Genotypic and Phenotypic Heterogeneity in Alicyclobacillus acidoterrestris: A Contribution to Species Characterization

Alicyclobacillus acidoterrestris is the main cause of most spoilage problems in fruit juices and acidic products. Since soil borne species often contaminate fruit juices and do not need strict extreme requirements for survival, it is a great concern to investigate whether and how soil species could evolve from their ecological niches in microbial community to new environments as fruit juices. In this study, 23 isolates of thermo-acidophilic, spore-forming bacteria from soil were characterized by cultural and molecular methods. In addition, 2 strains isolated from a spoilage incident in pear juice were typed. Strains phenotyping showed that they could be grouped into 3 different clusters, and some isolates showed identical or quite similar patterns. Analyzing pH and temperature ranges for growth, the majority of strains were able to grow at values described for many species of Alicyclobacillus. Qualitative utilization of lysine, arginine and indole production from tryptophan revealed, for the first time, deamination of lysine and decarboxylation of arginine. Resistance to 5% NaCl as well as the ability to hydrolyze starch and gelatin, nitrate reduction, catalase and oxidase activities confirmed literature evidences. Examining of 16S rRNA, showed that isolates were divided into three blocks represented by effectively soil species and strains that are moving from soil to other possible growing source characterized by parameters that could strongly influence bacterial survival. RAPD PCR technique evidenced a great variability in banding patterns and, although it was not possible to obtain genotypically well-distinguished groups, it was feasible to appreciate genetic similarity between some strains. In conclusion, the investigation of a microbial community entails a combination of metagenomic and classic culture-dependent approaches to expand our knowledge about Alicyclobacillus and to look for new subspecies.     

A hidden deadly venomous insect: First eco-epidemiological assessment and risk mapping of lonomism in Argentina

BackgroundEnvenomation by the South American Lonomia saturniid caterpillars, named lonomism, constitutes an emerging and somewhat neglected public health issue in Argentina and neighboring countries. Considering that there is an intricate relationship between environment and human health in such cases, this study aimed to analyze the eco-epidemiological profile of 40 accidents and 33 occurrences of Lonomia spp. in Misiones (Argentina) between January 2014 and May 2020.Methodology/Principal findingsWe described the eco-epidemiological variables and characterized the abiotic scenario of such cases. Additionally, we obtained a density map that shows the punctual intensity of Lonomia records throughout Misiones. Most of the accidents occurred in the Department of Guaraní and involved male victims younger than 20 years old. The accidental/occasional occurrence of Lonomia spp. (considering both adult and caterpillar stages together) was significantly higher in the rural area, whereas only adult specimens were found in urban areas. We determined that the presence of this insect in Misiones is positively related to higher temperatures and solar radiation, and larger precipitation and evapotranspiration throughout the year.Conclusion/SignificanceThis study represents an initial step towards the global understanding of lonomism as a public health problem in Argentina. It provides a map of the risk level for this envenomation in Misiones, which could help authorities address public health policy efforts to implement sustainable strategies for prevention and response to this threat in Northeastern Argentina and neighboring regions.     

Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca²⁺-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with K_d values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.     

Yeasts isolated from olive mill wastewaters from southern Italy: technological characterization and potential use for phenol removal

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682—C. boidinii and 625, 642, 647, and 941—P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10–30°C) and (NH₄)₂SO₄ (0.0–6.0 g l⁻¹) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10°C with 6.0 g l⁻¹ of (NH₄)₂SO₄ added.     

