Unravelling the molecular interactions behind the formation of PEG/PPG aqueous two-phase systems.

The understanding of molecular interactions that control phase separation in polymer/polymer aqueous two-phase systems (ATPS) has been a subject of debate up to this day. In light of this, we set out to investigate the molecular interactions occurring in ternary mixtures composed of polyethylene glycol (PEG600), polypropylene glycol (PPG400) and water. The ternary phase diagram was plotted at two temperatures (298 K and 323 K), revealing a transition from a type 0 to a type I diagram. Molecular dynamics (MD) simulations were performed to elucidate the polymer-polymer and polymer-water interactions occurring at different temperatures and water concentrations. COnductor-like Screening Model for Realistic Solvents (COSMO-RS) was used to assess the thermodynamic properties of the polymer-water binary mixtures and their correlation with ATPS formation. The MD simulations clearly demonstrate the effect of segregation/separation with increasing water content and temperature, highlighting a significant reduction in PPG-water interactions compared to PEG-water counterparts. Polymer-water interactions were identified as those controlling the phase separation mechanism, and the thermodynamic properties determined with COSMO-RS for the polymer-water binary systems further support this view.

The path towards type V deep eutectic solvents: inductive effects and steric hindrance in the system tert-butanol + perfluoro tert-butanol.

The solid-liquid phase behaviour of two tertiary alcohols, perfluoro-tert-butanol and tert-butanol, was studied here using experimental (ITC, DSC and density measurements) and theoretical (MD simulations) approaches. The phase diagram of the binary mixture reveals highly negative deviations from ideality at low concentrations, as well as the formation of co-crystals and is characterized by two eutectic points, a congruent melting point and a peritectic reaction corresponding to TBH : TBF stoichiometries of 2 : 1 and 1 : 1 respectively. Excess molar enthalpies and volumes were calculated, showing negative and positive deviations from ideality, respectively. The effect of acidity, stereochemical hindrance and phobic effects and how they affect intermolecular interactions in these binary mixtures is discussed, with the aim of designing and fine-tuning type V deep eutectic solvents. The results showed that the fluorination of tertiary alcohols can be used for the tuning of the mixing properties and solid-liquid phase diagrams.

Acute Recovery after a Fatigue Protocol Using a Recovery Sports Legging: An Experimental Study.

Enhancing recovery is a fundamental component of high-performance sports training since it enables practitioners to potentiate physical performance and minimise the risk of injuries. Using a new sports legging embedded with an intelligent system for electrostimulation, localised heating and compression (completely embodied into the textile structures), we aimed to analyse acute recovery following a fatigue protocol. Surface electromyography- and torque-related variables were recorded on eight recreational athletes. A fatigue protocol conducted in an isokinetic dynamometer allowed us to examine isometric torque and consequent post-exercise acute recovery after using the sports legging. Regarding peak torque, no differences were found between post-fatigue and post-recovery assessments in any variable; however, pre-fatigue registered a 16% greater peak torque when compared with post-fatigue for localised heating and compression recovery methods. Our data are supported by recent meta-analyses indicating that individual recovery methods, such as localised heating, electrostimulation and compression, are not effective to recover from a fatiguing exercise. In fact, none of the recovery methods available through the sports legging tested was effective in acutely recovering the torque values produced isometrically.

Identification of 45S rDNA in Passiflora using low coverage sequencing: analysis of GC content and chromosomal localization.

