Longer Incubation Times Research Articles

A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation

In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)32+) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena.

Cell cycle is disturbed in mucopolysaccharidosis type II fibroblasts, and can be improved by genistein

Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by mutations resulting in deficiency of one of enzymes involved in degradation of glycosaminoglycans (GAGs). These compounds accumulate in cells causing their dysfunctions. Genistein is a molecule previously found to both modify GAG metabolism and modulate cell cycle. Therefore, we investigated whether the cell cycle is affected in MPS cells and if genistein can influence this process. Fibroblasts derived from patients suffering from MPS types I, II, IIIA and IIIB, as well as normal human fibroblasts (the HDFa cell line) were investigated. MTT assay was used for determination of cell proliferation, and the cell cycle was analyzed by using the MUSE® Cell Analyzer. While effects of genistein on cell proliferation were similar in both normal and MPS fibroblasts, fractions of cells in the G0/G1 phase were higher, and number of cells entering the S and G2/M phases was considerably lower in MPS II fibroblasts relative to control cells. Somewhat similar tendency, though significantly less pronounced, could be noted in MPS I, but only at longer times of incubation. However, this was not observed in MPS IIIA and MPS IIIB fibroblasts. Genistein (5, 7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) was found to be able to partially correct the disturbances in the MPS II cell cycle, and to some extent in MPS I, at higher concentrations of this compound. The tendency to increase the fractions of cells entering the S and G2/M phases was also observed in MPS IIIA and IIIB fibroblasts treated with genistein. In conclusion, this is the first report indicating that the cell cycle can be impaired in MPS cells. The finding that genistein can improve the MPS II (and to some extent also MPS I) cell cycle provides an input to our knowledge on the molecular mechanisms of action of this compound.

Near infrared fluorescent nanoparticles based on hyaluronic acid: Self-assembly, optical properties, and cell interaction

Fluorescent imaging agents that can specifically highlight tumor cells could have a significant impact on image-guided tumor removal. Here, fluorescent nanoparticles (NPs) derived from hyaluronic acid (HA) are investigated. HA is a ligand for the receptor CD44, which is a common biomarker present on many primary tumor cells, cancer-initiating cells, and tumor-associated fibroblasts. In addition, a family of enzymes that degrade HA, called hyaluronidases (HYALs), are also overexpressed with increased activity in many tumors. We report the design and development of a panel of targeted imaging agents using the near-infrared (NIR) dye, Cy7.5, that was directly conjugated to hydrophobically-modified HA. Two different molecular weights of HA, 10kDa and 100kDa, and three different degrees of hydrophobic moiety conjugation (0, 10, and 30mol%) were utilized to develop a panel of NPs with variable size that ranged from 50 to 400nm hydrodynamic diameter (HD) depending HA molecular weight, extent of fluorescence quenching (25–50%), kinetics of cellular uptake, and targeting to CD44+ cells. The kinetics and energy-dependence of cellular uptake in breast and prostate cancer cell lines, MDA-MB 231 and PC-3 cells, respectively, showed increased uptake with longer incubation times (at 4 and 8h compared to 1h), as well as uptake at 37°C but not 4°C, which indicated energy-dependent endocytosis. NP uptake studies in the presence of excess free HA showed that pre-treatment of cells with excess high molecular weight (MW) free HA decreased NP uptake by up to 50%, while no such trend was observed with low MW HA. These data lay the foundation for selection of optimized HA-derived NPs for image-guided surgery. Statement of SignificanceHere, hyaluronic acid (HA), a well-studied biomacromolecule, is modified with a near infrared fluorophore and a hydrophobic moiety. The significance of this work, especially for imaging applications, is that the impact of HA molecular weight and the hydrophobic moiety conjugation degree on fluorescence and cell interaction can be predicted. With respect to existing literature, the eventual use of these HA-based NPs is image-guided surgery; thus, we focus on the dye, Cy7.5, for conjugation, which is more NIR than most existing HA literature. Furthermore, HA is a ligand for CD44, which is associated with cancer and tumor microenvironment cells. Systematic studies in this work highlight that HA can be tuned to maximize or minimize CD44 binding

Targeted Mesoporous Silica Nanoparticles Delivering Arsenic Trioxide with Environment Sensitive Drug Release for Effective Treatment of Triple Negative Breast Cancer.

