Engineering of PA-IIL lectin from Pseudomonas aeruginosa – Unravelling the role of the specificity loop for sugar preference

<p>Abstract</p> <p>Background</p> <p>Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from <it>Pseudomonas aeruginosa</it>, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose – a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action.</p> <p>Results</p> <p><it>In vitro </it>site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22–23–24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions – both of afore mentioned have strong effects on the saccharide preferences.</p> <p>Conclusion</p> <p>Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.</p

TOWARDS BETTER UNDERSTANDING EUROPEAN ICE AGE POLLEN RECORDS: A LESSON FROM MODERN POLLEN ASSEMBLAGES OBTAINED FROM YAKUTIAN LAKES

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SHAPE VARIATIONS OF GRASS PHYTOLITHS IN EURASIA: TOWARDS IMPROVED INTERPRETATION OF FOSSIL PHYTOLITH RECORD USING NEW METHODS OF GEOMETRIC MORPHOMETRY

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Comparison of different methods of antioxidant activity evaluation of green and roast C. Arabica and C. Robusta coffee beans

Coffee beans contain a large amount of antioxidants, which are subjected to various changes during roasting. In this study, antioxidant potential of raw and roasted to different degree (light, medium, dark) C. arabica and C. robusta coffee beans was evaluated by the four antioxidant assay methods, TPC, FRAP, TEAC, and DPPH˙. The obtained results revealed significant differences between the coffee types, roasting degree, and antioxidant activity assessment methods. FRAP and TPC appeared to be the most appropriate methods for revealing the differences in antioxidant potential of different coffee types and the effects of roasting. The results obtained by these methods were in good correlation. ABTS and DPPH? methods are not enough sensitive for the determination of roasting degrees. In general, based on statistical data evaluation, antioxidant activity is more dependent on the coffee type than on the degree of roasting, however, the selection of analytical method may also be significant

The Shaqadud Archaeological Project (Sudan): exploring prehistoric cultural adaptations in the Sahelian hinterlands

The authors present preliminary results from a new research project based in Jebel Shaqadud, Sudan. Their findings highlight the potential for this region's archaeological record to expand our understanding of the adaptation strategies used by human groups in arid north-east African environments away from rivers and lakes during the Holocene. Furthermore, they present exceptionally early radiocarbon dates that push postglacial human occupation in the eastern Sahel back to the twelfth millennium BP

A new methodology to assess the performance and uncertainty of source apportionment models II: The results of two European intercomparison exercises

The performance and the uncertainty of receptor models (RMs) were assessed in intercomparison exercises employing real-world and synthetic input datasets. To that end, the results obtained by different practitioners using ten different RMs were compared with a reference. In order to explain the differences in the performances and uncertainties of the different approaches, the apportioned mass, the number of sources, the chemical profiles, the contribution-to-species and the time trends of the sources were all evaluated using the methodology described in Belis et al. (2015). In this study, 87% of the 344 source contribution estimates (SCEs) reported by participants in 47 different source apportionment model results met the 50% standard uncertainty quality objective established for the performance test. In addition, 68% of the SCE uncertainties reported in the results were coherent with the analytical uncertainties in the input data. The most used models, EPA-PMF v.3, PMF2 and EPA-CMB 8.2, presented quite satisfactory performances in the estimation of SCEs while unconstrained models, that do not account for the uncertainty in the input data (e.g. APCS and FA-MLRA), showed below average performance. Sources with well-defined chemical profiles and seasonal time trends, that make appreciable contributions (>10%), were those better quantified by the models while those with contributions to the PM mass close to 1% represented a challenge. The results of the assessment indicate that RMs are capable of estimating the contribution of the major pollution source categories over a given time window with a level of accuracy that is in line with the needs of air quality management

Standardizing the classification of skin tears: validity and reliability testing of the International Skin Tear Advisory Panel Classification System in 44 countries

Background: Skin tears are acute wounds that are frequently misdiagnosed and under‐reported. A standardized and globally adopted skin tear classification system with supporting evidence for diagnostic validity and reliability is required to allow assessment and reporting in a consistent way. Objectives:To measure the validity and reliability of the International Skin Tear Advisory Panel (ISTAP) Classification System internationally. Methods: A multicountry study was set up to validate the content of the ISTAP Classification System through expert consultation in a two‐round Delphi procedure involving 17 experts from 11 countries. An online survey including 24 skin tear photographs was conducted in a convenience sample of 1601 healthcare professionals from 44 countries to measure diagnostic accuracy, agreement, inter‐rater reliability and intrarater reliability of the instrument. Results:A definition for the concept of a ‘skin flap’ in the area of skin tears was developed and added to the initial ISTAP Classification System consisting of three skin tear types. The overall agreement with the reference standard was 0·79 [95% confidence interval (CI) 0·79–0·80] and sensitivity ranged from 0·74 (95% CI 0·73–0·75) to 0·88 (95% CI 0·87–0·88). The inter‐rater reliability was 0·57 (95% CI 0·57–0·57). The Cohen's Kappa measuring intrarater reliability was 0·74 (95% CI 0·73–0·75). Conclusions: The ISTAP Classification System is supported by evidence for validity and reliability. The ISTAP Classification System should be used for systematic assessment and reporting of skin tears in clinical practice and research globally.info:eu-repo/semantics/publishedVersio

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent “a hypothetical ancestral super-sex chromosome” or random distribution?

Background Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. Results We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. Conclusions The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes