Tag-mediated single-step purification and immobilization of recombinant proteins toward protein-engineered advanced materials.

Background: The potential applications of protein-engineered functional materials are so wide and exciting that the interest in these eco-friendly advanced materials will further expand in the future. Tag-mediated protein purification/immobilization technologies have emerged as green and cost-effective approaches for the fabrication of such materials. Strategies that combine the purification and immobilization of recombinant proteins/peptides onto/into natural, synthetic or hybrid materials in a single-step are arising and attracting increasing interest. Aim of Review: This review highlights the most significant advances of the last 5 years within the scope of tag-mediated protein purification/immobilization and elucidates their contributions for the development of efficient single-step purification and immobilization strategies. Recent progresses in the field of protein-engineered materials created using innovative protein-tag combinations and future opportunities created by these new technologies are also summarized and identified herein. Key Scientific Concepts of Review: Protein purification/immobilization tags present a remarkable ability to establish specific non-covalent/covalent interactions between solid materials and biological elements, which prompted the creation of tailor-made and advanced functional materials, and of next-generation hybrid materials. Affinity tags can bind to a wide range of materials (of synthetic, natural or hybrid nature), being most suitable for protein purification. Covalently binding tags are most suitable for long-term protein immobilization, but can only bind naturally to protein-based materials. Hybrid affinity-covalently binding tags have allowed efficient one-step purification and immobilization of proteins onto different materials, as well as the development of innovative protein-engineered materials. Self-aggregating tags have been particularly useful in combination with other tags for generating protein-engineered materials with self-assembling, flexible and/or responsive properties. While these tags have been mainly explored for independent protein purification, immobilization or functionalization purposes, efficient strategies that combine tag-mediated purification and immobilization/functionalization in a single-step will be essential to guarantee the sustainable manufacturing of advanced protein-engineered materials.

Is Sotolon Relevant to the Aroma of Madeira Wine Blends?

Madeira wine (MW) oxidative aging results in the formation of several key aromas. Little is still known about their odor relevance to the aroma of the most commercialized MWs. This report presents an in-depth study of the odor impact of sotolon in MW blends. First, its odor perception was estimated in MWs according to ASTM E679, testing different 3-year-old (3-yo) commercial blends. The odor relevance of sotolon in the aroma of 3-, 5-, and 10-yo commercial blends (89 MWs) was then appraised by calculating its Odor Activity Value (OAV), after determining its content by RP-HPLC-MS/MS. The sotolon odor perception in MW was as low as 23 µg/L, although it was found that little differences in the wine matrix influenced its perception. OAVs varied between 0.1 and 22, increasing with the blend age. Considering that 16% of the OAVs are higher than 10 (mostly ≥ 10-yo), sotolon was found to be a key contributor to the overall aroma MW blends.

Comparative analysis between biofilm formation and gene expression in Staphylococcus epidermidis isolates.

AIM: To understand the relationship between ica, aap and bhp gene expression and the implications in biofilm formation in selected clinical and commensal Staphylococcus epidermidis isolates. MATERIAL & METHODS: Isolates were analyzed regarding their biofilm-forming capacity, biochemical matrix composition, biofilm spatial organization and expression of biofilm-related genes. RESULTS: On polysaccharide intercellular adhesin-dependent biofilms, aap and bhp contributions for the biofilm growth were negligible, despite very high levels of expression. In contrast, smaller increases in icaA expression contributed significantly to biofilm growth. Interestingly, no biological differences were observed between clinical and commensal strains. CONCLUSION: These results reinforce the concept that S. epidermidis is an 'accidental pathogen,' and that the ica operon is the main mechanism of biofilm formation in clinical and commensal isolates.

Synergistic Association of Genetic Variants with Environmental Risk Factors in Susceptibility to Essential Hypertension.

AIMS: Essential hypertension (EH) is a disease in which both environment and genes have an important role. This study was designed to identify the interaction model between genetic variants and environmental risk factors that most highly potentiates EH development. METHODS: We performed a case-control study with 1641 participants (mean age 50.6 ± 8.1 years), specifically 848 patients with EH and 793 controls, adjusted for gender and age. Traditional risk factors, biochemical and genetic parameters, including the genotypic discrimination of 14 genetic variants previously associated with EH, were investigated. Multifactorial dimensionality reduction (MDR) software was used to analyze gene-environment interactions. Validation was performed using logistic regression analysis with environmental risk factors, significant genetic variants, and the best MDR model. RESULTS: The best model indicates that the interactions among the ADD1 rs4961 640T allele, diabetes, and obesity (body mass index ≥30) increase approximately four-fold the risk of EH (odds ratio = 3.725; 95% confidence interval: 2.945-4.711; p < 0.0001). CONCLUSION: This work showed that the interaction between the ADD1 rs4961 variant, obesity, and the presence of diabetes increased the susceptibility to EH four-fold. In these circumstances, lifestyle adjustment and diabetes control should be intensified in patients who carry the ADD1 variant.

Optimizing a qPCR gene expression quantification assay for S. epidermidis biofilms: a comparison between commercial kits and a customized protocol.

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmß1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.

RAS gene polymorphisms, classical risk factors and the advent of coronary artery disease in the Portuguese population.