In this study, we report a novel modality of using a mesoporous silica nanoparticles (MSNs)-based drug delivery system with RGD peptide as a targeting ligand to load arsenic trioxide (ATO) (ATO-MSNs-RGD) for treating MDA-MB-231 triple-negative breast cancer. The MSNs, ATO-MSNs, and ATO-MSNs-RGD were characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA), transmission electron microscopy (TEM), and Brunauer-Emmet-Teller (BET) method. The data indicated that the MSNs possessed MCM-41 type mesopores with high surface area of ∼1021 m2/g and pore diameter of ∼2.2 nm. However, both values dramatically decreased after MSNs were encapsulated with ATO or modified with RGD. The amount of surface anchored RGD peptide was determined to be 0.20 mmol/g. Glutathione (GSH) greatly enhanced ATO release from MSNs. Confocal laser microscopy images demonstrated that both ATO-MSNs and ATO-MSNs-RGD had good cellular uptake that improved with longer incubation time and nanoparticle concentration and the ATO-MSNs-RGD showed clearly improved cellular uptake compared with ATO-MSNs. The MSNs, ATO-MSNs, ATO-MSNs-RGD, and ATO were used to treat mice bearing MDA-MB-231 breast tumors every 5 days and the findings suggested that ATO-MSNs-RGD provided superior therapeutic ability over MSNs, ATO-MSNs, and ATO.

Cryo-soft X-ray tomography as a quantitative three-dimensional tool to model nanoparticle:cell interaction.

BackgroundRecent advances in nanoparticle design have generated new possibilities for nano-biotechnology and nano-medicine. Here we used cryo-soft X-ray tomography (cryo-SXT) to collect comprehensive three-dimensional (3D) data to characterise the interaction of superparamagnetic iron oxide nanoparticles (SPION) with a breast cancer cell line.ResultsWe incubated MCF-7 (a human breast cancer cell line) from 0 to 24 h with SPION (15 nm average diameter, coated with dimercaptosuccinic acid), a system that has been studied previously using various microscopy and bulk techniques. This system facilitates the validation and contextualization of the new 3D data acquired using the cryo-SXT-based approach. After vitrification, samples tested by correlative cryo-epifluorescent microscopy showed SPION accumulation in acidic vesicles related to the endocytic pathway. Microscopy grids bearing MCF-7 cells were then analysed by cryo-SXT to generate whole cell volume 3D maps. Cryo-SXT is an emerging technique that benefits from high X-ray penetration into the biological material to image close-to-native vitrified cells at nanometric resolution with no chemical fixation or staining agents. This unique possibility of obtaining 3D information from whole cells allows quantitative statistical analysis of SPION-containing vesicle (SCV) accumulation inside cells, including vesicle number and size, distances between vesicles, and their distance from the nucleus.ConclusionsCorrelation between fluorescent microscopy, cryo-SXT and transmission electron microscopy allowed us to identify SCV and to generate 3D data for statistical analysis of SPION:cell interaction. This study supports continuous transfer of the internalized SPION from the plasma membrane to an accumulation area near the cell nucleus. Statistical analysis showed SCV increase in number and size concomitant with longer incubation times, and therefore an increase in their accumulated volume within the cell. This cumulative effect expands the accumulation area and cell organelles such as mitochondria are consequently displaced to the periphery. Our 3D cryo-SXT approach demonstrates that a comprehensive quantitative description of SPION:cell interaction is possible, which will serve as a basis for metal-based nanoparticle design and for selection of those best suited for hyperthermia treatment, drug delivery and image diagnosis in nanobiomedicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0170-4) contains supplementary material, which is available to authorized users.

Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum

This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50°C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35⿿40°C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme.

Early Activation of Apoptosis and Caspase-independent Cell Death Plays an Important Role in Mediating the Cytotoxic and Genotoxic Effects of WP 631 in Ovarian Cancer Cells.

The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline,?WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of WP631 and DOX suggest that this bisanthracycline can be an effective alternative in ovarian cancer treatment.

