Optimized methodology for extraction of (1 → 3)(1 → 6)-β-d-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative

The cell wall of Saccharomyces cerevisiae is an important source of β-d-glucan, a glucose homopolymer with immunostimulant properties. The standard methodologies described for its extraction involve acid and alkaline washings, which degrade part of its glucose chains and reduce the final yield. In the present study, an optimized methodology for extraction of β-d-glucan from S. cerevisiae cells, involving sonication and enzyme treatment, with a yield of 11.08 ± 0.19%, was developed. The high-purity (1 → 3)(1 → 6)-β-d-glucan was derivatized to carboxymethyl-glucan (CM-G). In vitro tests with CM-G in Chinese hamster epithelial cells (CHO-k1) did not reveal any cytotoxic or genotoxic effects or influences of this molecule on cell viability. The method described here is a convenient alternative for the extraction of (1 → 3)(1 → 6)-β-d-glucan under mild conditions without the generation of wastes that could be potentially harmful to the environment.     

The effect of dissolved oxygen concentrations on (1 → 3)- and (1 → 6)-β-glucanase production by Acremonium sp. IMI 383068 in batch culture

The influence of dissolved oxygen tension (DOT) on the production of extracellular (1 → 3)- and (1 → 6)-β-glucanases by the fungus Acremonium sp. IMI 383068 was investigated in batch culture. The controlled DOT levels at which cultures were grown affected the measured (1 → 3)-β-glucanase specific activities, but these effects were less marked on the measured (1 → 6)-β-glucanase specific activities. There appeared to be no direct link between the branching frequency of the fungus as reflected by its hyphal growth unit, and changing DOT levels. Whether pustulan or scleroglucan were used as the sole carbon source also affected production of these enzymes and their response to varying DOT. The measured (1 → 3)-β-glucanase specific activities were generally higher with scleroglucan, mainly because of the production of an extra active (1 → 3)-β-glucanase whereas pustulan grown cells produced a corresponding inactive protein with an identical electrophoretic mobility by SDS-PAGE and N-terminal amino acid sequence. With pustulan grown cells, DOT levels had comparatively little influence on the optimal measured specific activities of the (1 → 3)-β-glucanases, while with scleroglucan, they increased as DOT increased.     

Synthesis of deoxygenated α(1 → 5)-linked arabinofuranose disaccharides as substrates and inhibitors of arabinosyltransferases of Mycobacterium tuberculosis

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf α(1 → 5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The α(1 → 5)-linked disaccharides were produced by coupling n-octyl α-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H₃₇Ra and M. avium complex strains.     

Paenibacillus curdlanolyticus B-6 xylanase Xyn10C capable of producing a doubly arabinose-substituted xylose, α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp,from rye arabinoxylan

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α-l-arabinofuranosidase Axh43A indicated that U1 was α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp and U2 was α-l-Araf-(1 → 2)-d-Xylp, suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the −1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite −1. This is the first report of a xylanase capable of producing α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp from highly arabinosylated xylan.     

SYNE1-QK1 SNPs,G × G and G × E interactions on the risk of hyperlipidaemia

This study aimed to assess the relationship of 3 spectrin repeat containing nuclear envelope protein 1 (SYNE1) and 4 KH domain containing RNA binding (QK1) single nucleotide polymorphisms (SNPs), their haplotypes, gene-gene (G × G), gene-environment (G × E) interactions and hypercholesterolaemia (HCH) and hypertriglyceridaemia (HTG) in the Chinese Maonan minority. The genetic make-up of the SYNE1-QK1 SNPs in 1932 unrelated subjects (normal, 641; HCH, 649; and HTG, 642) was obtained by next-generation sequencing technologies. The genotypic frequencies of following SNPs were suggestively distinctive between the control and HCH groups (rs2623963, rs7745725, rs9459317, rs16897566), or between the control and HTG groups (rs2623963, rs1358317, rs7745725, rs1923608, rs16897566 SNPs; P < .05, respectively). Multiple-locus linkage disequilibrium analysis indicated that the identified SNPs were not inherited independently. Several haplotypes and gene-gene interaction haplotypes among the detected SNPs may be related with an increased morbidity of HCH (C-G-A, C-G-G and C-G-G-T-C-A-T) and HTG (C-G-G, G-T-G-C, C-G-G-G-T-G-C and C-G-G-T-C-A-T), whereas others may be related with an decreased risk of HCH (G-A-A, G-C-A-T, C-A-A-T-C-A-T and G-A-A-G-C-A-T) and HTG (G-A-A, G-C-A-T, C-A-A-T-C-A-T and G-A-A-G-C-A-T). The association evaluation based on haplotypes and gene-gene interactions could improve the power of detecting the risk of dyslipidaemia than anyone of SNP alone. There was significant three-locus model involving SNP-SNP, haplotype-haplotype/environment and G × G interactions (P < .05-0.001) that were detected by GMDR in HCH and HTG groups. Different interactions between genetic and environmental factors would produce different redundancy or synergy effects on the morbidity of HCH and/or HTG.     