Influence of Cross-linked Waxy Maize Starch on the Aggregation Behavior of Casein Micelles During Acid-induced Gelation

The influence of cross-linked waxy maize starch on the aggregation behavior of casein micelles was investigated using a combination of physico-chemical techniques. Milk was homogenized at two different temperatures (55 and 65 °C) and then heated at 95 °C for 5 min in a pilot scale system. The possible interactions between modified starch and milk proteins during lactic acid fermentation were evaluated. While 1% starch did not show differences in the whey protein complexes formed during heating compared to milk with no starch (as measured by size exclusion chromatography), a higher (2.5%) concentration of starch clearly showed an increased amount of heat-induced whey protein aggregates. The gelation pH also increased significantly with 2.5% starch compared to that of the control samples. The storage modulus (G′) increased with increasing levels of starch, and confocal microscopy confirmed that the microstructure of the casein gels was altered by the presence of modified starch. Milk-starch mixtures preheated and homogenized at 55 or 65 °C exhibited similar physico-chemical behavior during acidification. The results suggested a lack of interaction between starch granules and casein micelles during acidification, and scanning electron microscopy images collected with a self-assembled monolayer technique also confirmed that starch granules were not attached to milk caseins but only embedded in the protein gel matrix.     

Phase Separation Behavior of Caseins in Milk Containing Flaxseed Gum and κ-Carrageenan: A Light-Scattering and Ultrasonic Spectroscopy Study

The physico-chemical properties of skim milk containing κ-carrageenan (in the concentration range 0–0.06% w/v), flaxseed gum (in the concentration range 0–0.40% w/v), or a mixture of both polysaccharides were studied using dynamic light scattering, under diluted conditions, as well as in situ, undiluted, using diffusing wave spectroscopy (DWS) and ultrasonic spectroscopy. Flaxseed gum causes phase separation in milk mixtures, because of thermodynamic incompatibility between the casein micelles and the polysaccharide chains. Confocal microscopy and ultrasonic spectroscopy showed that while the addition of 0.01% κ-carrageenan was not sufficient to hinder phase separation, when 0.03% was added, the helix–helix interactions between κ-carrageenan molecules were sufficient to form a network and stabilize the system. DWS clearly demonstrated that clusters of casein micelles still form even at very low concentrations of polysaccharides (below the visible phase separation threshold) and that κ-carrageenan hinders visible phase separation by decreasing the mobility of the casein micelles.     

Accumulation of Eu chelates in cells expressing or not P-glycoprotein: Implications for blood-brain barrier crossing

Alzheimer’s disease (AD) is the most commonly form of dementia in the elderly. The development of molecules able to detect biomarkers characteristic of AD is critical to its understanding and treatment. However, such molecules must be able to pass blood-brain barrier (BBB) which is a major impediment to the entry of many therapeutic drugs into the brain. Such a limitation applies to the development of magnetic resonance imaging molecular neuroimaging agents using biomarkers of AD-like β-amyloid deposits, as the common extracellular contrast agents (CAs) are not able to cross an intact BBB. In this work, we have studied the ability of a series of simple Eu³⁺ complexes to enter cells overexpressing or not the ABCB1 (P-gp or P-glycoprotein) protein, which is expressed at the BBB and in human embryonic astrocytes. The intracellular uptake of the Eu³⁺ complexes of linear and macrocyclic polyaminocarboxylate ligands with different charges and lipophilicities was followed by atomic absorption spectrometry. Based on biochemical argument, we propose that lipophilic contrast agents can be efficiently taken up by cells and accumulate inside mitochondria when they are positively charged. The important point is that they are not P-gp substrates, which is one of the major obstacles for them to cross the BBB.     

Triterpene glycosides from Agrostemma gracilis

Four triterpene saponins, agrostemmosides A–D were isolated from the methanol extract of Agrostemma gracilis. The structures of the compounds were determined as 3-O-β-d-xylopyranosyloleanolic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-xylopyranosyloleanolic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-β-d-xylopyranosylechinocystic acid 28-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-β-d-xylopyranosylechinocystic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester by a combination of one- and two-dimensional NMR techniques, and mass spectrometry. To the best of our knowledge this is the first phytochemical report on A. gracilis, and echinocystic acid saponins were encountered for the first time in Caryophyllaceae family.