Recently, the TCGA research network identified 193 prognostic gene signatures predictive of OS, but the gene association with chemotherapy response remains unexplored. Here we used a large sample set for identification of molecular and morphologic signatures that are associated with chemotherapy response. The predictive model on the basis of gene signature revealed an accuracy of 87.9% in correctly classifying refractory from responsive tumors in the TCGA training set and stratified patients in both the TCGA validation set and the Australian data set into groups that demonstrated significant discrepancy in tumor progression, suggesting the capacity of the gene signature to serve as a mechanism to stratify patients with respect to MK-2206 2HCl treatment. The imaging approach stratifies the cells into 10 bins based on nuclear size and accounts for the heterogeneity of cells in a tumor population. Our stratification revealed that most significant morphologic features differed between the chemosensitive and chemoresistant groups in the larger nuclei. However, nuclei within this size range account for a very small percentage, and the majority of the nuclei do not show a significant difference in chemotherapy response. This observation not only is consistent with the Goldie-Coldman hypothesis that only a small cell population may contribute to differential response to chemotherapy, but also suggests the difficulty of a conventional approach of simply correlating the overall morphologic differences with chemotherapy response, owing to the “dilution” effect. Therefore, our imaging approach allows us to interrogate different cell populations separated on the basis of nuclear size in a high throughput and automated fashion. In addition, none of the image features calculated from the entire nucleus per sample, the way similar to those used in other studies, show significant difference between the chemoresistant and chemosensitive patients. This discrepancy from the previous studies likely results from the number of nuclei used in the feature calculation. We used approximately 4000 nuclei per sample for feature value calculation, almost 80 times more than the amount used in the other studies. Taken together, our approach of binning the nucleus size and then assessing the image feature in each individual bin improves the image feature resolution and enhances the discriminating power. Furthermore, our approach of calculating the morphologic features in separate bins is capable of alleviating the size dependence of some of the features, such as circularity and roundness. Aside from the potentially practical value, the morphologic features also provide insights into cancer morphogenesis. The chemosensitive patients exhibit a smaller value of nuclear roundness in Bin 8, but with a larger variability and a larger aspect ratio. Such morphologic differences likely result from the active response of the cells to their environment and heightened cellular metabolism, that is contributable from different molecular regulations. This is further corroborated by pathway analysis, which revealed the gene enrichment in the morphologic function at cellular, tissue, and tumor levels. The gene content of this table offers potential insight into the structural and molecular mechanisms of the chemotherapy response.

A complementary approach that we have developed is to treat multistimulus or time point data as a coexpression network and then use the topology of the network to identify points of constriction, or bottlenecks. Bottlenecks are predicted to represent points of control for transitions between system states that are important to the underlying conditions being studied. Though the term bottleneck is used in various ways we here define a functional bottleneck to be a gene whose inactivation causes a measurable effect in the expression of downstream targets, acting either directly or indirectly. Identification of and validation of functional bottlenecks predicted by network analysis should provide insight into the LY2109761 TGF-beta inhibitor dynamics of the disease-relevant biological processes and their regulation, and potentially serve as targets for clinical intervention. Neuroprotection against stroke can be induced by preconditioning with Toll-like receptor ligands that activate the innate immune system prior to stroke. Preconditioning with systemic administrations of the TLR4 agonist lipopolysaccharide or the TLR9 agonist CpG-oligonucleotide provides robust neuroprotection against stroke in mice and nonhuman primates. The responses produced by TLR activation depends on many factors such as the TLR ligand, the cell type, and the environment and these responses set off complex signaling cascade that ultimately affect other cell types and systems. Genomic analysis of the response to preconditioning with LPS, CpG-ODN, or brief ischemia shows that TLR signaling pathway is highly regulated. To identify functional bottlenecks with potential roles in TLRmediated responses and neuroprotection, we have gathered temporal high-throughput transcriptomic responses in the brain and blood using microarrays that simultaneously evaluate the expression of,40,000 genes. By analyzing these large datasets together, it is possible to identify genes of regulatory importance TLR signaling in the system that may be missed by examining a single dataset individually. Additionally, inferred networks provide an abstraction of the system in terms of functional modules that are active at different times and/or under different conditions, which allows placement of bottlenecks in the context of the functional dynamics of the system. Previously several studies have used computational and experimental approaches to define the regulatory structure of immune cells responding to TLR stimulus and to identify important players in these systems. We have used inferred networks to characterize macrophage response to TLR agonists and neuroprotection in a stroke model. Ramsey, et al. used a large set of microarray experiments and bioinformatics approaches to define functional modules and the regulatory structure of macrophage response to TLR agonists. Amit, et al. used a microarray experiments followed by high-throughput siRNA perturbation of a large panel of regulators to define a regulatory network in dendritic cells. Finally, Calvano, et al. constructed networks based on the effect of LPS stimulation on leukocytes from human patients. These networks were based on existing knowledge of protein-protein interactions and regulatory relationships and the authors used these networks to identify important subnetworks using differential expression overlaid on the network.