Effects of antimicrobial activity of silver nanoparticles on in vitro establishment of G × N15 (hybrid of almond × peach) rootstock

In the present investigation were evaluated the antifungal and antibacterial activities of Nano-silver (NS). Two separate experiments were done to evaluate the potential of silver nanoparticles in controlling the contamination of G × N15 micro-propagation. In the first experiment, a factorial experiment based on a completely randomized design with 15 treatments including five different NS concentrations (0, 50, 100, 150 and 200 ppm) and three soaking time of explants (3, 5 and 7 min) with five replications was conducted. In the other experiment, NS was added to Murashige and Skoog (MS) medium with concentrations of 0, 50, 100, 150 and 200 ppm in a completely randomized design. Results showed that the application of 100 and 150 ppm NS both as an immersion and as added directly to the culture medium significantly reduces internal and external contaminations compared with the control group. Using NS in culture medium was more efficient to reduce fungal and bacterial contamination than immersion. High concentrations of NS had an adverse effect on the viability of G × N15 single nodes and this effect was more serious in immersed explants in NS than directly added NS ones regarding the viability of buds and plantlet regeneration. In conclusion, due to high contamination of woody plants especially fruit trees and also adverse environmental effects of mercury chloride, the NS solution can be used as a low risk bactericide in micro-propagation of G × N15 and can be an appropriate alternative to mercury chloride in the future.     

Coupled electron and proton transfer reactions during the O → E transition in bovine cytochrome c oxidase

A combined DFT/electrostatic approach is employed to study the coupling of proton and electron transfer reactions in cytochrome c oxidase (CcO) and its proton pumping mechanism. The coupling of the chemical proton to the internal electron transfer within the binuclear center is examined for the O → E transition. The novel features of the His291 pumping model are proposed, which involve timely well-synchronized sequence of the proton-coupled electron transfer reactions. The obtained pK_as and E_ms of the key ionizable and redox-active groups at the different stages of the O → E transition are consistent with available experimental data. The PT step from E242 to H291 is examined in detail for various redox states of the hemes and various conformations of E242 side-chain. Redox potential calculations of the successive steps in the reaction cycle during the O → E transition are able to explain a cascade of equilibria between the different intermediate states and electron redistribution between the metal centers during the course of the catalytic activity. All four electrometric phases are discussed in the light of the obtained results, providing a robust support for the His291 model of proton pumping in CcO. This article is part of a Special Issue entitled: Respiratory oxidases.     

When did Cuon reach Java? – Reinvestigation of canid fossils from Homo erectus faunas

The presence of the genus Cuon during the Pleistocene of Java is attested by the endemic species C. sangiranensis and C. crassidens, sometimes classified as C. alpinus, a species that is also part of the Holocene fauna and has persisted until today. The current study revises the fossil canid material from the Sangiran Dome, previously assigned variously to C. sangiranensis, C. crassidens, C. alpinus, C. javanicus, and C. trinilensis. It aims to reinvestigate the arrival of Cuon in Java, in particular in relation to the late early Pleistocene endemic canid of Java: the Cuon-sized Xenocyon trinilensis, which had evolved in situ on Java from the large X. merriami, and which could be a potential competitor to early Cuon in case of contemporaneity. We found that a mandible fragment from Pandejan (holotype of C. crassidens) shows a morphology typical for C. alpinus. However, a maxilla and a mandible from the Bapang Formation at Sangiran, including the holotype of C. sangiranensis, is here assigned to the early dhole species C. priscus. Our results indicate that Cuon was already present in Java during the early middle Pleistocene and probably arrived as part of the immigration events during the Kedung Brubus faunal stage. Since X. trinilensis and Cuon are both hypercarnivorous medium-sized dogs, similar ecological niches are expected for both species. Thus, X. trinilensis probably was outcompeted and replaced by C. priscus. In turn, the latter species was replaced by the extant species, C. alpinus, during the Nangdong faunal stage, likely as part of the next immigration phase, which then disappeared during the tropical rainforest fauna of the Punung faunal stage and re-entered at the onset of the Holocene.     