BACKGROUND: The 45S rDNA is considered the most useful chromosomal marker for cytogenetic analysis of Passiflora. Amplification of 45S rDNA sequence via PCR are more advantageous than sequence maintenance in vectors for chromosomal hybridization via FISH. We aimed both to identify 45S rDNA by sequencing data for chromosomal localization and to verify the relationship between GC content and CMA3/DAPI banding. METHODS AND RESULTS: Low-coverage sequencing of Passiflora alata, P. cincinnata, and P. edulis was performed, and 45S rDNA units were identified using RepeatExplorer. The 45S rDNA units were used to construct a neighbor-joining tree to verify the similarities between the three species' 18S and 26S rDNA sequences. Clusters (CL)116 (P. alata), CL71 (P. cincinnata), and CL116 (P. edulis) were remarkably similar among the three species, and the 26S rDNA sequences of the clusters were similar to those of Populus tremuloides, Salix interior, and Averrhoa carambola (98% identity). The 26S rDNA was cytologically localized in the chromosomes of P. edulis, P. bahiensis, and the backcrossed hybrid (P. sublanceolata vs. HD13). The hybridization transfer capacity was evaluated in Citrus sunki and Cucumis melo. Finally, a chromosomal pair with a heteromorphic 26S rDNA site was observed in P. edulis, which was the same to that observed for CMA3. CONCLUSION: The amplification of the 26S rDNA in Passiflora via PCR and the chromosomal localization in Passiflora and other plant species was successfully achieved. The CMA3 bands were found to be related not only to the amount of GC but also to its structure and the number of repetitions.

Karyotyping of commercial cultivars of melon (Cucumis melo L.).

BACKGROUND: This study on cultivars of melon (Cucumis melo L.) marketed in Brazil was conducted to obtain information to be used in breeding programs of this species. Little is known about the karyotype variability among C. melo L. cultivars targeted at the consumer market. The objective of the present study was to verify the karyotype variability in eight commercial melon cultivars used in the Brazilian market. METHODS AND RESULTS: Slides were stained with 2% Giemsa and assembled with Neomount to perform chromosomal morphometry. GC-rich heterochromatin was observed by CMA3/DAPI staining. 5 S rDNA, centromeric satellite DNA (SatDNA), and telomeric sites were visualized using fluorescence in situ hybridization. All images were captured on an Olympus BX41 microscope equipped with a 5 M Olympus DP25 digital camera and DP2-BSW software. The cultivars showed symmetrical karyotypes with significant differences in total chromosome length and average chromosome size. Heterochromatic CMA3+ blocks were observed in terminal regions related to satellites (secondary constrictions), as well as in centromeric and pericentromeric regions. A single chromosomal pair of 5 S rDNA sites was observed in all cultivars, but at distinct locations. Centromeric satellite sequences, tested for the first time in melon, revealed only centromeric sites. Telomeric sites were observed in all the chromosomes of the cultivars. CONCLUSIONS: Karyotype variation was observed in cultivars of melon, which were analyzed for chromosomal morphology and localization of GC-rich heterochromatin, as well centromeric SatDNA, rDNA, and telomeric chromosomal markers. Hence, these cultivars can be used in future breeding programs.

Spontaneous self-assembly and structure of perfluoroalkylalkane surfactant hemimicelles by molecular dynamics simulations.

Fully atomistic molecular-dynamics (MD) simulations of perfluoroalkylalkane molecules at the surface of water show the spontaneous formation of aggregates whose size and topography closely resemble the experimentally observed hemimicelles for this system. Furthermore, the grazing incidence X-ray diffraction (GIXD) pattern calculated from the simulation trajectories reproduces the experimental GIXD spectra previously obtained, fully validating the MD simulation results. The detailed analysis of the internal structure of the aggregates obtained by the MD simulations supports a definite rational explanation for the spontaneous formation, stability, size, and shape of perfluoroalkylalkane hemimicelles at the surface of water.

Experimental validation of a computational fluid dynamics model using micro-particle image velocimetry of the irrigation flow in confluent canals.

AIM: This study aimed to experimentally validate a computational fluid dynamics (CFD) model, using micro-particle image velocimetry (micro-PIV) measurements of the irrigation flow velocity field developed in confluent canals during irrigation with a side-vented needle. METHODOLOGY: A microchip with confluent canals, manufactured in polydimethylsiloxane was used in a micro-PIV analysis of the irrigation flow using a side-vented needle placed 3 mm from the end of the confluence of the canals. Velocity fields and profiles were recorded for flow rates of 0.017 and 0.1 ml/s and compared with those predicted in CFD numerical simulations (using a finite volume commercial code - FLUENT) for both laminar and turbulent regimes. RESULTS: The overall flow pattern, isovelocity and vector maps as well as velocity profiles showed a close agreement between the micro-PIV experimental and CFD predicted data. No relevant differences were observed between the results obtained with the laminar and turbulent flow models used. CONCLUSIONS: Results showed that the laminar CFD modelling is reliable to predict the flow in similar domains.

