Application of comet assay for the assessment of DNA damage caused by chemical genotoxins in the dairy yeast Kluyveromyces lactis

Kluyveromyces lactis, also known as dairy yeast, has numerous applications in scientific research and practice. It has been approved as a GRAS (Generally Recognized As Safe) organism, a probiotic, a biotechnological producer of important enzymes at industrial scale and a bioremediator of waste water from the dairy industry. Despite these important practical applications the sensitivity of this organism to genotoxic substances has not yet been assessed. In order to evaluate the response of K. lactis cells to genotoxic agents we have applied several compounds with well-known cyto- and genotoxic activity. The method of comet assay (CA) widely used for the assessment of DNA damages is presented here with new special modifications appropriate for K. lactis cells. The comparison of the response of K. lactis to genotoxins with that of Saccharomyces cerevisiae showed that both yeasts, although considered close relatives, exhibit species-specific sensitivity toward the genotoxins examined.     

New structure–activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR)

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure–activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis).The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I]_0.5) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a–d and 6d, showed excellent efficacy with a α_max close to 1. Selected compounds (2d, 3a, 3b, 5a–d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells.The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site.In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,²⁹ are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.     

Characterization of the host-defense peptides from skin secretions of Merlin's clawed frog Pseudhymenochirus merlini: Insights into phylogenetic relationships among the Pipidae

The family Pipidae comprises the genera Hymenochirus, Pipa, Pseudhymenochirus, Silurana, and Xenopus but phylogenetic relationships within the family are unclear. Peptidomic analysis of norepinephrine-stimulated skin secretions from Pseudhymenochirus merlini Chabanaud, 1920, the single species within the genus Pseudhymenochirus, led to identification of 13 host-defense peptides with antimicrobial activity. Two peptides (hymenochirin-1Pa and -1Pb) show structural similarity to hymenochirin-1B from Hymenochirus boettgeri and eight peptides (hymenochirin-5Pa, -5Pb, -5Pc, -5Pd, -5Pe, -5Pf, 5Pg and -5Ph) are structurally similar to each other and to hymenochirin-5B from H. boettgeri. Two peptides differing by a single amino acid (IKIPSFFRNILKKVGKEAVSLM/I AGALKQS), termed pseudhymenochirin-1Pa and -1Pb, and pseudhymenochirin-2Pa (GIFPIFAKLLGKVIKVASSLISKGRTE) do not resemble host-defense peptides previously isolated from pipid frogs. Hymenochirin-5Pe was the most abundant peptide in the secretions and hymenochirin-1Pa the most potent against Staphylococcus aureus (MIC = 2.5 μM) and Escherichia coli (MIC = 10 μM). The data support a close phylogenetic relationship between Hymenochirus and Pseudhymenochirus that is distinct from the Xenopodinae (Xenopus + Silurana) clade with Pipa sister-group to all other extant pipids.     

Quality of Visual Cue Affects Visual Reweighting in Quiet Standing

Sensory reweighting is a characteristic of postural control functioning adopted to accommodate environmental changes. The use of mono or binocular cues induces visual reduction/increment of moving room influences on postural sway, suggesting a visual reweighting due to the quality of available sensory cues. Because in our previous study visual conditions were set before each trial, participants could adjust the weight of the different sensory systems in an anticipatory manner based upon the reduction in quality of the visual information. Nevertheless, in daily situations this adjustment is a dynamical process and occurs during ongoing movement. The purpose of this study was to examine the effect of visual transitions in the coupling between visual information and body sway in two different distances from the front wall of a moving room. Eleven young adults stood upright inside of a moving room in two distances (75 and 150 cm) wearing a liquid crystal lenses goggles, which allow individual lenses transition from opaque to transparent and vice-versa. Participants stood still during five minutes for each trial and the lenses status changed every one minute (no vision to binocular vision, no vision to monocular vision, binocular vision to monocular vision, and vice-versa). Results showed that farther distance and monocular vision reduced the effect of visual manipulation on postural sway. The effect of visual transition was condition dependent, with a stronger effect when transitions involved binocular vision than monocular vision. Based upon these results, we conclude that the increased distance from the front wall of the room reduced the effect of visual manipulation on postural sway and that sensory reweighting is stimulus quality dependent, with binocular vision producing a much stronger down/up-weighting than monocular vision.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