Mechanisms of mechanosensing — mechanosensitive channels,function and re-engineering

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Anaerobic microbial dehalogenation of organohalides — state of the art and remediation strategies

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Metal–organic chemical vapor deposition of Bi2Mn4O10 films on SrTiO3 〈1 0 0〉

Bi₂Mn₄O₁₀ films were deposited on SrTiO₃ (1 0 0) substrates via metal–organic chemical vapor deposition (MOCVD) from the Bi(phenyl)₃ and Mn(tmhd)₃ (Htmhd = 2,2,6,6-tetramethyl-3,5-heptanedione) precursors. The films were deposited in the temperature range of 600–800 °C. The X-ray diffraction (XRD) characterization indicates that the Bi₂Mn₄O₁₀ phase is stable within the investigated range, but the temperature plays a crucial role in determining the out-of-plane orientation of the films. The SEM shows very homogeneous surfaces with a fiber texture morphology at the highest deposition temperature. The AFM data indicate a textured surface with a root mean square roughness of 77.67 nm for films deposited at 800 °C.     

Genotype × region and genotype × production level interactions in Holstein cows

The identification of the presence of genotype by environment interaction effects on important traits in Holstein cattle allows for the use of international genetic evaluations and a more efficient design of regional genetic evaluation programmes. The aim of this study was to determine the genotype × environment interaction effects in Chilean Holstein dairy cattle through the analysis of records corresponding to calvings between 1998 and 2015. Herds were classified in the central and southern regions of Chile based on herd location as well as by high and low levels of production environments based on the fat plus protein yield averages per herd within each region. The central region has a Mediterranean climate and a confined production system while the southern region has a humid temperate climate and a production system based on grazing with supplementation. Traits studied were milk yield (MY), fat yield (FY), protein yield (PY), fat content (FC) and protein content (PC) by lactation, age at first calving (AFC) and calving interval (CI). Several four-trait mixed animal models were applied to environmental category data as different traits, which included herd-year-calving season (herd-year-birth season for AFC) and lactation number as fixed effects, and animal additive genetic, sire-herd, permanent environment and residual effects as random effects. Genetic correlations (r_g) for MY, FY, FC, PC and CI were found to decrease as differences between environmental categories increased. The r_g between the most extreme environmental categories considered in this study for AFC (0.26) was the only one found statistically lower than 0.60. Genetic correlation values statistically lower than 0.80 (P < 0.05) were observed for AFC, CI, MY, FY and PY between some environmental categories. If separate genetic evaluations are adopted as practical criteria when the value of r_g is lower than 0.60, the consequence of improving a multi-trait economic breeding objective in this population is likely to be small unless extreme environmental categories are considered. However, a moderate decrease in selection response and re-ranking of selection candidates is expected for AFC, CI and yield traits when selection is performed in different environmental conditions. Genotype × environment interaction effects involving production systems in a Mediterranean climate and confinement vs. Temperate Oceanic climate and grazing with supplementation, and between two fat plus protein yield level categories within each environment, were at most moderate for the studied traits.     

The Iron Gates Mesolithic – a brief review of recent developments

The Iron Gates section of the Lower Danube valley along the border between Romania and Serbia has an unparalleled record of Mesolithic and Early Neolithic settlement spanning the period from ca. 12,700 to 5600 cal BC. Over 50 cave and open-air sites were identified during archaeological surveys in advance of dam construction in the 1960s and 1980s, and follow-up rescue excavations revealed numerous burials and architectural remains and produced rich inventories of faunal material and portable artifacts including artworks and ornaments of bone, shell and stone. Most sites are no longer accessible, submerged beneath the reservoirs created by the Iron Gates I and II dams. Since 1990, new excavations have been conducted at Aria Babi and Vlasac in Serbia, and Schela Cladovei in Romania, while detailed studies of the finds from both new and old excavations have been undertaken by researchers based in Romania, Serbia and the UK fueled by developments in archaeological science. In this paper, we review the main advances in knowledge of the Mesolithic and the transition to farming in the Iron Gates over the past 25 years, and especially the period since 2005. The paper is divided into sections dealing with chronology, mortuary practices, isotopic studies of subsistence and mobility patterns, and the nature and timing of the transition from Mesolithic to Neolithic in the Iron Gates region. The review concludes with a forward look at research in progress.     

Cryoelectron Microscopy Structure of Purified γ-Secretase at 12 Å Resolution

γ-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of the amyloid β-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human γ-secretase in digitonin has a total molecular mass of ∼ 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the γ-secretase complex at 12 Å resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.     

