Prognostic impact of Metadherin–SND1 interaction in colon cancer

The interaction between Metadherin (MTDH) and Staphylococcal nuclease homology domain containing 1 (SND1) is involved in tumorigenesis and tumor progression of several human malignancies. However, its roles in colon cancer are still unclear. To investigate the clinical value of MTDH and SND1 expression in colon cancer. Immunohistochemical staining was performed to detect the expression of MTDH and SND1 using human colon cancer and their corresponding non-cancerous colon tissues from 196 patients’ biopsies. Positive expression of MTDH and SND1 were both increased in colon cancer tissues compared to paired non-cancerous colon tissues. There was a positive correlation between MTDH and SND1 expression in colon cancer tissues (r?=?0.86, p?<?0.001). In addition, their positive expression were both significantly associated with nodal status (both p?=?0.02), pathological stage (p?=?0.006 and 0.008, respectively) and differentiation (both p?=?0.03). Moreover, the overall survival in colon cancer patients with positive expression of MTDH and SND1 were significantly shorter than those without their expression (both p?=?0.01). Furthermore, multivariate Cox regression analysis suggested that positive expression of MTDH and SND1 was an independent poor prognostic predictor in colon cancer. Our data suggest that the increased expression of MTDH and/or SND1 is closely related to carcinogenesis, progression, and prognosis of colon cancer. The co-expression of MTDH/SND1 may be a novel distinctive marker to benefit us in prediction of the prognosis in colon cancer.     

Role of high-mobility group box-1 protein in disruption of vascular barriers and regulation of leukocyte–endothelial interactions

Abstract

High-mobility group box-1 protein (HMGB1) is a highly conserved non-histone DNA-binding protein present in the nuclei and cytoplasm of nearly all cell types. The results from recent research provide evidence that HMGB1 is secreted into the extracellular milieu and acts as a pro-inflammatory cytokine and exhibits angiogenic effects to fire the immunological response against the pathological effects. Recently, a great deal of evidence has indicated the critical importance of HMGB1 in mediating vascular barriers dysfunction by modulating the expression of adhesion molecules, such as intercellular adhesion molecule-1, vascular cell adhesion protein 1 and E-selectin on the surface of endothelial cells. Such process promotes the adhesion and migration of leukocytes across the endothelium, leading to breakdown of vascular barriers (blood–brain barrier and blood–retinal barrier) via modulating the expression, content, phosphorylation, and distribution of tight junction proteins. Therefore, here we give an abridged review to understand the mechanistic link between HMGB1 and vascular barriers dysfunction, including interaction with cell-surface receptors and intracellular signaling pathways.     

The effect of CD137–CD137 ligand interaction on phospholipase C signaling pathway in human endothelial cells

We previously reported the emerging role of CD137–CD137L interaction in inflammation and atherosclerosis. The mechanism of CD137–CD137L interaction may be related to a variety of signaling pathways. The most important signaling pathway involves the activation of phospholipase C(PLC) which induces the diacylglycerol–protein kinase C(DAG–PKC) and the inositol trisphosphate-intracellular free calcium (IP₃-[Ca²⁺]i) pathway. In the current study, we investigated whether CD137–CD137L interaction can stimulate the PLC signaling pathway in human umbilical vein endothelial cells (HUVEC). The diacylglycerol (DAG) and inositol trisphosphate (IP₃) levels in HUVEC were measured by radioenzymatic assay. The activity of protein kinase (PKC) was detected by its ability to transfer phosphate from [γ-³²P]ATP to lysine-rich histone. The [Ca²⁺]i concentrations were measured by flow cytometric analysis. The DAG level and PKC activity were increased in a concentration-dependent, biphasic manner in HUVEC induced by anti-CD137. PKC activity was mainly in the cytosol at rest, and then translocated to the membrane when stimulated by anti-CD137. Similarly, rapid IP₃ formation induced by anti-CD137 coincided with the peak of the DAG level. Moreover, anti-CD137 induced peak [Ca²⁺]i responses including the rapid transient phase and the sustained phase. However, anti-CD137L suppressed the activation of the DAG–PKC and IP₃-[Ca²⁺]i signaling pathway, which was stimulated by anti-CD137 in HUVEC. In conclusion, the data suggested that CD137–CD137L interaction induces robust activation of the PLC signaling pathway in HUVEC.     

