On the Adaptive Design Rules of Biochemical Networks in Evolution

Biochemical networks are the backbones of physiological systems of organisms. Therefore, a biochemical network should be sufficiently robust (not sensitive) to tolerate genetic mutations and environmental changes in the evolutionary process. In this study, based on the robustness and sensitivity criteria of biochemical networks, the adaptive design rules are developed for natural selection in the evolutionary process. This will provide insights into the robust adaptive mechanism of biochemical networks in the evolutionary process

Raf Activation Is Regulated by Tyrosine 510 Phosphorylation in Drosophila

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved

A 3D Model of the Membrane Protein Complex Formed by the White Spot Syndrome Virus Structural Proteins

Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus), is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process.In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument) proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers.From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented

Vitamin D₃-Inducible Mesenchymal Stem Cell-Based Delivery of Conditionally Replicating Adenoviruses Effectively Targets Renal Cell Carcinoma and Inhibits Tumor Growth

Cell-based carriers were recently exploited as a tumor-targeting tool to improve systemic delivery of oncolytic viruses for cancer therapy. However, the slow clearance of carrier cells from normal organs indicates the need for a controllable system which allows viral delivery only when the carrier cells reach the tumor site. In this study, we sought to develop a pharmaceutically inducible cell-based oncolytic adenovirus delivery strategy for effective targeting and treatment of renal cell carcinoma (RCC), which is one of the most malignant tumor types with an unfavorable prognosis. Herein, we demonstrated the intrinsic tumor homing property of human bone marrow-derived mesenchymal stem cells (hMSCs) to specifically localize primary and metastatic RCC tumors after systemic administration in a clinically relevant orthotopic animal model. The platelet derived growth factor AA (PDGF-AA) secreted from RCC was identified as a chemoattractant responsible for the recruitment of hMSCs. Like endogenous osteocalcin whose barely detectable level of expression was dramatically induced by vitamin D₃, the silenced replication of human osteocalcin promoter-directed Ad-hOC-E1 oncolytic adenoviruses loaded in hMSCs was rapidly activated, and the released oncolytic adenoviruses sequentially killed cocultured RCC cells upon vitamin D₃ exposure. Moreover, the systemic treatment of RCC tumor-bearing mice with hMSC cell carriers loaded with Ad-hOC-E1 had very limited effects on tumor growth, but the loaded hMSCs combined with vitamin D₃ treatment induced effective viral delivery to RCC tumors and significant tumor regression. Therapeutic effects of hMSC-based Ad-hOC-E1 delivery were confirmed to be significantly greater than those of injection of carrier-free Ad-hOC-E1. Our results presented the first preclinical demonstration of a novel controllable cell-based gene delivery strategy that combines the advantages of tumor tropism and vitamin D₃-regulatable human osteocalcin promoter-directed gene expression of hMSCs to improve oncolytic virotherapy for advanced RCC

Loss of Let-7 MicroRNA Upregulates IL-6 in Bone Marrow-Derived Mesenchymal Stem Cells Triggering a Reactive Stromal Response to Prostate Cancer

<div><p>Bone marrow-derived mesenchymal stem cells (MSCs) are able to migrate to tumors, where they promote tumorigenesis and cancer metastasis. However, the molecular phenotype of the recruited MSCs at the tumor microenvironment and the genetic programs underlying their role in cancer progression remains largely unknown. By using a three-dimensional rotary wall vessel coculture system in which human MSCs were grown alone or in close contact with LNCaP, C4-2 or PC3 prostate cancer cell lines, we established <i>in</i><i>vitro</i> matched pairs of normal and cancer-associated MSC derivatives to study the stromal response of MSCs to prostate cancer. We observed that prostate cancer-associated MSCs acquired a higher potential for adipogenic differentiation and exhibited a stronger ability to promote prostate cancer cell migration and invasion compared with normal MSCs both <i>in vitro</i> and in experimental animal models. The enhanced adipogenesis and the pro-metastatic properties were conferred by the high levels of IL-6 secretion by cancer-associated MSCs and were reversible by functionally inhibiting of IL-6. We also found that IL-6 is a direct target gene for the let-7 microRNA, which was downregulated in cancer-associated MSCs. The overexpression of let-7 via the transfection of let-7 precursors decreased IL-6 expression and repressed the adipogenic potential and metastasis-promoting activity of cancer-associated MSCs, which was consistent with the inhibition of IL-6 3′UTR luciferase activity. Conversely, the treatment of normal MSCs with let-7 inhibitors resulted in effects similar to those seen with IL-6. Taken together, our data demonstrated that MSCs co-evolve with prostate cancer cells in the tumor microenvironment, and the downregulation of let-7 by cancer-associated MSCs upregulates IL-6 expression. This upregulation triggers adipogenesis and facilitates prostate cancer progression. These findings not only provide key insights into the molecular basis of tumor-stroma interactions but also pave the way for new treatments for metastatic prostate cancer.</p></div