BACKGROUND: Several polymorphisms within the renin-angiotensin system cluster of genes have been associated with the advent of coronary artery disease (CAD) or related pathologies. We investigated the distribution of 5 of these polymorphisms in order to find any association with CAD development and distinguish if any of the biochemical and behavioural factors interact with genetic polymorphisms in the advent of the disease. METHODS: ACE I/D (rs4340), ACE A11860G (rs4343), AT1R A1166C (rs5186), AGT T174M (rs4762) and AGT M235T (rs699) gene polymorphisms were PCR-RFLP analysed in 298 CAD patients and 510 controls from Portugal. Several biochemical and behavioural markers were obtained. RESULTS: ACE I/D DD and ACE11860 GG genotypes are risk factors for CAD in this population. The simultaneous presence of ACE I/D I and ACE11860 A alleles corresponds to a significant trend towards a decrease in CAD incidence. We found several synergistic effects between the studied polymorphisms and classical risk factors such as hypertension, obesity, diabetes and dyslipidaemia: the presence of the DD genotype of ACE I/D (and also ACE11860 GG) increases the odds of developing CAD when associated to each one of these classical risk factors, particularly when considering the male and early onset CAD subgroup analysis; AGT235 TT also increases the CAD risk in the presence of hypertension and dyslipidaemia, and AT1R1166 interacts positively with hypertension, smoking and obesity. CONCLUSION: ACE polymorphisms were shown to play a major role in individual susceptibility to develop CAD. There is also a clear interaction between RAS predisposing genes and some biochemical/environmental risk factors in CAD onset, demonstrating a significant enhancement of classical markers particularly by ACE I/D and ACE11860.

Methylenetetrahydrofolate reductase gene, homocysteine and coronary artery disease: the A1298C polymorphism does matter. Inferences from a case study (Madeira, Portugal).

Elevated levels of plasma homocysteine, an independent risk factor and a strong predictor of mortality in patients with coronary artery disease (CAD), can result from nutritional deficiencies or genetic errors, including methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms. The contribution of these polymorphisms in the development of CAD remains controversial. We analysed the impact of MTHFR C677T and A1298C on fasting homocysteine and CAD in 298 CAD patients proved by angiography and 510 control subjects from the Island of Madeira (Portugal). After adjustment for other risk factors, plasma homocysteine remained independently correlated with CAD. Serum homocysteine was significantly higher in individuals with 677TT and 1298AA genotypes. There was no difference in the distribution of MTHFR677 genotypes between cases and controls but a significant increase in 1298AA prevalence was found in CAD patients. In spite of the clear effect of C677T mutation on elevated homocysteine levels we only found an association between 1298AA genotype and CAD in this population. The simultaneous presence of 677CT and 1298AA genotypes provides a significant risk of developing the disease, while the 1298AC genotype, combined with 677CC, shows a significant trend towards a decrease in CAD occurrence. The data shows an independent association between elevated levels of homocysteine and CAD. Both MTHFR polymorphisms are associated with increased fasting homocysteine (677TT and 1298AA genotypes), but only the 1298AA variant shows an increased prevalence in CAD group. Odds ratio seem to indicate that individuals with the MTHFR 1298AA genotype and the 677CT/1298AA compound genotype had a 1.6-fold increased risk for developing CAD suggesting a possible association of MTHFR polymorphisms with the risk of CAD in Madeira population.

Human paraoxonase gene polymorphisms and coronary artery disease risk.

BACKGROUND: Complex diseases such as coronary artery disease (CAD), hypertension and diabetes are usually caused by individual susceptibility to multiple genes, environmental factors, and the interaction between them. The paraoxonase 1 (PON1) enzyme has been implicated in the pathogenesis of atherosclerosis and CAD. Two common polymorphisms in the coding region of the PON1 gene, which lead to a glutamine (Q)/arginine (R) substitution at position 192 and a leucine (L)/methionine (M) substitution at position 55, influence PON1 activity. Studies have investigated the association between these polymorphisms and CAD, but with conflicting results. AIMS: 1) To evaluate the association between PON1 polymorphisms and CAD risk; and 2) to study the interaction between PON1 polymorphisms and others in different candidate genes. METHODS: We evaluated the risk of CAD associated with PON1 Q192R and L55M polymorphisms in 298 CAD patients and 298 healthy individuals. We then evaluated the risk associated with the interaction of the PON1 polymorphisms with ACE DD, ACE 8 GG and MTHFR 1298AA. Finally, using a logistic regression model, we evaluated which variables (genetic, biochemical and environmental) were linked significantly and independently with CAD. RESULTS: We found that the PON1 55MM genotype was more common in the CAD population, but this did not reach statistical significance as a risk factor for CAD, while PON1 192RR presented an 80% higher relative risk compared to the population without this polymorphism. The interaction between PON1 192RR and MTHFR 1298AA, sited in different genes, increased the risk for CAD, compared with the polymorphisms in isolation (OR=2.76; 95% CI=1.20-6.47; p=0.009), as did the association of PON1 192RR with ACE DD, which presented a 337% higher risk compared to the population without this polymorphic association (OR=4.37; 95% CI=1.47-13.87; p=0.002). Similarly, the association between PON1 192RR and ACE 8 GG was linked to an even higher risk (OR=6.23; 95% CI=1.67-27.37; p<0.001). After logistic regression, smoking, family history, fibrinogen, diabetes, Lp(a) and the association of PON1 192RR + ACE 8 GG remained in the regression model and proved to be significant and independent risk factors for CAD. In the regression model the latter association had OR=14.113; p=0.018. CONCLUSION: When analyzed separately, the PON1 192RR genotype presented a relative risk for CAD 80% higher than in the population without this genotype. Its association with other genetic polymorphisms sited in different genes, coding for different enzymes and belonging to different physiological systems, always increased the risk for CAD. After correction for other conventional and biochemical risk factors, the PON1 192RR + ACE 8 GG association remained a significant and independent risk factor for CAD.