Component of several enzyme complexes such as the branched chain a-keto acid dehydrogenase involved in the catabolism of leucine, isoleucine and valine. Cystathionine gamma-lyase was upregulated and is involved in the last step in the trans-sulfuration pathway from methionine to cysteine and can be both involved in the degradation of methionine and cysteine. Finally, upregulated glutamate dehydrogenase 1 is involved in the biosynthesis or catabolism of glutamate and catalyzes the oxidative deamination of glutamate to 2-ketoglutarate which is an important intermediate in the TCA cycle. The observed increase in amino acids catabolism may be necessary to compensate for the lack of energy available caused by the embryonic undernutrition. As the albumen removal decreased the protein availability at the end of the incubation, changes in the expression of enzymes involved in amino acid metabolism could be foreseen. Indeed, a previous study also found indications of an altered protein metabolism in broilers treated by albumen removal before incubation, suggesting a transient increase in muscle proteolysis. Metabolic programming caused by prenatal protein undernutrition was revealed by the observed hepatic proteome changes related with glucose metabolism. Upregulation of L-lactate dehydrogenase B chain suggests an increased conversion of pyruvate to lactate in the absence of oxygen in the liver. This lactate can subsequently be converted to glucose by the gluconeogenesis. Two of the regulatory enzymes of the latter pathway were dysregulated in the liver. Fructose-1,6-biphosphatase 1 was downregulated, whereas phosphoenolpyruvate 2 was upregulated. FBP1 catalyzes the conversion of D-fructose-1,6-biphosphate to D-fructose-6-phosphate, whereas PCK2 catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, the rate-limiting step. Upregulated a-enolase is also involved in both the gluconeogenesis and glycolysis and catalyzes the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate. In addition, glucose transporter 1, a facilitative low capacity/high affinity glucose transporter across the plasma membrane was upregulated. Hepatic GLUT1 is primarily involved in cellular uptake of glucose from the plasma into the hepatocytes when nutrients are in reduced supply. As no differences in plasma glucose or lactate levels were found, it is not clear whether the gluconeogenesis pathway in general is up- or downregulated. Furthermore, a general upregulation of the TCA pathway was observed, from the upregulation of several enzymes involved such as DLD, aconitate hydratase and malate dehydrogenase 1. DLD is a component of the pyruvate dehydrogenase complex, which converts pyruvate, originating from the breakdown of carbohydrates to acetyl-CoA, the input for the TCA cycle. ACO2 catalyzes the isomerization of citrate to isocitrate via cis-aconitate, and MDH1 the conversion of malate to oxaloacetate.

The IFN-b-inducing effect of c-di-AMP contributes, at least in part, to the reported proliferation of antigen-specific T cell types and the evolvement of the proposed balanced TH1/TH2/TH17 cell response. DC subset targeting was investigated also with regard to the c-di-AMPinduced IFN-b production. Conventional DCs showed a much more pronounced IFN-b production than plasmacytoid DCs. This finding was somewhat surprising because plasmacytoid DCs are known to be specialized in IFN type I production and usually produce these IFNs in much higher amounts than conventional DCs, which are specialized in antigen presentation. By targeting conventional DCs, c-di-AMP evokes the secretion of a rather limited amount of IFN-b which may be important to fine tune immune responses. Interestingly, also the IFN-b response to L. monocytogenes was reported not to be mediated by plasmacytoid DCs either. Since it is known that the bacterium secretes c-di-AMP in the course of infection, our results further strengthen the suggestion that c-di-AMP is indeed the mediator of the L. monocytogenes-induced IFN-b. The modes of action described here do not exclude additional, not yet identified mechanisms of c-di-AMP-mediated immune response modulation. For example, other co-stimulatory molecules or secreted immune signaling molecules could be regulated in a cdi-AMP-dependent manner to enhance or modulate antigeninduced immune responses by acting on either effector or bystander cells. However, our results further elucidate intermediate steps of the immune response cascade leading to the immune modulatory activity of c-di-AMP observed in immunization studies on mice. They advance the knowledge on modes of adjuvant action toward the regulation of effective immunization responses. Transcription Activator-Like Effectors technology comprises a rapidly developing tool for targeted genome manipulation. Deciphering of the TALE DNA recognition code in 2009 led to the development of a series of novel engineered TALE chimeras for a variety of purposes. For example, TALEs have been engineered to modulate gene expression, reprogram epigenetic modifications, repair or disrupt genes using TALENs or chimeric TALE recombinases, and promote targeted gene transposition using TALE-directed piggyBac. TALEs offer an attractive advantage over traditional zinc finger-based technologies due to their inherently simple and predictable DNA recognition code attributed to its novel type DNA-binding domain. The TALE DBD is highly repetitive and contains a tandem array of repeat monomers with each monomer consisting of,34 amino acids with the exception of the last repeat unit consisting of,20 amino acids. Relative to TALENs, the potential of TALEs as activators has not been fully explored until more recently. Initial studies showed that a single TALE activator was able to drive the expression of a reporter gene linked to a synthetic promoter derived from different cellular genes.

