Recombinant protein

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Production of a heterologous recombinant protein using fragments of the glyceraldehyde 3 phosphate dehydrogenase promoter from Penicillium camemberti

Production of a heterologous recombinant protein using fragments of the glyceraldehyde 3 phosphate dehydrogenase promoter from Penicillium camemberti

The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter is an ideal candidate for recombinant protein expression. As a key enzyme in glycolysis, glyceraldehyde- 3-phosphate dehydrogenase protein (GAPDH) can repre- sent up 5% of cellular proteins in higher eukaryotes, which correlates with a high level of gpd mRNA (Holland and Holland 1978; Piechaczyk et al.1984). Therefore, fungal gpd promoters are highly active, being successfully used for the expression of heterologous genes in several yeasts and fungi (for recent examples see Gonzalez et al. 2010; Sharma and Kuhad 2010). However, and despite their potential applications, little is known about critical parts of gpd promoters necessary to express recombinant proteins. The fungal gpd promoters usually contain two elements (called ‘‘boxes’’) that could be important for their activity (Punt et al. 1990): the gpd-box (a region highly conserved in Aspergillus gpd promoters) and the ct-box (a proximal C ? T rich region). Although in almost all the fungal promoters analyzed only the ct-box is found (Kuo et al. 2004; Nitta et al. 2004; Vastag et al. 2004; Fei et al. 2006; De Maeseneire et al. 2008), both boxes have been found in few of them (Punt et al. 1990; Liao et al. 2008). In these scarce cases, different fragment promoters have been used to produce recombinant proteins, concluding that although the proximal ct-box alone seems enough to drive the expression of a protein, the maximal expression levels would be reached in the presence of the additional gpd-box (Punt et al. 1990, 1992; Liao et al. 2008).

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Bioprocess engineering and metabolic modeling as a roadmap for enhanced recombinant protein production in Pichia pastoris

Bioprocess engineering and metabolic modeling as a roadmap for enhanced recombinant protein production in Pichia pastoris

Here, we present a robust dynamic genome-scale metabolic model of P. pastoris in glucose-limited, aerobic batch and fed-batch cultivations. To assemble the dynamic modeling framework, we started by selecting one of the available genome-scale metabolic models (Chung et al., 2010) and manually curated it to yield realistic flux distributions. Then, we included it in a set of mass balances representing the main compounds present in culture supernatant. Once assembled, the model was calibrated using experimental data from eight batch and three fed-batch cultivations. Next, we employed pre/post regression diagnostics to determine sensitivity, significance and identifiability problems in the model. In order to avoid the aforementioned statistical limitations, problematic parameters were fixed (i.e. removed from the adjustable parameter set) based on the pre/post regression diagnostics, yielding reduced and potentially robust model structures. Potentially robust model structures consisted in the original model formulation with less adjustable parameters. After evaluating these reduced models for each type of cultivation, we chose the one that presented fewer parametric limitations after being re-calibrated with the available data. These reduced models yielded no (or just a few) significance, sensitivity or identifiability problems when calibrating new data and they could predict bioreactor dynamics in conditions like the ones used for their determination. Finally, we carried out simulations to assess the potential of the model to study P. pastoris metabolism under industrially relevant conditions, and to select molecular and process engineering strategies to improve recombinant protein production.

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Recent Contributions of Elastin-Like Recombinamers to Biomedicine and Nanotechnology

Recent Contributions of Elastin-Like Recombinamers to Biomedicine and Nanotechnology