P66Shc knockout mice exhibit increased longevity due to their resistance to oxidative stress-induced DNA damage, apoptosis and disease. Herein this study we examined the mechanistic role of p66Shc in controlling the oxidative stress response of early preimplantation stage embryos. Utilizing RNA interference -mediated knockdown of p66Shc in bovine zygotes, we demonstrate that p66Shc regulates intracellular ROS-induced DNA damage that leads to permanent embryo arrest and apoptosis. P66Shc knockdown embryos are stress resistant exhibiting reduced intracellular ROS levels, during in vitro embryo development. The aim of this study was to elucidate the functional role of p66Shc throughout early embryo development. We have previously determined that elevated p66Shc mRNA and protein abundance in early embryos are associated with reduced developmental potential. We have also shown that the early embryo’s response to reactive oxygen species is developmental regulated and that activated p66Shc is linked to increased intracellular ROS levels, reduced antioxidant expression and to permanent embryo arrest. Previous microinjections of short hairpin RNAi molecules targeting p66Shc in immature bovine oocytes successfully knockdown p66Shc and alleviated the 2–4 cell block observed after fertilization, but also resulted in reduced blastocyst development suggesting a role for p66Shc beyond early cleavage arrest. Herein, we mechanistically demonstrate for the first time that RNAi-mediated knockdown of p66Shc at the zygote stage confers oxidative stress resistance throughout early bovine embryo development. P66Shc knockdown embryos are more resistant to oxidant treatment due to increased Perifosine side effects levels of MnSOD and a reduction in intracellular ROS and DNA damage content that diminishes the frequencies of both permanent embryo arrest and apoptosis resulting in improved blastocyst development in vitro. According to a series of criteria, two non-overlapping siRNA sequences were designed to specifically target the unique CH2 domain of bovine p66Shc. These siRNAs were different than the previous p66Shc shRNA sequence utilized in our previous study. The use of multiple target sequences of the same RNA molecule is an effective means of verifying a genuine loss-of-function effect. While both siRNA molecules induced a significant decrease in p66Shc mRNA and protein, RNAi-E was the more effective of the two. The accessibility to the target sequence may be greater for RNA-E molecules than for RNAi-A siRNAs. A difference in the internal stability of the two siRNA molecules could manifest as different rates of strand selection and entry into the RNA-induced silencing complex leading to differential knockdown efficiencies. A significant increase in p66Shc mRNA and protein was observed in embryos injected with negative control siRNAs compared to noninjected embryos. The microinjection process, which involves prolonged exposure to ambient temperature and oxygen tensions, as well as disruption of the cell membrane and components of the cytoskeleton, is a significant stress upon the early bovine embryo. This upregulation may mask the true knockdown effect of p66Shc siRNA molecules, which may prove more effective if introduced in a less traumatic manner. Attempts introducing siRNAs into bovine zygotes using lipofectamine were futile.

In the LCCH group at the predefined time points after surgery. In addition, the number of FG-labeled motoneurons and sensory neurons in the L-CCH+OW group was as well significantly higher than that in the L-CCH group. These mean that the “axon-SC dance” may be supported to some extent by application of omentum, and more neurons may be supported survival and more neurites may be generated by regenerating neurons. The introduction of omentum-wrapping led to improved motor functional recovery. The amplitude of CMAP as well as the histological appearance of target muscles reflects the reinnervation of distal target muscles. In the present study, both the amplitude of CMAP and the histological appearance of gastrocnemius muscles in the L-CCH+OW group were significantly higher than those in the L-CCH group. This indicates that more axons may successfully proceed through the omentum-wrapped scaffold into the distal stumps and reinnervate target muscles, therefore the atrophy of target muscles was partially reversed. In addition, walking track analysis was performed and scored by SFI which provides a reliable measure for evaluating the recovery of motor function. In the present study, the SFI values were in the similar range between the L-CCH+OW and the Reversine autograft groups. Nerve autograft is the most frequently used positive control in studies of nerve defects reconstruction. In many previous studies, incorporating neurotrophic factors or introducing supportive cells into nerve scaffolds can achieve a similar performance to nerve autograft in promoting nerve regeneration and functional recovery. Therefore, it is reasonable to speculate that the efficacy of the omentum-wrapped L-CCH scaffold in promoting nerve regeneration might be further improved by introducing supportive cells and incorporating neurotrophic factors into the scaffold. It is reported that an omentum on contact with a foreign body or activated by injury expands rapidly in size and mass, and the tissue growth is paralleled by the increase in blood vessel density, thereby supporting increased angiogenesis. Vascular endothelial growth factor is the primary angiogenic factor produced by omentum, which may facilitate the growth of new blood vessels and accelerate tissue repair. In the present study, a significantly higher blood vessel density was observed in the L-CCH+OW group compared with that in the LCCH group. Further investigation found that the protein levels of VEGF in the L-CCH+OW group were significantly higher than those in the L-CCH group at 2 and 4 weeks after surgery, which might be responsible for the formation of higher blood vessel density, thereafter providing adequate blood supply to the regenerating axons and consequently resulting in improved nerve regeneration and functional recovery. In addition, VEGF has been reported to have neurotrophic and mitogenic activity on growth cones and SCs, hence stimulating axonal outgrowth, survival and proliferation of SCs independent of the increased vascularization. Therefore, the angiogenic activity and neurotrophic property of VEGF might contribute to the beneficial effect of omentum on axonal regeneration and functional recovery. Brain-derived neurotrophic factor and nerve growth factor hold great potential in promoting nerve regeneration by providing an appropriate environment for axonal outgrowth.

In the present study, both the protein levels of BDNF and NGF were significantly higher in the L-CCH+OW group than those in the L-CCH group at 2 weeks after surgery, which might be, at least in part, responsible for the improved nerve regeneration in the L-CCH+OW group. Despite the finding that elevated protein levels of BDNF and NGF was observed in the L-CCH+OW group, the source of BDNF and NGF was not identified in the present study. SCs regain the ability of synthesizing neurotrophic factors after peripheral nerve injury, thus are probably the main source of BDNF and NGF. In addition, omentum might be served as a pool for BDNF. It has been shown that BDNF expression was noted in vascular endothelial cells, which were abundantly found in omentum. The omentum implanted at the local site of nerve scaffold might contribute to the up-regulation of BDNF observed in the LCCH+OW group. The mechanism underlying the up-regulated expression of BDNF and NGF needs to be clarified in the future studies. Large peripheral nerve defects are frequently caused by trauma, and patients with those injuries should be advised to seek emergency surgery immediately. Otherwise, the dispersed axonal growth would lead to neuroma formation, and the atrophy of denervated target organs would increase the risk of permanent disability. Autologous omentum is not only free of ethical issues but also easily harvested through laparoscopic techniques without many intraabdominal complications associated with laparotomy. In addition, omentum is easy to integrate with local sites and avoid questions regarding immunogenicity, thus exhibits therapeutic potential in the immediate repair of large nerve defects while combined with nerve scaffolds. The encouraging outcomes in the present study indicate that the combined usage of omentum and nerve scaffolds, if further confirmed in larger animals and even humans, may serve as a potent alternative to nerve autografts. Moreover, nerve autograft contains Schwann cells and basal lamina micro-channels, which are responsible for axonal regeneration achieved by nerve autograft, while the longitudinally oriented micro-channels within the L-CCH scaffold and omentum wrapped around the channels might largely account for axonal regeneration achieved by the omentum-wrapped LCCH scaffold. Although the nerve regeneration achieved by omentum-wrapped L-CCH scaffold is not superior to that by nerve autograft, it is still encouraging that these two grafts achieved similar performance in promoting nerve regeneration in the present study. It can be hypothesized that seeding SCs and incorporating neurotrophic factors into the omentum-wrapped LCCH scaffold may achieve better nerve regeneration and functional recovery than nerve autograft, which will be investigated in our future studies. In conclusion, the combined usage of omentum and the L-CCH scaffold described here has several potential advantages over other strategies for promoting large nerve defect regeneration. Firstly, the L-CCH scaffold is relatively easy to prepare, handle, store, and sterilize. Secondly, the longitudinally oriented micro-channels in the L-CCH scaffold are capable of guiding the linear growth of Reversine regenerated axons, and the interconnected porous structure may facilitate penetration of blood vessels.