Chlorella vulgaris as a lipid source: Cultivation on air and seawater‐simulating medium in a helicoidal photobioreactor

The freshwater microalga Chlorella vulgaris was cultured batchwise on the seawater‐simulating Schlösser medium either in a 1.1‐L‐working volume helicoidal photobioreactor (HeP) or Erlenmeyer flask (EF) as control and continuously supplying air as CO₂ source. In these systems, maximum biomass concentration reached 1.65 ± 0.17 g L^?1 and 1.25 ± 0.06 g L^?1, and maximum cell productivity 197.6 ± 20.4 mg L^?1 day^?1 and 160.8 ± 12.2 mg L^?1 day^?1, respectively. Compared to the Bold's Basal medium, commonly employed to cultivate this microorganism on a bench‐scale, the Schlösser medium ensured significant increases in all the growth parameters, namely maximum cell concentration (268% in EF and 126% in HeP), maximum biomass productivity (554% in EF and 72% in HeP), average specific growth rate (67% in EF and 42% in HeP), and maximum specific growth rate (233% in EF and 22% in HeP). The lipid fraction of biomass collected at the end of runs was analyzed in terms of both lipid content and fatty acid profile. It was found that the seawater‐simulating medium, despite of a 56–63% reduction of the overall biomass lipid content compared to the Bold's Basal one, led in HeP to significant increases in both the glycerides‐to‐total lipid ratio and polyunsaturated fatty acid content compared to the other conditions taken as an average. These results as a whole suggest that the HeP configuration could be a successful alternative to the present means to cultivate C. vulgaris as a lipid source. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:279–284, 2016     

Chemical and Morphological Inter‐ and Intrapopulation Variability in Natural Populations of Gentiana pneumonanthe L.

Inter‐ and intrapopulation variability in six natural populations of the rare species Gentiana pneumonanthe was examined based on morphological and chemical data. Population size and linear morphometric parameters differed significantly among populations, but without a clear connection to habitat conditions, i. e. water supply and light availability. Leaf shape varied from ovate to lanceolate in all populations, and one population was distinctive in having the largest number of leaves of transitional shape. HPLC analyses of six secondary metabolites were performed separately for belowground parts, and aboveground vegetative and reproductive parts of individual plants (6 populations ×7 individuals ×3 plant parts, n=126) in order to examine differences at the population and individual levels. Three secoiridoids (swertiamarin (SWM), sweroside (SWZ), and gentiopicrin (GP)), one xanthone (mangiferin (MGF)), and two flavones (isoorientin (IO) and isovitexin (IV)) were detected and quantified in the analyzed samples: sweroside dominated in the aboveground reproductive part, mangiferin in the aboveground vegetative part, and gentiopicrin in the belowground part. At the population level, differences in contents of the analyzed chemicals among populations were significant only for a few metabolites. At the individual level, a pronounced organ‐dependent distribution of secondary metabolites was revealed. The results of this study contribute to a better understanding of natural variability within populations of the rare and threatened G. pneumonanthe, and provide data on the contents and within‐plant distribution of secondary metabolites, which are important as pharmacologically active compounds and may be useful for further biotechnological procedures regarding this species.     

The art of vector engineering: towards the construction of next-generation genetic tools

When recombinant DNA technology was developed more than 40 years ago, no one could have imagined the impact it would have on both society and the scientific community. In the field of genetic engineering, the most important tool developed was the plasmid vector. This technology has been continuously expanding and undergoing adaptations. Here, we provide a detailed view following the evolution of vectors built throughout the years destined to study microorganisms and their peculiarities, including those whose genomes can only be revealed through metagenomics. We remark how synthetic biology became a turning point in designing these genetic tools to create meaningful innovations. We have placed special focus on the tools for engineering bacteria and fungi (both yeast and filamentous fungi) and those available to construct metagenomic libraries. Based on this overview, future goals would include the development of modular vectors bearing standardized parts and orthogonally designed circuits, a task not fully addressed thus far. Finally, we present some challenges that should be overcome to enable the next generation of vector design and ways to address it.