Sweet Cherries as Anti-Cancer Agents: From Bioactive Compounds to Function.

Sweet cherries (Prunus avium L.) are among the most appreciated fruits worldwide because of their organoleptic properties and nutritional value. The accurate phytochemical composition and nutritional value of sweet cherries depends on the climatic region, cultivar, and bioaccessibility and bioavailability of specific compounds. Nevertheless, sweet cherry extracts are highly enriched in several phenolic compounds with relevant bioactivity. Over the years, technological advances in chemical analysis and fields as varied as proteomics, genomics and bioinformatics, have allowed the detailed characterization of the sweet cherry bioactive phytonutrients and their biological function. In this context, the effect of sweet cherries on suppressing important events in the carcinogenic process, such as oxidative stress and inflammation, was widely documented. Interestingly, results from our research group and others have widened the action of sweet cherries to many hallmarks of cancer, namely metabolic reprogramming. The present review discusses the anticarcinogenic potential of sweet cherries by addressing their phytochemical composition, the bioaccessibility and bioavailability of specific bioactive compounds, and the existing knowledge concerning the effects against oxidative stress, chronic inflammation, deregulated cell proliferation and apoptosis, invasion and metastization, and metabolic alterations. Globally, this review highlights the prospective use of sweet cherries as a dietary supplement or in cancer treatment.

Sweet Cherry Extract Targets the Hallmarks of Cancer in Prostate Cells: Diminished Viability, Increased Apoptosis and Suppressed Glycolytic Metabolism.

The present work evaluated the anticancer properties of sweet cherry (Prunus avium) extract on human prostate cells. Several sweet cherry cultivars from Fundão (Portugal) were methanol-extracted and their phytochemical composition characterized. The Saco "late harvest" extract was highly-enriched in anthocyanins and selected for use in biological assays. Non-neoplastic (PNT1A) and neoplastic (LNCaP and PC3) human prostate cells were treated with 0-2,000 µg/ml of extract for 48-96 h. Cell viability was evaluated by the MTT assay. Apoptosis, oxidative stress, and glycolytic metabolism were assessed by Western blotting and enzymatic assays. Glucose consumption and lactate production were measured spectrophotometrically. Saco cherry extract diminished the viability of neoplastic and non-neoplastic cells, whereas enhancing apoptosis in LNCaP. Cherry extract-treatment also diminished oxidative damage and suppressed glycolytic metabolism in LNCaP cells. These findings widened the knowledge on the mechanisms by which cherry extract modulate cell physiology, demonstrating their broad action over the hallmarks of cancer.

Reviving the local second-order boundary approach within the two-relaxation-time lattice Boltzmann modelling.

This work addresses the Dirichlet boundary condition for momentum in the lattice Boltzmann method (LBM), with focus on the steady-state Stokes flow modelling inside non-trivial shaped ducts. For this task, we revisit a local and highly accurate boundary scheme, called the local second-order boundary (LSOB) method. This work reformulates the LSOB within the two-relaxation-time (TRT) framework, which achieves a more standardized and easy to use algorithm due to the pivotal parametrization TRT properties. The LSOB explicitly reconstructs the unknown boundary populations in the form of a Chapman-Enskog expansion, where not only first- but also second-order momentum derivatives are locally extracted with the TRT symmetry argument, through a simple local linear algebra procedure, with no need to compute their non-local finite-difference approximations. Here, two LSOB strategies are considered to realize the wall boundary condition, the original one called Lwall and a novel one Lnode, which operate with the wall and node variables, roughly speaking. These two approaches are worked out for both plane and curved walls, including the corners. Their performance is assessed against well-established LBM boundary schemes such as the bounce-back, the local second-order accurate CLI scheme and two different parabolic multi-reflection (MR) schemes. They are all evaluated for 3D duct flows with rectangular, triangular, circular and annular cross-sections, mimicking the geometrical challenges of real porous structures. Numerical tests confirm that LSOB competes with the parabolic MR accuracy in this problem class, requiring only a single node to operate. This article is part of the theme issue 'Fluid dynamics, soft matter and complex systems: recent results and new methods'.