One of the main drawbacks of physical ELR-based hy- drogels is the relatively weak nature of the physical interac- tion mediated by the hydrophobic domains of the elastomeric motifs. Several new alternatives have been explored to over- come this problem. Thus, a further step in the design of multiblock ELRs for the formation of physical hydrogels involves using cross-linking domains from peptide motifs other than elastin [99]. For example, silk fibroin domains, the primary sequence of which follows patterns such as GAGAGS, GAGAGY and GAGAGVGY, have been incor- porated into the peptide backbone of the ELR, thus giving rise to so-called SELPs (silk elastin-like polymers) [112]. SELP-based hydrogels display better mechanical properties than their ELR-based counterparts due to the stability and irreversibility provided by the presence of the silk motifs, which adopt a characteristic anti-parallel -sheet secondary structure [113]. SELP hydrogels have been found to exhibit in vivo biocompatibility since no clinical signs of toxicity were observed up to one month after intradermal or subcuta- neous injection [114]. As is the case with ELR-polymer solu- tions, SELP ones can be mixed with drugs, thus allowing homogenous entrapment of the compound once the gelling process occurs. Thus, the release profiles for different com- pounds, such as cytochrome c, theophylline, vitamin B12, the recombinant protein mitotoxin and several fluorescently labeled probes, have been explored and several parameters, such as the molecular weight of the drug, the concentration of the SELP and its specific composition, have been found to influence drug release [114, 115]. The application of SELPs has not been limited exclusively to drug carriers as their use for the localized matrix-mediated delivery of viral vectors has also been explored. For example, an SELP matrix em- bedded with an adenovirus containing both thymidine kinase-1 and luciferase genes has been intratumorally in- jected into a nude mouse model of head and neck tumor, thus resulting in a fivefold reduction in tumor volume with re- spect to intra-tumoral injection of adenovirus in saline. Moreover, whereas expression in the animal injected with the SELP-based virus was confined to the tumor site, 50% of animals treated with the virus in saline showed liver dis- semination [116]. In order to further assess the safety of this viral delivery system, a non-tumor bearing immunocompe- tent mouse model was used. The ability of the mice to regain their normal weight and blood count was assessed, and it was shown that matrix-mediated gene delivery with the SELP offers a significant reduction in toxicity compared to the injection of free virus [117].

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Identificación de epítopes T citotóxicos restringidos a la molécula HLA A2 1 en la proteína HSP70 de T  cruzi

Identificación de epítopes T citotóxicos restringidos a la molécula HLA A2 1 en la proteína HSP70 de T cruzi

Cloning, expression and purification of the T. cruzi HSP70 recombinant protein. For the clo- ning of the T. cruzi HSP70 protein, the HSP70 genomic unit was PCR-amplified from phage Tc70.6 DNA (12), using primers 70-U (5’ - TCCTTTCTCCCTATTC-3’ ) and 70-L (5’ -CG- CACACACTACGCTCTA-3’ ), and cloned in the pGEM-T vector (Promega). The DraI-SspI frag- ment containing the HSP70 coding region was subcloned in the SmaI site of pQE32 expression vector (Quiagen) in phase with a poly-histidine tract. The recombinant protein was overexpres- sed in E. coli M15 strain after induction for 3 hours at 37ºC with 0.1 mM IPTG. The protein was solubilized in mild sonication conditions in 50 mM phosphate buffer, 30 mM NaCl, 0.025% SDS, pH=8. Soluble extracts were adjusted to reach final concentrations of 300 mM NaCl, 10 mM α-mercaptoethanol, 1 mM PMSF, 5 mM MgCl 2 , 10% glycerol. The soluble HSP70 recom- binant protein was purified by Ni 2+ -NTA-agaro-

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"Expression of the Recombinant Human Granulocyte-Colony Stimulating Factor (rhG-CSF) with Zera® in NT1 Cells, and Suppression of Gene Silencing by the p19 Protein of Tombusvirus."-Edición Única

"Expression of the Recombinant Human Granulocyte-Colony Stimulating Factor (rhG-CSF) with Zera® in NT1 Cells, and Suppression of Gene Silencing by the p19 Protein of Tombusvirus."-Edición Única

Recombinant protein plant production systems still present the disadvantage of low expression levels, when compared to bacterial, primitive eukaryotic, insect and animal cells. For this reason, many researchers have focused on improvement of the recombinant protein expression levels in plant cells. Once, the protein expression levels become competitive, plant systems could be considered as protein production platforms, like the animal cells platforms. Other aspects that must be considered for the development of a competitive plant system are: culture maintenance lower cost, easier protein purification and relatively low possibility of product contamination (51). In order to improve the expression levels of recombinant proteins in plants, it is mandatory to study the diverse factors that drive the expression of the protein. Among those factors, strong promoters, enhancers, and preferential codons for the host cell, are important elements which can improve the expression of a foreign gene. However, other mechanisms are also responsible for the silencing of the genetic expression. These must be seriously considered in order to obtain high expression levels. Some silencing mechanisms are localized at the mRNA level or at the post- translational level (52, 53).

