A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999     

Evaluating the Impact of Different Sequence Databases on Metaproteome Analysis: Insights from a Lab-Assembled Microbial Mixture

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge.This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.     

Evaluation of intra-specific diversities in Oenococcus oeni through analysis of genomic and expressed DNA

In winemaking Oenococcus (O.) oeni is the most frequent species of lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). Several studies have demonstrated that O. oeni is a quite homogeneous species and strains are difficult to differentiate especially when isolates from the same region are analyzed. In this study, the molecular biodiversity of O. oeni isolated from wines of the same region (Aglianico produced in Basilicata Region, Southern Italy) was evaluated with the aim of designing a molecular approach for discrimination and characterization of the isolates at the strain level. Three molecular techniques were applied: random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), restriction endonucleases analysis-pulsed field gel electrophoresis (REA-PFGE) and differential display PCR (DD-PCR). The results obtained by RAPD-PCR confirmed the difficulty in differentiating isolates. By means of REA-PFGE a higher polymorphism, often related to the origin (winery) of strains, was revealed. However, on analyzing strains isolated from the same winery, only in some cases was more than one REA-PFGE pattern obtained. By analyzing dendrograms constructed on the basis of DD-PCR profiles differentiation of strains isolated from the same winery, in some cases, could be accomplished. The reliability of the DD-PCR in the differentiation of closely related strains suggests that this method could represent an alternative and/or additional tool to other molecular methods, such as REA-PFGE, for fine characterization of oenococcal strains.     

The process of thermodialysis in bioremediation of waters polluted by endocrine disruptors

Endocrine disruptors are chemicals able to induce adverse effects into wildlife and humans owing to their ability of interfering with the endocrine system. Bisphenol A (BPA) has been chosen as model of endocrine disruptors. To reduce the BPA pollution in waters we proposed the employment of the process of thermodialysis. Two different catalytic membranes have been prepared by covalently immobilizing laccase (from Trametes versicolor) by means of a diazotation process or tyrosinase (from mushroom) by condensation. The support was a nylon membrane. The bioremediation power of both catalytic membranes has been analysed under isothermal and non-isothermal conditions.The advantages in using non-isothermal bioreactors were discussed in terms of reduction of the bioremediation times.     

Pyrrolizidine alkaloids from Anchusa strigosa and their antifeedant activity

The pyrrolizidine alkaloid (PA) content of flowers, leaves, and roots of Anchusa strigosa (Boraginaceae) was analysed by ESI-LC-MS. Six PAs, including two new natural compounds, were detected, characterized by NMR spectroscopy, and quantified in each plant organ. The results indicated that the highest total concentration of PAs was in the leaves (23.63 mg/g of dried part), followed by the flowers (19.77 mg/g), and finally by the roots (1.80 mg/g). All PAs isolated were subjected to Spodoptera exigua and Pieris brassicae larvae. Feeding activity by both herbivore species using a bioassay was inhibited up to circa 75% depending on PA and applied concentration.     

Grasping behavior in tufted capuchin monkeys (Cebus apella): grip types and manual laterality for picking up a small food item

This study investigates prehension in 20 tufted capuchins (Cebus apella) in a reaching task requiring individuals to grasp a small food item fixed to a tray. The aim was twofold: 1) to describe capuchins' grasping techniques in detail, focusing on digit movements and on different areas of contact between the grasping fingers; and 2) to assess the relationship between grip types and manual laterality in this species. Capuchins picked up small food items using a wide variety of grips. In particular, 16 precision grip variants and 4 power grip variants were identified. The most frequently used precision grip involved the distal lateral areas of the thumb and the index finger, while the most preferred kind of power grip involved the thumb and the palm, with the thumb being enclosed by the other fingers. Immature capuchins picked up small food items using power grips more often than precision grips, while adult individuals exhibited no significant preference for either grip type. The analysis performed on the time capuchins took to grasp the food and withdraw it from the tray hole revealed that 1) precision grips were as efficient as power grips; 2) for precision grips, the left hand was faster than the right hand; and 3) for power grips, both hands were equally quick. Hand preference analysis, based on the frequency for the use of either hand for grasping actions, revealed no significant hand bias at group level. Likewise, there was no significant relationship between grip type and hand preference.