Physical and Chemical Characteristics of Jackfruit (Artocarpus Heterophyllus Lamk.) Seeds Flour Produced Under Fermentation Process by Lactobacillus Plantarum

Abstract Jackfruit seed is a potential local food source, which can be derived as flour. However, this seed contains several oligosaccharides such as raffinose and stachyose, which can cause flatulence for humans, and these substances will also create a darker color during flour processing. Effort to reduce this type of oligosaccharides is reported here by introducing fermentation technique using Lactobacillus plantarum. The aim of this study is to determine the physical and chemical characteristics of jackfruit seed flour fermented by fermentation process for 32 hours incubation time. The results showed that the jackfruit seed flour after fermentation processes have different characteristics compare to that of non-fermented one. The degree of whiteness of jackfruit flour produced under longer time of incubation during fermentation were higher compare to that of shorter duration. Based on the result of FTIR analysis, starch structure differences could not be seen between flour produced from fermentation process and without fermentation. It means that the fermentation process by L. plantarum were not able to change the structure or the degree of polymerization of the starch molecules. The only changes observed were the differences of the oligosaccharides content. The more incubation time assigned, the higher the amount of raffinose detected in the flour, followed by the decrease of the bigger oligosaccharides content such as stachyose and verbascose, as has been shown by HPLC chromatogram, which indicate that enzyme produced by L. plantarum during fermentation process is able to degrade bigger oligosaccharides rather than raffinose.

CdSe/ZnS quantum dots induce photodynamic effects and cytotoxicity in pancreatic cancer cells.

To investigate the photodynamic effect of CdSe/ZnS quantum dots (QDs) on pancreatic cancer cells and elucidate the probable mechanisms. The pancreatic cancer cell line SW1990 was treated with different concentrations of CdSe/ZnS QDs (0, 0.5, 1.0, 1.5, 2.0, 2.5 μmol/L), with or without illumination. The viability of SW1990 cells was tested using the Cell Counting Kit-8 (CCK-8) assay. The ultrastructural changes of SW1990 cells were observed by transmission electron microscopy. Apoptosis was detected by nuclear staining and flow cytometry (FCM). Reactive oxygen species (ROS) were measured by dichlorofluorescein diacetate via fluorescence microscopy. Expression of Bax, Bcl-2 and caspase-3 was measured by real-time polymerase chain reaction (PCR) and protein immunoblotting 24 h after SW1990 cells were treated with CdSe/ZnS QDs and illuminated. The CCK-8 assay results showed that both CdSe/ZnS QDs with and without illumination suppressed SW1990 cell proliferation. Cell viability was significantly lower when illuminated or with a longer incubation time and a higher light dose. CdSe/ZnS QDs with illumination caused ultrastructural changes in SW1990 cells, such as organelle degeneration and chromatin condensation and aggregation at the periphery of the nucleus. Fluorescence microscopy and FCM showed that CdSe/ZnS QDs (1.5 μmol/L) with illumination increased SW1990 cell apoptosis (53.2%) and ROS generation compared with no illumination. Real-time PCR showed that expression of Bax and caspase-3 was upregulated and Bcl-2 was downregulated. Immunoblotting results were consistent with real-time PCR results. Inhibition of ROS and apoptosis both attenuated QD-photodynamic-therapy-induced cell death. CdSe/ZnS QDs can be used as a photosensitizer to inhibit SW1990 cell proliferation through ROS generation and apoptotic protein expression regulation.

Quantification of Protein-Derived Thiols during Atmosphere-Controlled Brewing in Laboratory Scale

An atmosphere-controlled brewing system was built to study thiol oxidation during brewing in laboratory scale under conditions with limited oxygen exposure. Quantification of free and total thiols and protein showed that thiols were lost during wort boiling possibly owing to protein precipitation. Fermentation caused an increase in free thiols, and the balance between free and total thiols was shifted toward a higher degree of free thiols. This was explained by either a reducing effect of fermentation or secretion of thiol-containing compounds from yeast. The efficiency of sulfite to reduce reversibly oxidized thiols was determined by incubating boiled wort obtained from a pilot scale trial with various concentrations of sulfite (0–2,000 μM) in an anaerobic chamber for up to 7 days. Addition of sulfite increased the concentration of free thiols in a concentration-dependent manner, and the effect was more pronounced at longer incubation times. However, the reduction of the pool of oxidized thiols by sulfite was inefficient for sulfite concentrations typically found in beer, and the reaction was found to be relatively slow compared with reduction by tris(carboxyethyl)phosphine.