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Expression of the recombinant human granulocyte colony stimulating factor (rhg (rhG CSF) with zera in NT1 cells, and suppression of gene silencing by the p19 protein of tombusvirus

Expression of the recombinant human granulocyte colony stimulating factor (rhg (rhG CSF) with zera in NT1 cells, and suppression of gene silencing by the p19 protein of tombusvirus

Recombinant protein plant production systems still present the disadvantage of low expression levels, when compared to bacterial, primitive eukaryotic, insect and animal cells. For this reason, many researchers have focused on improvement of the recombinant protein expression levels in plant cells. Once, the protein expression levels become competitive, plant systems could be considered as protein production platforms, like the animal cells platforms. Other aspects that must be considered for the development of a competitive plant system are: culture maintenance lower cost, easier protein purification and relatively low possibility of product contamination (51). In order to improve the expression levels of recombinant proteins in plants, it is mandatory to study the diverse factors that drive the expression of the protein. Among those factors, strong promoters, enhancers, and preferential codons for the host cell, are important elements which can improve the expression of a foreign gene. However, other mechanisms are also responsible for the silencing of the genetic expression. These must be seriously considered in order to obtain high expression levels. Some silencing mechanisms are localized at the mRNA level or at the post- translational level (52, 53).

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The Lipocalin Protein Family: Protein Sequence, Structure and Relationship to the Calycin Superfamily

The Lipocalin Protein Family: Protein Sequence, Structure and Relationship to the Calycin Superfamily

adopts a nonnative α-helical conformation different to that in the fully folded protein. These results also help to explain the long-standing observation that polar solvents, such as water– alcohol mixtures, tend to increase the α-helical structure apparent in Blg, as observed by circu- lar dichroism spectroscopy or NMR. Their observation fits well with an emerging consensus on the fundamental mechanisms underlying protein folding: a multi-dimensional energy land- scape, sometimes described as a folding funnel, allows a large number of unequally populated alternative routes from the unfolded protein to the native state. For all but the simplest pro- teins, some of these routes will involve intermediates, or local minima, and may lead to kineti- cally trapped misfolded proteins.

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Surfactant protein A recognizes outer membrane protein OprH on Pseudomonas aeruginosa chronic infection isolates

Surfactant protein A recognizes outer membrane protein OprH on Pseudomonas aeruginosa chronic infection isolates

Binding of surfactant protein A to P. aeruginosa chronic infection isolates. To study the direct SP-A-binding capacity of a collection of chronic infection isolates sequentially isolated from different CF patients, we performed binding assays with Infrared Dye 800CW (IRD800CW) labeled SP-A. We observed a wide range of binding efficiencies among isolates, although the results obtained in different experiments were quite reproducible. We detected a significant reduction in the binding of SP-A in four of the late isolates compared to the early ones (Fig. 1). Thus, the late isolates of patients FQSE5, FQSE11, FQSE12 and FQSE15 bound 1.5 to 2.5 fold less SP-A than their respective early isolates. This phenomenon was predominant among the chronic isolates but not general, because we did not observe differences in the SP-A binding capacity between the early and late isolates of the rest of the patients.

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TítuloDecrease in plasma cholesterol, triglycerides and CPK levels in rats fed on the marine microalga Dunaliella tertiolecta

TítuloDecrease in plasma cholesterol, triglycerides and CPK levels in rats fed on the marine microalga Dunaliella tertiolecta

The test diet with 25 % of dried D. tertiolecta supplies 12 % of protein in the feed, which covers the requirements of the weaning rats. The amino acid profile of D. tertiolecta covers the amino acids requirements of the rat except for methionine (14, Planta medica 1988 12, 13) and, therefore, the microalgal diet was supplemented with 0.3 % of methionine. In this way, the essential amino acids requirement of the rats are covered. The control diet is supplemented with 0.5 % of methionine because casein is deficient in this amino acid. Both diets were adjusted a t4 % of crude fibre and 8% of oil by appropriate additions of cellulose and olive oil.

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Prediction of Protein Oxidation Sites

Prediction of Protein Oxidation Sites

Different proteomic approaches have recently been inspirited by the realisa- tion that methionine oxidation may provide a mechanism to the redox-dependent modulation of protein activity and cellular mechanisms. In this way, proteome- wide studies of methionine oxidation have identified, in both Arabidopsis [11] and human [12], a large number of proteins as potential targets of oxidative signals. Moreover, these proteomic approaches have pointed out the precise sites of oxida- tion on the target proteins. However, these experimental efforts are considerably labor-intensive, time-consuming and expensive, thus making the development of in silico methods for predicting methionine oxidation sites highly desirable. In the field of protein phosphorylation, which can be considered as the most widely studied PTM, computational methods for prediction of phosphorylation sites in proteins have become very popular approaches [13]. Unfortunately, to the best of our knowledge, there are no such methods for methionine oxidation site prediction. Thus, in the current study we have addressed this issue by devel- oping predictive models based on computational intelligence, aimed at accurate prediction of methionine oxidation sites.

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Activation of the double-stranded RNA-dependent protein kinase PKR by small ubiquitin-like modifier (SUMO).

Activation of the double-stranded RNA-dependent protein kinase PKR by small ubiquitin-like modifier (SUMO).

phosphorylation of both PKR and eIF2 ␣ in those cells trans- fected with siRNA control (siC), as expected. Phosphorylation of PKR or eIF2 ␣ was clearly reduced in those cells transfected with siUbc9 (Fig. 4B). To further prove a positive role of SUMO on the activity of PKR, we decided to evaluate the ability of PKR-SUMOmut to induce eIF2 ␣ phosphorylation in response to dsRNA. PKR-deficient immortalized fibroblasts were trans- fected with PKR-WT, PKR-SUMOmut, or GFP, and 36 h after transfection, cells were incubated or not with dsRNA for 8 h. Western blot analysis of the protein extracts revealed that incu- bation with dsRNA induced a strong phosphorylation of eIF2 ␣ in those cells expressing PKR-WT protein, as expected (Fig. 4C). The level of phosphorylated eIF2 ␣ protein detected in cells expressing PKR-SUMOmut was slightly higher than that detected in cells transfected with GFP and clearly lower than that detected in cells expressing the WT protein (Fig. 4C), sug- gesting that SUMOylation of PKR contributes to the inhibition of protein synthesis resulting from dsRNA treatment. To eval- uate this hypothesis, HEK-293 cells or PKR-deficient cells were co-transfected with PGL3-control and increasing doses of PKR-WT or PKR-SUMOmut, as indicated, and 48 h after trans- fection, luciferase expression was measured. As shown in Fig. 4D, we detected a decrease in the luciferase reporter synthesis in a PKR-WT dose-dependent manner in both HEK-293 and PKR-deficient cells. However, transfection of PKR-SUMOmut did not induce a significant decrease in the luciferase expres- sion in any of the cells analyzed (Fig. 4D), indicating that a PKR SUMOylation mutant was unable to inhibit protein synthesis in the absence of stimulus. We then evaluated the consequences of increasing the SUMOylation machinery on the activity of PKR-WT or PKR-SUMOmut using this assay. HEK-293 or PKR-deficient cells were co-transfected with PGL3-control and PKR-WT or PKR-SUMOmut together with pcDNA, SUMO1, SUMO2, Ubc9 and SUMO1, or Ubc9 and SUMO2, and then treated or not with dsRNA, as indicated. The luciferase expres- sion levels detected after PKR-WT transfection were not signif- icantly affected by co-transfection of SUMOylation machinery components, either in HEK-293 or in PKR-deficient cells, in the absence of stimulus. Co-transfection of PKR-deficient cells with SUMOylation machinery components induced a signifi- cant reduction in the luciferase levels detected after transfec- tion of PKR-WT but did not affect the luciferase expression detected in PKR-SUMOmut-transfected cells, upon stimula- tion with dsRNA (Fig. 4E). Similarly, overexpression of SUMOylation machinery components also potentiated the inhibition of protein synthesis induced by PKR-WT trans-

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The Protein DNA Interface database

The Protein DNA Interface database

ing the original structure such as the resolution, source species and whether the structure has water molecules (Figure 6, panel B). This section also contains direct links to the following important and related databases: PDB, PUBMED, CATH, SCOP, COPS, PDBSum [19], BIPA and 3D-Footprint. The Protein Features section shows the classification of the protein part of the interface and all features previously described above (see previous sec- tion). Analogously, the DNA Features section displays graphically all DNA features previously described above (see previous section). Both Protein and DNA Features sections have a link through the chain ID (Figure 6, panel B), which displays a pop-up window with the sequences in FASTA format, highlighting the contacting residues and linking the sequences to a query ready for BLAST analysis at the NCBI website server (Figure 6, panel E). The Interface Features section describes the detailed composition of the atomic contacts in the effective inter- face (Figure 6, panel B). This information is displayed as

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NEU - intraamygdala injections-protein s

NEU - intraamygdala injections-protein s

The present findings raise several questions regarding the generality of these findings as well as the mechanisms underlying the effects of anisomycin on memory and on neurotransmitter release. Although NE participates significantly in modulation of memory processes, this is a role shared by DA and 5-HT (24–26). In addition, the neurochemical results reported here may extend to neurotransmitters beyond the biogenic amines, a possibility that must also be tested directly in the future. The effects of anisomycin on release of multiple neurotransmitters may explain the partial, rather than full, reversal of amnesia attained with the ␤ -adrenergic receptor agents used in the present experiment. Additional assessments of the generality of these findings to other inhibitors of protein synthesis are also needed. Such experiments would help to determine whether anisomycin- induced effects on neurotransmitter release are a consequence of protein synthesis inhibition generally or are a secondary effect restricted to anisomycin. Moreover, it is unclear whether the neurochemical responses shown here will be evident in brain areas other than the amygdala.

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GRh1-dependent unconventional protein secretion

GRh1-dependent unconventional protein secretion

3.1.1 Re-localization of Grh1 upon nutrient starvation Grh1 is essential for the unconventional secretion of Acb1 (Duran et al., 2010; Manjithaya et al., 2010), however, its exact role in this process still needs to be unraveled. To monitor its localization un- der nutrient-rich conditions and nutrient-starvation, the condition known to promote unconventional release of Acb1, endogenous Grh1 was tagged at the C terminus with GFP. In rich medium, Grh1-GFP was found in several punctae equally distributed throughout the cytoplasm whereas upon starvation, Grh1-GFP re- localized to 1-3 larger structures and occasionally smaller ones. These structures were clearly visible within 2h of starvation and remained stable for up to 8h. When 4h-starved yeast were further cultured in rich medium, Grh1-GFP was found to redistribute into numerous small punctae, resembling the growth condition pheno- type. These starvation-dependent changes in localization were in- dependent of new protein synthesis as cycloheximide treatment had no effect on them (Figure 7A).

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New molecular mechanisms of Arabidopsis thaliana resistance
to necrotrophic pathogens = New molecular mechanisms of Arabidopsis thaliana resistance to necrotrophic pathogens

New molecular mechanisms of Arabidopsis thaliana resistance to necrotrophic pathogens = New molecular mechanisms of Arabidopsis thaliana resistance to necrotrophic pathogens

Pathogens have developed strategies to overcome PTI that consist in the releasing of effectors. These effectors have different functions as PTI suppression, nutrient release or pathogen dispersal promotion. These effectors can be specifically recognized by de plant R (Resistance) proteins, and be named avirulent (avr) factors (Flor, 1971; Jones and Dangl, 2006; de Wit, 2007). R proteins recognize avirulent factors either directly or indirectly, consistently with the guard hypothesis (Van der Biezen and Jones, 1998). In indirect recognitions, pathogen effectors target and modify host proteins in order to suppress defence responses or gain nutrients. An R protein guards particular host proteins and perceives modifications induced by the effector (Avr), thereby triggering defence activation (Mackey et al. , 2002). Recognition of avr factors by R proteins leads to the so called effector triggered immunity, a rapid and strong defence response (Feys and Parker, 2000; Glazebrook, 2005). This response causes at the infection site a rapid production of reactive oxygen species (ROS), which may have a direct antimicrobial role or be a signal for other defence responses; the hypersensitive response (HR), a controlled plant cell death around the infection site, which is preferentially active against biotrophic pathogens as it limits their water and nutrient uptake (Greenberg and Yao, 2004); and finally the expression of several pathogenesis related ( PR ) genes (Durrant and Dong, 2004). Subsequently, in uninfected leaves, an increased expression of PR genes and a long-lasting enhanced disease resistance to future pathogen attack is induced. This resistance is known as Systemic Acquired Resistance (SAR) and is effective against a broad range of pathogens and depends on SA-signalling pathway (Durrant and Dong, 2004).

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Hormonal regulation during initial berry development in grapevine

Hormonal regulation during initial berry development in grapevine

families related to gibberellin biosynthesis (Serrani et al., 2008). Also, transgenic tomato lines silenced for Indole Acetic Acid 9 (IAA9), a gene that encodes an Aux/IAA protein, presented parthenocarpic fruits. In pollination-dependent fruitset a high number of transcription factors were differentially expressed. Additionally, when expression of genes involved in hormone responses was analyzed in pollination-dependent fruitset, auxins and ethylene were the most predominant categories (Wang et al., 2009). All the fruitset regulators described so far act as repressors, that is, when this genes are downregulated, fruitset occurs in absence of pollination and fertilization. This indicates that the genetic program which triggers the ovary growth is tightly controlled (Goetz et al., 2006, Lora et al., 2011, Wang et al., 2005). Also, several global gene expression assays have been performed in tomato and other species in order to determine key genes related to pollination, fertilization and parthenocarpy (Wang 2009, Jiang et al., 2013, Kang et al., 2013).

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Mejora del valor proteico para rumiantes de la harina de girasol mediante tratamientos combinados con ácidos (málico y fosfórico) y calor = Improvement of the protein value for ruminants of sunflower meal by combined treatments with acids (malic and ortho

Mejora del valor proteico para rumiantes de la harina de girasol mediante tratamientos combinados con ácidos (málico y fosfórico) y calor = Improvement of the protein value for ruminants of sunflower meal by combined treatments with acids (malic and orthophosphoric) and heat

the CP digestibility in the small intestine. Additional studies are needed to determine the effects of these treatments on intestinal digestion. In situ results on sunflower meal treated with 400 ml/kg of solutions of orthophosphoric acid (0.67 M) or malic acid (1 M) and then heated in an oven at 1508C for 6 h showed a reduction of a little bit more than 50% of the ruminal degradability using either acid associated with an increase (between 10% and 16%) of the intestinal digest- ibility (Arroyo and Gonza´lez, 2009). In this way, the treat- ment with any of both acids doubled the protein value estimates for this meal, measured as the total (microbial and dietary) supply of intestinal digestible amino acids. Another benefit of these last treatments was to multiply by three times the intestinal digestible supply from this meal of lysine and methionine and by 3.7 that of cysteine (Gonza´lez et al ., 2009). Similarly, Ouarti et al . (2006) protecting soybean meal with malic acid at 0.9 and 1.8 eq/kg and drying temperatures of 1178C for 6 h did not find negative effects on the intestinal digestibility of the undegraded protein, which was nearly complete. Besides, the inclusion at 5% of these treated meals in an alfalfa hay-concentrate diet did not alter the pH, the concentration of volatile fatty acids or the fibrolytic ability of the rumen, but reduced the excessive ammonia concentration resulting from the diet fermentation as well as the acetic acid/propionic acid ratio, attributing this latest effect to the incorporation of malate to the diet.

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A phase I/II trial of the safety and clinical activity of a HER2-protein based immunotherapeutic for treating women with HER2-positive metastatic breast cancer

A phase I/II trial of the safety and clinical activity of a HER2-protein based immunotherapeutic for treating women with HER2-positive metastatic breast cancer

In the second-line cohort, the rate of response during the immunization period peaked at 50 % for dHER2 and at 25 % for HER2-ECD (Fig. 3a). These low response rates are partly explained by the presence of anti-dHER2 or anti- HER2-ECD Abs due to the prior treatment with trastuzu- mab (knowing that the ECD of the HER2 protein com- prises the epitope targeted by trastuzumab) and the protocol definition of responders involving the baseline Ab concentration. During the follow-up, the rate of responders to dHER2 and HER2-ECD increased to above 70 %. All the patients became responders to HER2-ICD after dose 5 and after 1 year of follow-up, the rate of responders was still 50 %. As expected, 13/17 patients (76.5 %) in the second-line cohort had detectable anti-dHER2 and anti- HER2-ECD Abs at baseline and one had anti-HER2-ICD Abs.

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Functional study of potential sHSPs in Arabidopsis and tomato under environmental stress

Functional study of potential sHSPs in Arabidopsis and tomato under environmental stress

OMICS technologies such as proteomics and metabolomics are highly useful techniques to elucidate and explore changes occurring at a more global scale [Feussner, 2015]. These techniques provide a huge amount of data about metabolites and proteins being modified and thus can be extremely informative in the study of sHSP-M functions. The present work provides information on the differential protein and metabolites abundance between knockdown mutants and Col-0 plants. Down-regulation of the mitochondrial sHSPs produced profound proteome-wide changes as it can be assumed from the high number of proteins that significantly changed. Interestingly, most of the proteome accumulated to higher levels and only a few proteins decrease in the mutants. In seedlings grown under normal conditions, amiR26.5, amiR23.5/23.6, and amiR23.5/23.6/26.5 showed differential accumulation of more than 200 proteins compared to control plants. The number of changing proteins was considerably smaller in the amiR23.5 and amiR23.6. This indicates that, although no major changes were observed in the phenotype of amiR26.5, reduced level of sHSP26.5 produced wide effects on protein homeostasis. Similarly, simultaneously reduction of two and the three mitochondrial sHSPs lead to important changes in the basal proteome and consequently in the phenotype. On the other hand, individual reduction of sHSP23.5 and sHSP23.6 appeared to produce less severe effects in the proteome of the mutants. However, when compared the differential basal proteome with the differential proteome of heat-treated mutants, the number of proteins with modified abundances increased after heat only in the amiR23.5 and amiR23.6. In the rest of the analyzed mutants, a smaller number of differential proteins were observed in the treated compared to non-treated plants. These results imply that proteomes of single mutants amiR23.5 and amiR23.6 are more vulnerable and responsive to the heat shock stress, not like the other mutants. Proteomic analysis of the single amiR showed similarities in the gene ontology annotation of the changed proteins at the first level of ontology of Panther and by using String. It can be speculated though, from the low number of common changed proteins between single amiRs, that low abundance of the individual sHSP-M produced distinct proteomes response.

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Anti Human Endoglin (hCD105) Immunotoxin—Containing Recombinant Single Chain Ribosome Inactivating Protein Musarmin

Anti Human Endoglin (hCD105) Immunotoxin—Containing Recombinant Single Chain Ribosome Inactivating Protein Musarmin

Targeting endoglin over-expressed in tumor neovasculature has been used for therapeutic purposes with anti-endoglin antibodies [16], endoglin-targeted radioimmunotherapy [17], and anti-endoglin antibody–containing ITs [18]. Efficient conventional anti-endoglin ITs containing ricin A-chain [19] and the type 2 RIPs nigrin b [12,20] and ebulin l [21] have been constructed. In the present work, we report proof of the validity of the new IT (rMU1-44G4), containing rMU1 as a toxic moiety linked to an anti-human CD105 monoclonal antibody (44G4), on cultured mouse L929 fibroblasts transfected with the short form of CD105 [22]. rMU1, a recombinant form expressed in Escherichia coli, of musarmin 1 (MU1) present in bulbs of the plant Muscari armeniacum L. Miller was able to retain full activity when tested on mammalian ribosomes both in translation and in N-glycosidase activities [23].

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