Mojca Lunder

Phage Display: Selecting Straws Instead of a Needle from a Haystack

An increasing number of peptides with specific binding affinity to various protein and even non-protein targets are being discovered from phage display libraries. The power of this method lies in its ability to efficiently and rapidly identify ligands with a desired target property from a large population of phage clones displaying diverse surface peptides. However, the search for the needle in the haystack does not always end successfully. False positive results may appear. Thus instead of specific binders phage with no actual affinity toward the target are recovered due to their propagation advantages or binding to other components of the screening system, such as the solid phase, capturing reagents, contaminants in the target sample or blocking agents, rather than the target. Biopanning experiments on different targets performed in our laboratory revealed some previously identified and many new target-unrelated peptide sequences, which have already been frequently described and published, but not yet recognized as target-unrelated. Distinguishing true binders from false positives is an important step toward phage display selections of greater integrity. This article thoroughly reviews and discusses already identified and new target-unrelated peptides and suggests strategies to avoid their isolation.

Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition

Lipids are important components in human nutrition; however, their increased intake contributes to the development of obesity and can lead to multiple long‐term complications. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a key enzyme for the absorption of dietary triglycerides. Interference with fat hydrolysis results in the reduced utilization of ingested lipids, therefore inhibition of lipases decreases fat absorption. Extracts from 106 species of medicinal plants, vegetables and fruits were screened for potential lipase inhibitory activity. p‐Nitrophenylpalmitate and 5‐bromo‐4‐chloro‐3‐indoxylpalmitate were used as substrates in an in vitro test with crude porcine pancreatic lipase. Bearberry (Arctostaphylos uva‐ursi), garden pea (Pisum sativum), Norway spruce (Picea abies) and large‐leaved lime (Tilia platyphyllos) extracts were the most active. Additionally, the activity of selected extracts with removed polyphenols was measured. Extracts of bearberry, garden pea and large‐leaved lime are a promising source for developing functional foods or isolating active compounds. Copyright

Arterial Stiffness and Cardiovascular Therapy

The world population is aging and the number of old people is continuously increasing. Arterial structure and function change with age, progressively leading to arterial stiffening. Arterial stiffness is best characterized by measurement of pulse wave velocity (PWV), which is its surrogate marker. It has been shown that PWV could improve cardiovascular event prediction in models that included standard risk factors. Consequently, it might therefore enable better identification of populations at high-risk of cardiovascular morbidity and mortality. The present review is focused on a survey of different pharmacological therapeutic options for decreasing arterial stiffness. The influence of several groups of drugs is described: antihypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, beta-blockers, diuretics, and nitrates), statins, peroral antidiabetics, advanced glycation end-products (AGE) cross-link breakers, anti-inflammatory drugs, endothelin-A receptor antagonists, and vasopeptidase inhibitors. All of these have shown some effect in decreasing arterial stiffness. Nevertheless, further studies are needed which should address the influence of arterial stiffness diminishment on major adverse cardiovascular and cerebrovascular events (MACCE).

Subtherapeutic, low-dose fluvastatin improves functional and morphological arterial wall properties in apparently healthy, middle-aged males--a pilot study.

OBJECTIVE Early arterial wall changes are already present in the apparently healthy, middle-aged population and continuously progress with age. The aim of our study was to investigate whether 30 days low-dose fluvastatin treatment could improve and reverse these arterial changes that are primarily associated with ageing, in otherwise healthy middle-aged males. METHODS In a double blind, randomized study, 50 middle-aged males received either placebo or fluvastatin (10mg) for 30 days. Brachial artery flow-mediated dilation (FMD), pulse wave velocity (PWV) and β-stiffness of the common carotid artery were measured on the 1st, 14th and 30th day of the study using an Aloka instrument by integrated eTracking. RESULTS In 77% of subjects, impaired endothelial function was revealed at inclusion in the study. All the parameters were improved already after 14 days, and after 30 days of treatment FMD improved by 91.5 ± 15.6%, while PWV and β-stiffness improved by 6.2 ± 1.1% and 10.7 ± 1.5%, respectively (all P<0.001). After therapy discontinuation, the beneficial effects progressively decreased, but were still detectable after 5 months. During the study the lipid profile remained unchanged, thus the beneficial effects obtained were attributed to the pleiotropic effects of fluvastatin. CONCLUSIONS We found that subtherapeutic low-dose fluvastatin (10mg daily; 30 days) considerably improves and reverses early functional and morphological arterial wall impairments that are present in apparently healthy, middle-aged males. It might be supposed that such a new and original approach could be valuable in cardiovascular prevention.

The effects of low-dose fluvastatin and valsartan combination on arterial function: A randomized clinical trial

BACKGROUND Ageing progressively diminishes arterial functions, even in the absence of traditional risk factors. Our aim was to explore whether age-related arterial changes in middle-aged males could be reversed using short-term, low-dose fluvastatin/valsartan combination intervention. METHODS Forty apparently healthy, middle-aged males (43.3 ± 5.8 years) were recruited in a double-blind, randomised intervention. Individuals received either 10mg fluvastatin/20mg valsartan daily or placebo over 30 days. The brachial artery flow mediated dilation (FMD), pulse wave velocity (PWV) and common carotid artery β-stiffness were assessed at baseline and after 30 days, and again 5-10 months after therapy discontinuation. RESULTS Arterial function variables significantly improved after 30 days of intervention; FMD improved by 167.7% (P<0.001), PWV by 10.9% (P<0.05) and β-stiffness by 18.8% (P<0.01), whereas no changes were obtained in the placebo group. The favourable outcomes in the intervention group were accompanied by a significant decrease of high sensitivity-C reactive protein levels (1.8-fold; P<0.05). In contrast, lipids and blood pressure remained unchanged. Surprisingly, the beneficial arterial effects were still present to a substantial degree 7 months after completing intervention (remaining % of initial improvement: FMD 82.1%, PWV 69.5% and β-stiffness 68.5%), but declined substantially after 10 months. CONCLUSION Our results indicate that age-related arterial changes, at least in middle-aged males, can be reversed. Short-term treatment with a low-dose fluvastatin/valsartan combination resulted in a large and long lasting improvement of arterial function.