Performance of the Assurance GDS® Assay for the Detection of L. monocytogenes in Pure Cultures and Spiked Food Samples

Listeria monocytogenes is a foodborne pathogen with significant impacts on public health and economy worldwide. Reliable and fast detection of L. monocytogenes is of major importance for both diagnostic laboratories and the food industry. The current study evaluated the performance of the Assurance GDS® assay for the detection of L. monocytogenes in pure cultures and spiked food samples. In the pure culture experiments, the Assurance GDS® assay for Listeria monocytogenes accurately detected the target strains of different serotypes and was correctly negative for a variety of other Listeria species. For reliable detection of L. monocytogenes in pure culture experiments, colony counts >105 cfu/ml were required, which emphasizes the need for an adequate enrichment step. The challenge test experiments (steak tartare, bologna type sausage, Gorgonzola cheese) using a one-broth enrichment strategy showed that the Assurance GDS® assay reliably detected L. monocytogenes after 16 h of enrichment in Half-Fraser broth, provided that spiking levels of the different matrices were ≥102 cfu/g. Depending of the food matrix, longer incubation times of 24 h or 48 h were required when the initial spiking level was <102 cfu/g, as to be expected in a proportion of naturally contaminated food products. Thus, the Assurance GDS® Listeria monocytogenes assay has proven to be a reliable and easy to handle, rapid test system for the specific detection of L. monocytogenes. This system is a suitable tool for generating microbiological results used for a “positive batch release”, especially for RTE foods with short shelf lives. However, longer enrichment times (24 h or 48 h) are required in a one-broth enrichment strategy, when the contamination level of the food matrix is low (<102 cfu/g).

7-Keto-cholesterol and 25-hydroxy-1 cholesterol rapidly enhance ROS production in human neutrophils.

Oxysterols are cholesterol-oxygenated derivatives generated in the organism and also present in foods because of cholesterol oxidation during processing and storage. They are the natural ligands of liver X receptors (LXRs) and are generally recognized as hypocholesterolemic and anti-inflammatory molecules although this latter property is still controversial. Most oxysterol studies have been performed in macrophages, whereas the effects of oxysterols in neutrophils are poorly known. In this study, human neutrophils were exposed to two different oxysterols, 7-keto-cholesterol (7-k-chol) and 25-hydroxy-cholesterol (25-OH-chol), and their possible participation in inflammatory process was evaluated. Human neutrophils were incubated with 7-k-chol and 25-OH-chol, and ROS production, translocation of the NADPH oxidase cytosolic components, hemoxygenase-1 (HO-1) expression and lysozyme secretion were analyzed. An increase in ROS production was observed within a short period of time (minutes) with both molecules. These oxysterols also stimulated the cellular membrane translocation of the NADPH oxidase cytosolic components, p47phox and p67phox. On the other hand, HO-1 expression, a cytoprotector enzyme, is inhibited in human neutrophils upon oxysterols treatment. Moreover, both oxysterols were associated with high lysozyme enzyme secretion at 5 and 18h of incubation. The present paper describes for the first time that two oxysterols (7-k-chol and 25-OH-chol) enhance the ROS production within a short period of time in human neutrophils, stimulate the translocation of the cytosolic components of NADPH oxidase to the cellular membrane and increase lysozyme secretion. These data suggest that both oxysterols are able to activate pro-inflammatory effects in human neutrophils which contrasts with the role assigned to the oxysterols when they act through LXR at long time of incubation.