Correction to: low coverage sequencing for repetitive DNA analysis in Passiflora edulis Sims: citogenomic characterization of transposable elements and satellite DNA.

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Low coverage sequencing for repetitive DNA analysis in Passiflora edulis Sims: citogenomic characterization of transposable elements and satellite DNA.

BACKGROUND: The cytogenomic study of repetitive regions is fundamental for the understanding of morphofunctional mechanisms and genome evolution. Passiflora edulis a species of relevant agronomic value, this work had its genome sequenced by next generation sequencing and bioinformatics analysis performed by RepeatExplorer pipeline. The clusters allowed the identification and characterization of repetitive elements (predominant contributors to most plant genomes). The aim of this study was to identify, characterize and map the repetitive DNA of P. edulis, providing important cytogenomic markers, especially sequences associated with the centromere. RESULTS: Three clusters of satellite DNAs (69, 118 and 207) and seven clusters of Long Terminal Repeat (LTR) retrotransposons of the superfamilies Ty1/Copy and Ty3/Gypsy and families Angela, Athila, Chromovirus and Maximus-Sire (6, 11, 36, 43, 86, 94 and 135) were characterized and analyzed. The chromosome mapping of satellite DNAs showed two hybridization sites co-located in the 5S rDNA region (PeSat_1), subterminal hybridizations (PeSat_3) and hybridization in four sites, co-located in the 45S rDNA region (PeSat_2). Most of the retroelements hybridizations showed signals scattered in the chromosomes, diverging in abundance, and only the cluster 6 presented pericentromeric regions marking. No satellite DNAs and retroelement associated with centromere was observed. CONCLUSION: P. edulis has a highly repetitive genome, with the predominance of Ty3/Gypsy LTR retrotransposon. The satellite DNAs and LTR retrotransposon characterized are promising markers for investigation of the evolutionary patterns and genetic distinction of species and hybrids of Passiflora.

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface.

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody-protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.

Tissue culture and next-generation sequencing: A combined approach for detecting yam (Dioscorea spp.) viruses.

In vitro culture offers many advantages for yam germplasm conservation, propagation and international distribution. However, low virus titres in the generated tissues pose a challenge for reliable virus detection, which makes it difficult to ensure that planting material is virus-free. In this study, we evaluated next-generation sequencing (NGS) for virus detection following yam propagation using a robust tissue culture methodology. We detected and assembled the genomes of novel isolates of already characterised viral species of the genera Badnavirus and Potyvirus, confirming the utility of NGS in diagnosing yam viruses and contributing towards the safe distribution of germplasm.

Structure of Langmuir Monolayers of Perfluorinated Fatty Acids: Evidence of a New 2D Smectic C Phase.

Due to the characteristic chain rigidity and weak intermolecular interactions of perfluorinated substances, the phase diagram of Langmuir monolayer formed by perfluorinated molecules has been interpreted so far as displaying only two phases, a 2D gas (G) and a liquid condensed (LC). However, in this work, we presented Grazing Incidence X-ray Diffraction measurements, which exhibit two diffraction peaks on the transition plateau: One is the signature of the hexagonal structure of the LC phase, the second one is associated to the low-density fluid phase and is thus more ordered than expected for a 2D gas or a typical fluid phase. Atomistic molecular dynamics simulations, performed on the transition plateau, revealed the existence of clusters in which domains of vertical molecules organized in a hexagonal lattice coexist with domains of parallel lines formed by tilted molecules, a new structure that could be described as a "2D smectic C" phase. Moreover, the diffraction spectrum calculated from the simulation trajectories compared favorably with the experimental spectra, fully validating the simulations and the proposed interpretation. The results were also in agreement with the thermodynamic analysis of the fluid phase and X-ray Reflectivity experiments performed before and after the transition between these two phases.

Rapid detection of potyviruses from crude plant extracts.

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named 'Direct RT-RPA', detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.

Identification and characterization of karyotype in Passiflora hybrids using FISH and GISH.

BACKGROUND: A great interest exists in the production of hybrid plants of the genus Passiflora given the beauty and exotic features of its flowers which have ornamental value. Hybrid paternity confirmation is therefore important for assuring germplasm origin, and is typically carried out by molecular marker segregation. The aim of this study was to karyotypically characterize the chromosome heritance patterns of the progeny resultant from a cross of P. gardneri and P. gibertii using classical cytogenetics, chromosome banding, and molecular cytogenetics. RESULTS: All analyzed genotypes showed the same diploid chromosome number as the genitor species: 2n = 18. Classical and CMA3 and DAPI staining allowed for chromosome counting and satellite identification (secondary constrictions). Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used to characterize subgenomes by either identifying rDNA-specific genome patterns or parental genomes, respectively. CONCLUSIONS: The heritance of chromosomal markers presenting rDNA sites from each parent for genome identification confirmed that all obtained plants were hybrids. These results will improve breeding programs involving the species of this genus. Apart from confirming hybridization, GISH allowed the visualization of recombination between the homeologous chromosome and the introgression of sequences of interest.

Complete genome sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT virus 3, reveals the first evidence of recombination in yam badnaviruses.

Yams (Dioscorea spp.) host a diverse range of badnaviruses (genus Badnavirus, family Caulimoviridae). The first complete genome sequence of Dioscorea bacilliform RT virus 3 (DBRTV3), which belongs to the monophyletic species group K5, is described. This virus is most closely related to Dioscorea bacilliform SN virus (DBSNV, group K4) based on a comparison of genome sequences. Recombination analysis identified a unique recombination event in DBRTV3, with DBSNV likely to be the major parent and Dioscorea bacilliform AL virus (DBALV) the minor parent, providing the first evidence for recombination in yam badnaviruses. This has important implications for yam breeding programmes globally.

Chromogenic detection of yam mosaic virus by closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP).

A closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP) assay was developed for the detection of yam mosaic virus (YMV, genus Potyvirus) infecting yam (Dioscorea spp.). The assay uses a set of six oligonucleotide primers targeting the YMV coat protein region, and the amplification products in YMV-positive samples are visualized by chromogenic detection with SYBR Green I dye. The CT-RT-LAMP assay detected YMV in leaf and tuber tissues of infected plants. The assay is 100 times more sensitive in detecting YMV than standard RT-PCR, while maintaining the same specificity.

Antibody adsorption in protein-A affinity chromatography - in situ measurement of nanoscale structure by small-angle X-ray scattering.

Protein-A chromatography is the most widely used chromatography step in downstream processing of antibodies. A deeper understanding of the influence of the surface topology on a molecular/nanoscale level on adsorption is essential for further improvement. It is not clear if the binding is homogenous throughout the entire bead network. We followed the protein absorption process and observed the formation of a protein layer on fibers of chromatography resin in a time-resolved manner in nanoscale. To characterize the changes in the antibody-protein-A ligand complex, small angle X-ray scattering was employed using a miniaturized X-ray-transparent chromatography column packed with a MabSelect SuRe resin. Antibody-free MabSelect SuRe resin fiber had an average radius of 12 nm and the protein layer thickness resulting from antibody adsorption was 5.5 and 10.4 nm for fiber and junctions, respectively under applied native conditions. We hypothesize that an average of 1.2 antibodies were adsorbed per protein-A ligand tetramer bound to the outermost units. In contrast to previous studies, it was therefore possible for the first time to directly correlate the nanostructure changes inside the column, which is otherwise a black box, with the adsorption and elution process.