The endothelial plasma membrane transporter bilitranslocase mediates rat aortic vasodilation induced by anthocyanins

BACKGROUND AND AIMS Anthocyanins, a sub-class of flavonoids, induce endothelium-dependent vasorelaxation, by activating endothelial nitric oxide synthase and consequently increasing production of the vasorelaxant agent nitric oxide. It is not yet clear if anthocyanin-induced vasorelaxation starts with their interaction with plasma membrane receptors in the extracellular compartment, or with their membrane transport toward intracellular molecular targets. We therefore investigated the possible role of bilitranslocase (TC 2.A.65.1.1), an endothelial plasma membrane carrier that transports flavonoids, in the vasodilation activity induced by anthocyanins. METHODS AND RESULTS Vascular reactivity was assessed in thoracic aortic rings obtained from male Wistar rats. Pre-treatment of aortic rings with anti-sequence bilitranslocase antibodies targeting the carrier, decreased vasodilation induced by cyanidin 3-glucoside and bilberry anthocyanins. CONCLUSION Here we show for the first time that bilitranslocase mediates a critical step in vasodilation induced by anthocyanins. This offers new insights into the molecular mechanism involved in endothelium-dependent vasorelaxation by flavonoids, and the importance of their specific membrane carriers.

Engineered Lactic Acid Bacterium Lactococcus lactis Capable of Binding Antibodies and Tumor Necrosis Factor Alpha

ABSTRACT We have optimized the display of the B domain of staphylococcal protein A on the surface of Lactococcus lactis. The maximum binding capacity was estimated at 0.146 μg of antibody per 108 cells and was sustained at 86% after treatment with simulated gastric juice. A tumor necrosis factor alpha (TNF-α)-binding affibody was also displayed and bound TNF-α, which could be useful in the treatment of inflammatory bowel disease.

Coenzyme Q10 Supplementation Decreases Statin-Related Mild-to-Moderate Muscle Symptoms: A Randomized Clinical Study

Background Statin use is frequently associated with muscle-related symptoms. Coenzyme Q10 supplementation has yielded conflicting results in decreasing statin myopathy. Herein, we tested whether coenzyme Q10 supplementation could decrease statin-associated muscular pain in a specific group of patients with mild-to-moderate muscle symptoms. Material/Methods Fifty patients treated with statins and reporting muscle pain were recruited. The Q10 group (n=25) received coenzyme Q10 supplementation over a period of 30 days (50 mg twice daily), and the control group (n=25) received placebo. The Brief Pain Inventory (BPI) questionnaire was used and blood testing was performed at inclusion in the study and after 30 days of supplementation. Results The intensity of muscle pain, measured as the Pain Severity Score (PSS), in the Q10 group was reduced from 3.9±0.4 to 2.9±0.4 (P<0.001). The Pain Interference Score (PIS) after Q10 supplementation was reduced from 4.0±0.4 to 2.6±0.4 (P<0.001). In the placebo group, PSS and PIS did not change. Coenzyme Q10 supplementation decreased statin-related muscle symptoms in 75% of patients. The relative values of PSS and PIS significantly decreased (−33.1% and −40.3%, respectively) in the Q10 group compared to placebo group (both P<0.05). From baseline, no differences in liver and muscle enzymes or cholesterol values were found. Conclusions The present results show that coenzyme Q10 supplementation (50 mg twice daily) effectively reduced statin-related mild-to-moderate muscular symptoms, causing lower interference of statin-related muscular symptoms with daily activities.

Comparison of bacterial and phage display peptide libraries in search of target-binding motif

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-throughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better.

Reduction of age-associated arterial wall changes by low-dose valsartan

Background: Functional and morphological arterial wall impairment progresses with ageing. Angiotensin II in the arterial wall is involved in this process. Appropriate early intervention might theoretically slow the progress of age-related changes. Herein, we investigated a new approach to this issue: whether arterial wall changes present in middle-aged males could be reduced by low-dose valsartan intervention. Methods: Forty apparently healthy, middle-aged males (42.9 ± 0.9 years) were recruited for a double-blind randomized study and received either placebo or valsartan (20 mg daily) for 30 days. Brachial artery flow-mediated dilation (FMD), pulse wave velocity (PWV), and β-stiffness of the common carotid artery were measured using an Aloka alfa-10 Prosound with an integrated eTracking system at inclusion, after 30 days, and after 3 and 8 months. Results: Intervention resulted in FMD increase (154.2 ± 20.1 %; p < 0.001) and PWV and β-stiffness decrease compared to initial values (−6.9 ± 1.0 % and −13.2 ± 1.4 %; both p < 0.01) whereas values in the untreated group (p < 0.001 for all parameters) remained unchanged throughout the study. The advantageous effects decreased over the months following valsartan discontinuation, but were still significant after 3 months (largely in FMD and less in PWV and β-stiffness), and negligible after 8 months. The beneficial effects were ascribed to valsartans pleiotropic effects, as no blood pressure changes were recorded. Conclusions: We showed that age-related arterial wall changes in middle-aged males are reversible and could be reduced by a low-dose, short-term valsartan intervention. The new approach merits detailed investigation in future studies.