Pre‐infection processes of Botryosphaeriaceae spp.: adhesion of conidia to different substrata

Species of Botryosphaeriaceae are important wound pathogens of grapevines as causal agents of botryosphaeria dieback, but the behaviour of their conidia pre‐infection is unknown and may be important for disease development. Adhesion properties of conidia were investigated for Botryosphaeria dothidea, Neofusicoccum luteum and N. parvum on substrata with different affinities for water. Greatest adhesion on any surface was reached after 5 min for isolates N. luteum MM558, B. dothidea 007 and N. parvum G652 (53·1, 54·0 and 50·6%, respectively) and for N. luteum isolate CC445 after 20 min (61·4%). As conidia adhered well to all artificial substrata, it appeared as if the attachment process was nonspecific. Overall, surface wettability did not play a major role in the adhesion of conidia. Spore surface proteins appeared to play a role in the adhesion process because treatment of conidia of N. luteum MM558 with a protease completely prevented adhesion. Histochemical labelling of conidia and germlings with Coomassie brilliant blue (specific for proteins) was positive for all isolates, with a blue ‘halo’ often seen surrounding conidia or near the germ tube emergence point after incubation times conducive to germination. Alcian blue also stained material surrounding conidia after longer incubation times, which indicated that mucopolysaccharide and protein production may be involved in a second phase of adhesion.

Corrigendum

Terra NovaVolume 27, Issue 6 p. 479-479 CorrigendumFree Access Corrigendum This article corrects the following: Microbially influenced formation of Mg-calcite and Ca-dolomite in the presence of exopolymeric substances produced by sulphate-reducing bacteria Tomaso R. R. Bontognali, Judith A. McKenzie, Rolf J. Warthmann, Crisogono Vasconcelos, Volume 26Issue 1Terra Nova pages: 72-77 First Published online: October 17, 2013 First published: 21 November 2015 https://doi.org/10.1111/ter.12175AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Microbially influenced formation of Mg-calcite and Ca-dolomite in the presence of exopolymeric substances produced by sulphate-reducing bacteria by Tomaso R. R. Bontognali, Judith A. McKenzie, Rolf J. Warthmann and Crisogono Vasconcelos published in Terra Nova, 26, 72–77, 2014. The Editors of Terra Nova and the authors of the above article would like to draw the reader's attention to an error in Figure 4B. The label ‘20’ on the horizontal axis was misplaced, making it difficult to precisely measure the positions of the peaks shown in the figure. In the corrected figure, the position of the XRD spectra have been precisely aligned with respect to the main peak of the internal quartz standard. The corrected figure and its caption are reproduced below. Figure 4Open in figure viewerPowerPoint X-ray diffractograms of the carbonate precipitated in the presence of EPS, showing that no major crystallographic difference exists between the minerals formed with (Experiment B) and without (Experiment A) metabolizing microbes. The main peaks corresponding to the minerals are indicated as follows: high Mg-calcite (hmc), Mg-calcite (mc), Ca-dolomite (cd), quartz (q). Quartz powder was added as an internal standard. (A) Comparison between bulk precipitates collected from Experiments A and B after an incubation time of 30 days at 30°C. In both cases, the mineralogy corresponds to a solid solution of high Mg-calcite and disordered Ca-dolomite. (B) After a longer incubation time (i.e., about 16 months), both solid solutions evolve into two distinctive mineral phases consisting of Mg-calcite with less than 5 mol.% Mg2+ and disordered Ca-dolomite with more than 40 mol% Mg2+. The authors apologise for this error and any confusion it may have caused. Reference Bontognali, T.R.R., McKenzie, J.A., Warthmann, R.J. and Vasconcelos, C., 2014. Microbially influenced formation of Mg-calcite and Ca-dolomite in the presence of exopolymeric substances produced by sulphate-reducing bacteria. Terra Nova, 26, 72– 77. Volume27, Issue6December 2015Pages 479-479 FiguresReferencesRelatedInformation

Deciphering Community Structure of Methanotrophs Dwelling in Rice Rhizospheres of an Indian Rice Field Using Cultivation and Cultivation-Independent Approaches.