Species-specific interaction of seminal plasma on sperm–neutrophil binding

Bovine semen is naturally deposited in the vagina and spermatozoa migrate through the cervix into the uterus leaving the bulk of seminal plasma (SP) behind. In equine, both spermatozoa and SP are deposited directly in the uterus and SP reduces sperm binding to neutrophils and prevents the formation of DNA-based neutrophil extracellular traps (NETs). We investigated the role of bovine SP on sperm–neutrophil binding using the four most common bovine semen extenders. Contrary to equine, bovine spermatozoa removed from SP had low binding to neutrophils for up to 3 h, but as little as 10% SP increased sperm–neutrophil binding and NETs formation over time. Similar results were obtained with neutrophils isolated from peripheral blood or from the uterus. Scanning electron microscopy showed that the binding can be mediated by NETs or by direct attachment of the cell membranes for both species. The increased binding with SP reduced the number of free spermatozoa indicating that sperm transport to the site of fertilization (and thus fertility) may be hindered. Surprisingly, egg yolk negated the role of bovine SP on sperm–neutrophil binding compared to all the other semen extenders, but did not alter equine sperm binding to neutrophils. Current artificial insemination in bovine relies heavily on egg yolk extender and introduces variable amounts of SP into the uterus, which naturally remains in the vagina. Our results indicate a need to re-evaluate the composition of semen extenders and the semen processing procedures in relation to sperm transport, longevity and fertilizing ability.     

Effects of methylation of deiodinase 3 gene on gene expression and severity of Kashin–Beck disease

Kashin–Beck disease (KBD) is a complex endemic osteoarthropathy, which mainly occurs in the northeast to southwest China. Iodothyronine deiodinases 3 (DIO3) is one of the selenoproteins, which is closely related to bone metabolism and unclear to KBD. This study aims to investigate the role and associated mechanisms of methylation and expression of DIO3 with disease severity in patients with KBD. We performed a bioinformatics analysis first to identify the biological mechanisms involved in selenoproteins. The methylation status of the DIO3 gene and DIO3 gene expression, as well as DIO3-related regulatory genes in patients with KBD, were analyzed. We found that 15 CpG sites of six selenoproteins were hypomethylated with 5-azacytidine treatment. DIO3 hypermethylation was associated with an increased risk of KBD and may lead to downregulation of DIO3 gene expression as well as be an indicator of the severity of KBD, which may provide a new insight for gene–environment correlations and interactions in etiology and pathogenesis of KBD.     

The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism

Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity. ¹³C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by Bcl-2∆. Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase. This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD. Additionally, Bcl-2∆ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. This is the first comprehensive ¹³C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures.     

Discovery of novel inhibitors of vascular endothelial growth factor-A–Neuropilin-1 interaction by structure-based virtual screening

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A₁₆₅ to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A₁₆₅ binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A₁₆₅ binding to recombinant NRP-1 with K_i of about 10 μM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.     

A vitamin D pathway gene–gene interaction affects low-density lipoprotein cholesterol levels

Much evidence suggests an association between vitamin D deficiency and chronic diseases such as obesity and dyslipidemia. Although genetic factors play an important role in the etiology of these diseases, only a few studies have investigated the relationship between vitamin D-related genes and anthropometric and lipid profiles. The aim of this study was to investigate the association of three vitamin D-related genes with anthropometric and lipid parameters in 542 adult individuals. We analyzed the rs2228570 polymorphism in the vitamin D receptor gene (VDR), rs2134095 in the retinoid X receptor gamma gene (RXRG) and rs7041 in the vitamin D-binding protein gene (GC). Polymorphisms were genotyped by TaqMan allelic discrimination. Gene–gene interactions were evaluated by the general linear model. The functionality of the polymorphisms was investigated using the following predictors and databases: SIFT (Sorting Intolerant from Tolerant), PolyPhen-2 (Polymorphism Phenotyping v2) and Human Splicing Finder 3. We identified a significant effect of the interaction between RXRG (rs2134095) and GC (rs7041) on low-density lipoprotein cholesterol (LDL-c) levels (P=.005). Furthermore, our in silico analysis suggested a functional role for both variants in the regulation of the gene products. Our results suggest that the vitamin D-related genes RXRG and GC affect LDL-c levels. These findings are in agreement with other studies that consistently associate vitamin D and lipid profile. Together, our results corroborate the idea that analyzing gene–gene interaction would be helpful to clarify the genetic component of lipid profile.     

Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53–MDM2 interaction

Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable d-isomers to recapitulate biological activities of their parent l-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent l-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical l-peptide on its target protein binding has been estimated to be 3.0–3.4 kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX—the two negative regulators of p53. We also prepared the retro isomer and d-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of l-stingin weakened its MDM2 binding by 720 fold (3.9 kcal/mol); while enantiomerization of l-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural level.¹     

Insight on the interaction of polychlorobiphenyl with nucleic acid–base

The interaction between one polychlorobiphenyl (3,3′,4,4′,-tetrachlorobiphenyl, coded PCB77) and the four DNA nucleic acid–base is studied by means of quantum mechanics calculations in stacked conformations. It is shown that even if the intermolecular dispersion energy is the largest component of the total interaction energy, some other contributions play a non negligible role. In particular the electrostatic dipole-dipole interaction and the charge transfer from the nucleobase to the PCB are responsible for the relative orientation of the monomers in the complexes. In addition, the charge transfer tends to flatten the PCB, which could therefore intercalate more easily between DNA base pairs. From these seminal results, we predict that PCB could intercalate completely between two base pairs, preferably between Guanine:Cytosine pairs.