Identification of let-7 as an IL-6 targeting miRNA in cancer-associated MSCs.

<p>(A) Quantitative RT-PCR analysis of the expression levels of let-7 miRNA in normal 3A6^RWV and cancer-associated 3A6^LNCaP and 3A6^PC3 cell lines. Values are presented as the means ± SD of relative expression levels compared to the U6 internal control and normalized to the 3A6^RWV cells of each matched pair. *<i>P</i><0.005. (B) Regulation of IL-6 by let-7. 3A6 cells were transfected with the let-7c precursor (pre-miR let-7c) or let-7 family inhibitors (anti-miR let-7). Cells and conditioned medium were harvested 72 h after transfection, and IL-6 RNA and protein levels were determined by qRT-PCR and ELISA, respectively. *<i>P</i><0.005. (C) IL-6 response element reporter assay with let-7c precursor or let-7 family inhibitors. The seed region of the let-7c potential target sites in human IL-6 was predicted by microRNA.org. Top, wild-type IL-6 sequence; middle, let-7c sequence; bottom, mutant IL-6 sequence. 3′-UTR vectors with wild-type (pMIR-Luc-WT) or mutant IL-6 (pMIR-Luc-Mut) sequence were cotransfected with let-7c or Mir-199a precursors into HEK 293 cells. Luciferase activities were assayed 48 h after transfection and normalized to internal control β-galactosidase activity. Data are the means ± SD of three independent experiments, *<i>P</i><0.05.</p

Effects of IL-6 on the reactive stromal phenotypes of MSCs.

<p>(A) Cytokine expression profile of the conditioned medium obtained from the normal 3A6^RWV and the prostate cancer-associated 3A6^LNCaP, 3A6^C4-2, and 3A6^PC3 was analyzed by using a Human Cytokine Antibody Array C Series 2000. The autoradiograph of one set of Array VI containing IL-6 spots (rectangular boxes) is presented. (B) Quantitative detection of the expression level of IL-6 by ELISA assay. Data represent the means ± SD of triplicate determination. (C) Chemotactic response of PC3 cells induced by human recombinant IL-6 (rIL-6). Data are represented as the means ± SD of the number of cells per high-power field (100x) in triplicate experiments. *<i>P</i><0.05, **<i>P</i><0.001 <i>vs.</i> serum-free medium alone. (D) Inhibition of PC3 cell migration toward 3A6^PC3 conditioned medium by the anti-IL-6 antibody. Conditioned medium from 3A6^PC3 cells were pre-treated with the indicated concentration of mouse anti-IL-6 antibody and then loaded at the bottom chamber of transwells. Treatment with 30 µg/ml of mouse IgG (mIgG) was a negative control. *<i>P</i><0.05, **<i>P</i><0.001 <i>vs.</i> mouse IgG. (E) Induction of adipogenesis of 3A6^RWV cells by human recombinant IL-6, and (F) inhibition of adipogenesis of 3A6^PC3 cells by anti-IL-6 antibody at the indicated concentration. Cells were stained with Oil red O to visualize lipid droplets after 21 days incubation. The representative image (100x) of each condition is shown at top. The stained cells were quantified using a dye extraction method and data are represented as the means ± SD of three independent experiments. **<i>P</i><0.001 <i>vs.</i> mouse IgG control group.</p