Methanotrophs play a crucial role in filtering out methane from habitats, such as flooded rice fields. India has the largest area under rice cultivation in the world; however, to the best of our knowledge, methanotrophs have not been isolated and characterized from Indian rice fields. A cultivation strategy composing of a modified medium, longer incubation time, and serial dilutions in microtiter plates was used to cultivate methanotrophs from a rice rhizosphere sample from a flooded rice field in Western India. We compared the cultured members with the uncultured community as revealed by three culture-independent methods. A novel type Ia methanotroph (Sn10-6), at the rank of a genus, and a putative novel species of a type II methanotroph (Sn-Cys) were cultivated from the terminal positive dilution (10(-6)). From lower dilution (10(-4)), a strain of Methylomonas spp. was cultivated. All the three culture-independent analyses, i.e., pmoA clone library, terminal restriction fragment length polymorphism (T-RFLP), and metagenomics approach, revealed the dominance of type I methanotrophs. Only metagenomic analysis showed significant presence of type II methanotrophs, albeit in lower proportion (37 %). All the three isolates showed relevance to the methanotrophic community as depicted by uncultured methods; however, the cultivated members might not be the most dominant ones. In conclusion, a combined cultivation and cultivation-independent strategy yielded us a broader picture of the methanotrophic community from rice rhizospheres of a flooded rice field in India.

A Double Emulsion-Based, Plastic-Glass Hybrid Microfluidic Platform for Protein Crystallization

This paper reports the design and construction of a plastic-glass hybrid microfluidic platform for performing protein crystallization trials in nanoliter double emulsions. The double emulsion-based protein crystallization trials were implemented with both the vapor-diffusion method and microbatch method by controlling the diffusion of water between the inner and outer phases and by eliminating water evaporation. Double emulsions, whose inner and outer environments can be easily adjusted, can provide ideal conditions to explore protein crystallization with the advantages of a convection-free environment and a homogeneous interface. The property of the water-oil interface was demonstrated to be a critical factor for nucleation, and appropriate surfactants should be chosen to prevent protein adsorption at the interface. The results from the volume effect study showed a trend of fewer crystals and longer incubation time when the protein solution volume became smaller, suggesting that the nucleation in protein crystallization process can be controlled by changing the volume of protein solutions. Finally, sparse matrix screening was achieved using the double emulsion-based microbatch method. The double emulsion-based approach for protein crystallization is a promising tool for enhancing the crystal quality by controlling the nucleation process.

The mitochondrial carnitine/acylcarnitine carrier is regulated by hydrogen sulfide via interaction with C136 and C155

BackgroundThe carnitine/acylcarnitine carrier (CAC or CACT) mediates transport of acylcarnitines into mitochondria for the β-oxidation. CAC possesses Cys residues which respond to redox changes undergoing to SH/disulfide interconversion. MethodsThe effect of H2S has been investigated on the [3H]carnitine/carnitine antiport catalyzed by recombinant or native CAC reconstituted in proteoliposomes. Site-directed mutagenesis was employed for identifying Cys reacting with H2S. ResultsH2S led to transport inhibition, which was dependent on concentration, pH and time of incubation. Best inhibition with IC50 of 0.70μM was observed at physiological pH after 30–60min incubation. At longer times of incubation, inhibition was reversed. After oxidation of the carrier by O2, transport activity was rescued by H2S indicating that the inhibition/activation depends on the initial redox state of the protein. The observed effects were more efficient on the native rat liver transporter than on the recombinant protein. Only the protein containing both C136 and C155 responded to the reagent as the WT. While reduced responses were observed in the mutants containing C136 or C155. Multi-alignment of known mitochondrial carriers, highlighted that only the CAC possesses both Cys residues. This correlates well with the absence of effects of H2S on carriers which does not contain the Cys couple. ConclusionsAltogether, these data demonstrate that H2S regulates the CAC by inhibiting or activating transport on the basis of the redox state of the protein. General significanceCAC represents a specific target of H2S among mitochondrial carriers in agreement with the presence of a reactive Cys couple.