In agreement with our nutrigenomic results, a significant decreased adhesion of monocytes pre-exposed to MOF could be observed at the endothelial cells compared to control. The potential of grape seed extract to decrease adhesion of monocytes to HUVECs has been reported recently, however at concentrations ranging from 50 to 100 mg/ml. These concentrations are higher than the concentrations used in our study. Our results emphasize the capacity of grape seed– derived MOF to decrease the adhesion of immune cells to the vascular endothelium and potentially lower infiltration of these immune cells into the vascular wall, which is an initial step in atherosclerosis development. It has been reported that supplementation of the diet with catechin, a flavanol monomer present in MOF, results in lower atherosclerosis development in apolipoprotein E-deficient mice. It could be postulated that the regular consumption of MOF could decrease blood cell infiltration into the vasculature and potentially protect against atherosclerotic lesions in humans. Our bioinformatic data also revealed that over 30 differentially expressed genes are involved in different processes related to inflammation. The role of chronic inflammation in the promotion, initiation and development of chronic diseases, such as cancer, cardiovascular disease or osteoporosis has been described. In our study, among the differentially expressed genes involved in inflammatory processes is the gene coding for the proinflammatory cytokine IL2, of which the expression has been down-regulated by the MOF supplementation. Together with IL2, we also observed a decrease in the expression of a subunit of its receptor, the IL2RB gene. These inflammation-related genes, as well as some of the cell adhesion molecules, are regulated by the transcription factor NF-kB. Bioinformatic analyses of the nutrigenomic data identified several transcription factors potentially involved in the regulation of the expression of differentially expressed genes, among which is NF-kB. This suggests that NF-kB is a potential target by which MOF exert their anti-inflammatory effects in circulating blood cells. Interestingly, our nutrigenomic data also revealed an increase in the expression of the gene coding for NFKBIA, the endogenous inhibitor of NF-kB. A previous in vivo study in an atherosclerotic ApoE mouse model identified NF-kB as a major upstream regulator in leukocyte adhesion and transendothelial migration through the vascular endothelium. Moreover, NF-kB activity in peripheral blood mononuclear cells from smokers has been found to be significantly higher than in PBMCs from non-smokers. In order to corroborate a direct effect of MOF on NF-kB we performed NF-kB reporter gene studies in an inflammatory human monocyte/macrophage cell model. The experiments revealed a dose- and time-dependent repression of NF-kB luciferase expression in the presence of MOF.

