm), a Vmm–apple callus interaction system was developed to evaluate the induced resistance of an attenuated Vmm strain LXS081501 against further infection by a virulent Vmm strain LXS080601. The infection index was up to 97.32 for apple calli inoculated with LXS080601 alone at 15 days after inoculation whereas it was only 41.84 for calli pretreated with LXS081501 followed by LXS080601 inoculation. In addition, the maximum levels of free proline, soluble sugar, and protein in calli treated with LXS081501 plus LXS080601 were 2.14 to 3.47 times higher than controls and 1.42 to 1.75 times higher than LXS080601 treatment. The activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and catalase (CAT) as well as β-1,3-glucanase and chitinase in apple calli inoculated with LXS080601 alone or LXS081501 plus LXS080601 increased significantly 24 hai and peaked from 48 to 120 hpi. However, in the latter treatment, the maximum enzyme activities were much higher and the activities always maintained much higher levels than control during the experimental period. These results suggested the roles of osmotic adjustment substances and defense-related enzymes in induced resistance

Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-β1

Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor-β1 (TGF-β1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-β1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-β1-induced protein ig-h3 (BGH3) protein levels by TGF-β1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-β1, but calponin 3 was not significantly regulated by mechanical strain or TGF-β1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-β1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-β1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation