Escherichia coli expression and purification of four antimicrobial peptides fused to a family 3 carbohydrate-binding module (CBM) from Clostridium thermocellum

Antimicrobial peptides (AMPs) are molecules that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. Several hundreds of AMPs have been identified and characterized. These molecules are presently gaining increasing importance, as a consequence of their remarkable resistance to microorganism adaptation. Carbohydrate-binding modules (CBMs) are non-catalytic domains that anchor glycoside hydrolases into complex carbohydrates. Clostridium thermocellum produces a multi-enzyme complex of cellulases and hemicellulases, termed the cellulosome, which is organized by the scaffoldin protein CipA. Binding of the cellulosome to the plant cell wall results from the action of CipA family 3 CBM (CBM3), which presents a high affinity for crystalline cellulose. Here CipA family 3 CBM was fused to four different AMPs using recombinant DNA technology and the fusion recombinant proteins were expressed at high levels in Escherichia coli cells. CBM3 does not present antibacterial activity and does not bind to the bacterial surface. However, the four recombinant proteins retained the ability to bind cellulose, suggesting that CBM3 is a good candidate polypeptide to direct the binding of AMPs into cellulosic supports. A comprehensive characterization of the antimicrobial activity of the recombinant fusion proteins is currently under evaluation.Instituto de Tecnologia Química e Biológica,Fundação para a Ciência e a Tecnologia (FCT

Caffeine and Placebo Improved Maximal Exercise Performance Despite Unchanged Motor Cortex Activation and Greater Prefrontal Cortex Deoxygenation

Caffeine (CAF) is an ergogenic aid used to improve exercise performance. Independent studies have suggested that caffeine may have the ability to increase corticospinal excitability, thereby decreasing the motor cortex activation required to generate a similar motor output. However, CAF has also been suggested to induce a prefrontal cortex (PFC) deoxygenation. Others have suggested that placebo (PLA) may trigger comparable effects to CAF, as independent studies found PLA effects on motor performance, corticospinal excitability, and PFC oxygenation. Thus, we investigated if CAF and CAF-perceived PLA may improve motor performance, despite the likely unchanged MC activation and greater PFC deoxygenation. Nine participants (26.4 ± 4.8 years old, VO2MAX of 42.2 ± 4.6 mL kg-1 min-1) performed three maximal incremental tests (MITs) in control (no supplementation) and ∼60 min after CAF and PLA ingestion. PFC oxygenation (near-infrared spectroscopy at Fp1 position), MC activation (EEG at Cz position) and vastus lateralis and rectus femoris muscle activity (EMG) were measured throughout the tests. Compared to control, CAF and PLA increased rectus femoris muscle EMG (P = 0.030; F = 2.88; d = 0.84) at 100% of the MIT, and enhanced the peak power output (P = 0.006; F = 12.97; d = 1.8) and time to exhaustion (P = 0.007; F = 12.97; d = 1.8). In contrast, CAF and PLA did not change MC activation, but increased the PFC deoxygenation as indicated by the lower O2Hb (P = 0.001; F = 4.68; d = 1.08) and THb concentrations (P = 0.01; F = 1.96; d = 0.7) at 80 and 100% the MIT duration. These results showed that CAF and CAF-perceived PLA had the ability to improve motor performance, despite unchanged MC activation and greater PFC deoxygenation. The effectiveness of CAF as ergogenic aid to improve MIT performance was challenged

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

This work was supported in part by the European Union, Area NMP.2013.1.1–2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530, and by the EU Seventh Framework Programme (FP7 2007–2013) under the WallTraC project (Grant Agreement no 263916), and BioStruct-X (grant agreement no 283570). This paper reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein. CMGAF is also supported by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-PRO/103980/2008 and EXPL/BIA-MIC/1176/2012. EAB is also funded by a grant (No. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel and by a grant (No. 2013284) from the U.S.-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry.Peer reviewedPublisher PD

Generation of a library of carbohydrate-active enzymes for plant biomass deconstruction

Áreas de pesquisa: Biochemistry & Molecular Biology ; ChemistryIn nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sourcesinfo:eu-repo/semantics/publishedVersio

CORIOLUS VERSICOLOR BIOMASS INCREASES HIPPOCAMPAL DENTATE GYRUS NEWLY-GENERATED NEURONS COMPLEXITY IN MICE

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Endothelial progenitor cells in diabetic patients with myocardial infarction – Can statins improve their function?

The effect of statins on endothelial progenitor cells (EPCs) function derived from diabetic patients (DMpts) with acute myocardial infarction (AMI) is unknown. In this study we assess the response of early and late EPCs from diabetic versus non-diabetic patients (NDMpts) with AMI to statins. EPCs were obtained from 10 diabetic and 10 age-matched non-diabetic male patients with AMI. For each patient, cultures of early and late EPCs were performed under 4 different conditions: normal glucose concentration (control); high glucose concentration; normal glucose concentration with atorvastatin supplementation and normal glucose concentration with pravastatin supplementation. To compare the effect of these treatments on EPC function in DMpts versus NDMpts, we performed in vitro: EPC colony-forming units (CFU) assay; cell cycle analysis; viability assessment and expression of the surface markers CXCR4, CD133, CD34 and KDR. Under control conditions, CFU numbers were reduced in DMpts-derived EPCs when compared to those of NDMpts (1.4±0.8 vs 2.6±1.2 CFU/well, P=0.021). When early EPCs from DMpts were cultured in the presence of statins, CFU capacity was restored, surmounting that of NDMpts under control conditions. Statins significantly improved viability of early EPCs and delayed the onset of late EPCs senescence, even in cells from DMpts. In addition, statins induced approximately a 2-fold increase in the proportion of late EPCs in S-phase of the cell cycle (P<0.05). Statins have a beneficial effect on both early and late EPCs from DMpts with AMI. Despite the functional impairment of EPCs from DMpts, they exhibit similar responsiveness to statins as equivalent cells from NDMpts

Biochemical changes throughout grape berry development and fruit and wine quality

Wine is made up of more than one thousand compounds, the majority of which, such as vitamins and minerals, come from the grapes, while others, like ethanol and glycerol, are products of the winemaking process. Although sugars are either partially or completely transformed, sugar import and accumulation into the ripening berry is a major parameter of wine quality. Sugar status is directly related to the final alcoholic content of wine, and regulates several genes responsible for the development of its aromatic and organoleptic properties. Physiological ripeness is reached when the grapes achieve sufficiently high sugar levels without loosing too much acidity; however, aromatic and phenolic compound content must also be taken into account. Softening and water content are other essential characteristics of a ripe berry. From a winemaker point of view, optimal grape maturity is essential for wine quality, but is difficult to assess because it is under multifactorial control, involving grapevine cultivar variety and environmental parameters such as soil, temperature, exposure to sun, and hormonal regulation. Continued study of the key control points in grape ripening is crucial if we ultimately hope to improve grape and wine quality.Fundação para a Ciência e a Tecnologia (FCT)(research project ref. POCI/AGR/56378/2004; to C.C., grant ref. SFRH/BD/10689/2002, to P.S. grant ref. SFRH/BD/13460/2003, to N.F. grant ref. SFRH/BD/23169/2005, and to A.A., grant ref. SFRH/PBD/17166/2004