A key inhibitor of osteoclast activity, osteoprotegrin, is normally enriched in the cochlea relative to other sites in the body. In mice, OPG is produced at high levels by fibrocytes within the spiral ligament and secreted in the perilymph. Mice deficient in OPG show excessive remodeling throughout the middle and inner ear resulting in severe hearing loss. While there are cases of FD causing sensorineural hearing loss in humans, it has been hypothesized that this is due to auditory neural compression from the FD bone changes. The results in our mouse model with FD like lesions would support that mechanism, as well as possibly physical changes to the ossicular chain, as opposed to pathologic changes of sensory structures as seen in such lesions as cochlear otosclerosis. These results point to a role for peri-lacunar remodeling in the cochlea that may be disrupted in FD. In conclusion, our results show that the cochlea is a unique bony structure characterized by limited bone turnover that confers protection from proliferative and metabolically active FD bony lesions. The invasive fibro-osseous lesions seen in this mouse model cause conductive hearing loss through involvement of the ossicular chain, in a manner very similar to that seen in humans. These mechanisms could be new pharmacologic targets to treat the skeletal or hearing manifestations of FD or other skeletal diseases. The vitrification process plays a key role in cryopreservation for the long term conservation of plant genetic resources. Vitrification is defined as a physical process by which a concentrated aqueous solution solidifies into a stable amorphous glass without the formation of ice crystals when the temperature is decreased. Vitrification of plant specimens can be achieved in many ways, including air drying of embryos, and more recently through the use of highly concentrated plant vitrification solutions that readily form glasses on cooling and inhibit crystallization. However, exposure to PVS must be controlled to enable sufficient cellular dehydration whilst limiting injury from chemical toxicity and establishment of a simple and high-throughput cryopreservation method using cryoprotectant is highly desirable. Plant vitrification solutions combine cryoprotectants that vary in permeability, such that cellular water is replaced, cell viscosity is increased and the freezing behaviour of the remaining water is altered. PVS2 is probably the most commonly used cryoprotectant for plant cells, tissues and embryos; for example, the cryopreservation of embryonic axes of citrus and in vitro shoot-tips of Parkia speciosa, a tropical species with recalcitrant seeds. The conventional approach to vitrification generally involves a tissue pre-culture step on sucrose-enriched medium, before cooling, with highly concentrated vitrification solution for a period that varies with species, tissue and temperature.

Signal during exposition with a quantitative imaging system, as these systems are often calibrated to stop the exposure process before the strongest signal on the membrane is outside the linear range of detection. We, therefore, recommend to mask the non-specific signal at 25 kDa, or to cut the membrane prior to incubation with secondary anti-LC antibodies, or to do incremental exposure to improve signal detection. We also tested all of the monoclonal antibodies within a heat stable fraction. This method is routinely used by many groups for Western blotting or to measure total tau after passive immunization, as this procedure very effectively removes any mouse Igs. The heat-stable procedure is based on the fact that tau is an extremely heat stable protein. Here, the non-specific signal seen in TKO mice was completely absent in the heat stable fraction, as expected. However, this technique also seems to decrease the overall tau signal and resolution on a Western blot, as evidenced by the observation that some antibodies failed to reveal all the bands of murine tau in the HS fraction. Furthermore, our results show that there is a significant fraction of tau lost into the pellet, and that this technique is not for studying proteins other than tau, such as kinases, as they were absent from the HS fraction. We further tested two others techniques to clear Igs. We took advantages of the capacity of protein G and Dynabeads to bind the fragment crystallizable region of mouse Igs in order to clear the samples of endogenous Igs. Results with cleared samples showed that the non-specific band in TKO mice was completely eliminated, further confirming that the non-specificity is due to mouse Igs. Both methods can be used to remove the nonspecificity but they involve significant modification of Western blot procedures and are more time-consuming than the simple replacement of secondary antibodies. Furthermore, it is possible that some proteins of interest could form a complex with protein G or Dynabeads and be discarded in the pellet. Finally, we tested some polyclonal antibodies and, as expected, our results indicate that these antibodies were not associated with specificity-related problems secondary to endogenous Igs. Nevertheless, in our study, some polyclonal antibodies, particularly the pS262 and pS409 antibodies, led to high non-specificity that obscured the tau signal detection. These antibodies displayed high non-specificity mainly due to their cross-reactivity with others proteins in the samples. While the HS treatment improved the phospho-tau signal of pS262, it did not for pS409. Indeed some studies have shown that pS409 is not phosphorylated in the human and rat adult brain, which could explain this problem with detection. In conclusion, this study provides evidence that both monoclonal and polyclonal antibodies can display non-specificity in mouse samples during Western blot analysis for tau protein.