Effects of nutrient media,different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond × peach) vegetative rootstock

The objective of this research was to assess the effects of different media i.e. Murashige and Skoog (MS) and Quoirin and Lepoivre (QL), cytokinin type i.e. 6-Benzyladenin (BA) and 6-Benzylaminopurine (BAP) and cytokinin concentration on in vitro proliferation of the G × N15 rootstock. To evaluate the effects of different media and cytokinin type, two separate experiments were conducted as factorial based on completely randomized design, and single nodes were used as explants. The results showed that MS nutrient medium was found to be superior to QL nutrient medium. Regarding the interaction between media and growth regulators, the best interaction was found in MS medium supplemented with 1 mg l⁻¹ BAP resulting in 8.5 new micro shoots/explant while 7.75 shoots were observed in MS medium containing 1.25 mg l⁻¹ BA. The longest length of new micro-shoots (2.10 cm) was obtained in hormone-free MS medium. Findings of this study showed that there is a significant correlation between the hormone level and plantlet height and formed callus weight so that an increase in BAP and BA levels in both of MS and QL media resulted significantly in height decrease and callus weight increase. The results also suggest that the best and the worst plantlets in terms of quality were observed in hormone-free QL medium and MS medium supplemented with 1.25 mg l⁻¹, respectively. These results reflect the fact that the presence of high amounts of NH₄NO₃ and cytokinin especially BAP in culture medium triggered inhibitory effect on shoot growth.     

Novel screening methods — biosensors

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Response of the ArcCHECK® device at 6 MV and 15 MV for VMAT and IMRT quality control

PurposeTo study the response of the ArcCHECK® device as VMAT and IMRT verification system.MethodsVarious tests analyzing the linearity, the repeatability and the angular dependence of the device response, its dependence with the pulse repetition rate and the leakage losses were performed. The long-term response in dose measurements and the uniformity of the detectors conforming the system were controlled using a statistical process control program. The Elekta Infinity™ 6 and 15 MV photon beams were used.ResultsThe device showed excellent repeatability and linearity. The differences between the responses obtained for any pair of angular incidences were less than 2%. The absorbed dose increased by 3% when the pulse repetition rate varied from 50 to 600 MU/min. Results are in overall agreement with those found in previous works for the ArcCHECK®, in which a reduced number of the device diodes were analyzed, and for the MapCheck®, an older 2D device that used the same diodes. Charge losses were found to be negligible except for some of the diodes of the device. The statistical process control program is a very useful tool to control the correct functioning of the device in the long term.ConclusionsThe results of the analysis carried out indicate that the working and stability conditions of the ArcCHECK® device are adequate for its purpose. The dependence with the pulse repetition rate should be considered when VMAT or similar treatments are evaluated. A control program for the statistical monitoring of the device would be desirable and useful.     

In-vivo dosimetry for field sizes down to 6 × 6 mm2 in shaped beam radiosurgery with microMOSFET

The aim of this study is to evaluate microMOSFET as in-vivo dosimeter in 6 MV shaped-beam radiosurgery for field sizes down to 6 × 6 mm². A homemade build-up cap was developed and its use with microMOSFET was evaluated down to 6 × 6 mm². The study with the homemade build-up cap was performed considering its influence on field size over-cover occurring at surface, achievement of the overall process of electronic equilibrium, dose deposition along beam axis and dose attenuation. An optimized calibration method has been validated using MOSFET in shaped-beam radiosurgery for field sizes from 98 × 98 down to 18 × 18 mm². The method was detailed in a previous study and validated in irregular field shapes series measurements performed on a head phantom. The optimized calibration method was applied to microMOSFET equipped with homemade build-up cap down to 6 × 6 mm². Using the same irregular field shapes, dose measurements were performed on head phantom. MicroMOSFET results were compared to previous MOSFET ones. Additional irregular field shapes down to 8.8 × 8.8 mm² were studied with microMOSFET. Isocenter dose attenuation due to the homemade build-up cap over the microMOSFET was near 2% irrespective of field size. Our results suggested that microMOSFET equipped with homemade build-up cap is suitable for in-vivo dosimetry in shaped-beam radiosurgery for field sizes down to 6 × 6 mm² and therefore that the required build-up cap dimensions to perform entrance in-vivo dosimetry in small-fields have to ensure only partial charge particle equilibrium.     

The redox chemistry of the Alzheimer's disease amyloid β peptide

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid β peptide (Aβ), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Aβ from the brain. Aβ can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Aβ will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Aβ oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Aβ levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Aβ species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.