Effects of elevated ultraviolet-B radiation on a plant–herbivore interaction

Enhanced ultraviolet-B (UV-B) radiation may have multiple effects on both plants and animals and affect plant–herbivore interactions directly and indirectly by inducing changes in host plant quality. In this study, we examined combined effects of UV-B and herbivory on the defence of the mountain birch (Betula pubescens ssp. czerepanovii) and also the effects of enhanced UV-B radiation on a geometrid with an outbreak cycle: the autumnal moth (Epirrita autumnata). We established an experiment mimicking ozone depletion of 30% (a relevant level when simulating ozone depletion above Northern Lapland). Both arctic species responded only slightly to the enhanced level of UV-B radiation, which may indicate that these species are already adapted to a broader range of UV-B radiation. UV-B exposure slightly induced the accumulation of myricetin glycosides but had no significant effect on the contents of quercetin or kaempferol derivatives. Mountain birch seedlings responded more efficiently to herbivory wounding than to enhanced UV-B exposure. Herbivory induced the activities of foliar oxidases that had earlier been shown to impair both feeding and growth of moth larvae. In contrast, the contents of foliar phenolics did not show the same response in different clones, except for a decrease in the contents of tannin precursors. The induction of foliar phenoloxidase activities is a specific defence response of mountain birches against insect herbivory. To conclude, our results do not support the hypothesis that the outbreak cycle of the autumnal moth can be explained by the cycles of solar activity and UV-B.     

Structural–Functional Units of Epidermis

The available data suggest that epidermis is organized as a system of discrete structural–functional units (SFUs) that reproduce both in vivo and in vitro. SFUs are formed in the culture of epidermal keratinocytes via self-organization of the developing cellular elements. SFUs are capable of self-maintenance and form a niche for stem cells. At the same time, due to the maintenance of asymmetric proliferation kinetics of the stem cells, SFUs serve as a barrier to their uncontrolled replication.     

181 Integrative analysis of gene expression and protein–protein interaction networks in Glioblastoma

Glioblastoma multiforme (GBM) is the most malignant of all the brain tumors with very low median survival time of one year, as per Central Brain Tumor Registry of the USA, 2001. Efforts are ongoing to understand this disease pathogenesis in complete details. Global gene expression changes in GBM pathogenesis have been studied by several groups using microarray technology (e.g. Carro et al., 2010). One of the many approaches to ‘understand the control mechanisms underlying the observed changes in the activity of a biological process’ (Cline et al., 2007) is integration of gene expression and protein–protein interactions (PPI) datasets. Among several examples, aberrant activation of Wnt/β-catenin signaling pathway as well as sonic hedgehog (SHH) signaling pathway is reported in GBMs (Klaus & Birchmeier, 2008). Further, these two pathways are also involved in proliferation and clonogenicity of glioma cancer stem cells (Li et al., 2009), which are thought to play a role in glioma initiation, proliferation, and invasion, and are one of the important points of intervention. Hedgehog–Gli1 signaling is also found to regulate the expression of stemness genes. In this paper, analyses of the relationship between the significant differential expression of these and other genes and the connectivity as well as topological features of a PPI network would be discussed. This way, genes potentially overlooked when relying solely on expression profiles may be identified which can be biologically relevant as possible drug target/s or disease biomarker/s.     

The effect of nanoparticle size on endocytosis dynamics depends on membrane–nanoparticle interaction

Molecular simulation studies on the interaction between nanoparticles (NPs) and cell membranes have been limited by small NP size of several nanometres. In this work, by using a simplified lipid model, we study the endocytosis of large NPs with a size being enlarged to 37.5 nm. It is found that the effect of NP size on endocytosis dynamics depends on the membrane–NP interaction. As the interaction strength between NP and lipid changes, different wrapping modes are observed. For the system with weak membrane–NP attraction, the wrapping process is controlled by the membrane bending, and thus large size of NPs (within the range of NP size we studied) would promote the wrapping dynamics. While for the case with strong membrane–NP adhesion, the wrapping process is dominated by lipid diffusion and small NPs show a larger wrapping rate. In this wrapping mode, the membrane–NP adhesion drives small NPs to move towards the membrane as the wrapping process proceeds. For relatively larger NPs, however, the membrane moves towards the NPs instead. We also find that for the second wrapping mode, the rapid wrapping rate, especially with the hydrophobic ligands on the hydrophilic NP would impose significant perturbations on membrane stability, and consequently, membrane pores may be induced during the process of NP endocytosis.