In vivo and in vitro effects of a blend of essential oils on rumen methane mitigation

The effect of Agolin Ruminant, a blend of essential oils, on methane (CH4) emissions were investigated in two in vivo experiments and in four in vitro experiments. In the in vivo experiments, four lactating dairy cows and four beef heifers were supplemented 0.2g/d of the essential oils (ca. 2–4ppm m/v) during an eight-weeks period, where the first two weeks served as control (no essential oils supplementation). In dairy cattle, essential oils tended to decrease the daily CH4 emissions (g/d) and CH4 relative to dry matter intake (g/kg DMI) by 15% and 14%, respectively, after 6 weeks of supplementation (P=0.07), but no difference was observed for CH4 relative to milk production (g/kg milk) (P=0.64) or CH4 relative to bodyweight (g/kg BW) (P=0.12). In the in vivo experiment with beef cattle daily CH4 emissions and CH4 relative to DMI did not change when supplemented the essential oils at a dose of 0.2g/d (numerical decreases of 10 and 11% for g CH4/d and g CH4/kg DMI, respectively) but CH4 relative to body weight tended to decrease by 20% after 6 weeks of supplementation (P=0.07). The in vitro experiments were expected to replicate the results observed in vivo. However, no decrease in CH4 production was observed in 24h batch incubations at concentrations up to 30ppm (m/v). A longer contact time between the essential oils (15 and 30ppm) and the feedstuff (essential oils added ca. 16h prior the start of the incubation) did not elicit any effect on CH4 production and was not different from addition immediately prior to the start of the incubation. Longer incubation time (96h and 14d) and regular supply of both substrate and additive in a consecutive batch incubation system did not induce CH4 inhibition up to essential oils doses of 30ppm (m/v) and hence, also were not able to replicate in vivo results. Using the gas production technique (GPT) methane was inhibited by 17% with an essential oils dose of 30ppm after 24h, but this decrease was not constant across all times during the 72h incubation. The blend of essential oil was effective reducing daily emissions of methane in dairy cattle and emissions relative to body weight in beef cattle, interestingly, these effects were not observed in vitro regardless of the techniques used to replicate in vivo results. This might be due to differences in the mode of action of the essential oils in vitro and in vivo, which merits attention for future research.

Enzymatic preparation of oligosaccharides by transglycosylation: A comparative study of glucosidases

Five glucosidases were studied with respect to their ability to catalyse the transglycosylation of maltose or cellobiose. Experiments were carried out at high substrate concentrations to increase the ratio between transglycosylation and hydrolysis. The properties of the enzymes were quite different, and as a simple descriptor of transglycosylation ability we suggest the use of the acceptor concentration (in this case, the disaccharide concentration) required to achieve the same initial rate in transglycosylation as in hydrolysis. We call this descriptor T50. Aspergillus niger transglucosidase had the lowest T50 value (30mM) and produced panose as the major product from maltose with a 69% yield after 6h. With a longer incubation time, secondary hydrolysis occurred and isomaltose could be obtained with a 65% yield. Aspergillus niger glucosidase was the most efficient enzyme for the transglycosylation of cellobiose with a T50 value of 130mM. It produced mainly cellotriose with the 1,6-linked trisaccharide (6-O-β-glucopyranosyl-cellobiose) as a side product. Almond β-glucosidase had a T50 value much higher than 320mM (the highest concentration tested), and it produced 6-O-β-glucopyranosyl-cellobiose as the main transglycosylation product.

The effects of long-range residual stress, elastic follow-up and applied load on creep crack incubation and material toughness

Creep crack incubation of Type 316H stainless steel at 550 °C is explored in this article. Fracture mechanics specimens, subjected to combinations of residual and applied loads and in the presence of elastic follow-up, are tested. The design of two new test rigs is described. The rigs introduce planned levels of elastic follow-up together with combined residual and applied loading conditions to the specimens. A series of high-temperature elastic–plastic and elastic–plastic–creep experiments are undertaken to compare the experimentally determined values of elastic follow-up with the theoretical values. A further series of fracture mechanics tests are performed to measure creep crack incubation and material toughness for samples subjected to constant load and for tests under combined loading with elastic follow-up. It is demonstrated that for tests subjected to the same initial reference stresses, longer incubation times are attained for elastic follow-up tests compared to constant load tests. Also, combined loading tests exhibit longer creep crack incubation times based on the same measured material toughness obtained from constant load tests. This suggests that not all the available strain energy provided by combined loading to a specimen at high temperature contributes to creep crack incubation.