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Microscopic organisms maintained in long-term living monocultures undergo evolution, adapting to the stable laboratory environment. Culture adaptation can result in isolates no longer being represe...

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.

We present an evaluation of aircraft observations of the carbon and greenhouse gases CO2, CH4, N2O, and CO using a direct-absorption pulsed quantum cascade laser spectrometer (QCLS) operated during the HIPPO and CalNex airborne experiments. The QCLS made continuous 1 Hz measurements with 1σ Allan precisions of 20, 0.5, 0.09, and 0.15 ppb for CO2, CH4, N2O, and CO, respectively, over > 500 flight hours on 79 research flights. The QCLS measurements are compared to two vacuum ultraviolet (VUV) CO instruments (CalNex and HIPPO), a cavity ring-down spectrometer (CRDS) measuring CO2 and CH4 (CalNex), two broadband non-dispersive infrared (NDIR) spectrometers measuring CO2 (HIPPO), two onboard gas chromatographs measuring a variety of chemical species including CH4, N2O, and CO (HIPPO), and various flask-based measurements of all four species. QCLS measurements are tied to NOAA and WMO standards using an in-flight calibration system, and mean differences when compared to NOAA CCG flask data over the 59 HIPPO research flights were 100, 1, 1, and 2 ppb for CO2, CH4, N2O, and CO, respectively. The details of the end-to-end calibration procedures and the data quality assurance and quality control (QA/QC) are presented. Specifically, we discuss our practices for the traceability of standards given uncertainties in calibration cylinders, isotopic and surface effects for the long-lived greenhouse gas tracers, interpolation techniques for in-flight calibrations, and the effects of instrument linearity on retrieved mole fractions.

Background Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia–Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied. Methods We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p_gt1b). The boceprevir EC50 value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays. Results The boceprevir EC50 value for the gt6NS3p_gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p_gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir. Conclusions A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease.

The survival by pathogenic bacteria within the specific conditions of an anatomical niche is critical for their persistence. These conditions include the combination of toxic chemicals, such as reactive oxygen (ROS) and reactive nitrogen species (RNS), with factors relevant to cell growth, such as oxygen. Haemophilus influenzae senses oxygen levels largely through the redox state of the intracellular fumarate-nitrate global regulator (FNR). H. influenzae certainly encounters oxygen levels that fluctuate, but in reality, these would rarely reach a state that results in FNR being fully reduced or oxidized. We were therefore interested in the response of H. influenzae to ROS and RNS at moderately high or low oxygen levels and the corresponding role of FNR. At these levels of oxygen, even though the growth rate of an H. influenzae fnr mutant was similar to wild type, its ROS and RNS tolerance was significantly different. Additionally, the subtle changes in oxygen did alter the whole cell transcriptional profile and this was different between the wild type and fnr mutant strains. It was the changed whole cell profile that impacted on ROS/RNS defence, but surprisingly, the FNR-regulated, anaerobic nitrite reductase (NrfA) continued to be expressed and had a role in this phenotype.

We report a rare example of the malonamide functionality being used as a ligand in copper catalysis. We have ligated a homologous series of these O,O-chelating architectures to copper, investigated their structure and exploited them in azide–alkyne cycloaddition reactions for the step-growth synthesis of oligo(triazoles) and in the synthesis of small organic azoles.

Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species.

Small-angle neutron scattering measurements on a series of monodisperse linear entangled polystyrene melts in nonlinear flow through an abrupt 4:1 contraction have been made. Clear signatures of melt deformation and subsequent relaxation can be observed in the scattering patterns, which were taken along the centerline. These data are compared with the predictions of a recently derived molecular theory. Two levels of molecular theory are used: a detailed equation describing the evolution of molecular structure over all length scales relevant to the scattering data and a simplified version of the model, which is suitable for finite element computations. The velocity field for the complex melt flow is computed using the simplified model and scattering predictions are made by feeding these flow histories into the detailed model. The modeling quantitatively captures the full scattering intensity patterns over a broad range of data with independent variation of position within the contraction geometry, bulk flow rate and melt molecular weight. The study provides a strong, quantitative validation of current theoretical ideas concerning the microscopic dynamics of entangled polymers which builds upon existing comparisons with nonlinear mechanical stress data. Furthermore, we are able to confirm the appreciable length scale dependence of relaxation in polymer melts and highlight some wider implications of this phenomenon.

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [35S]-sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF-beta1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two-fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF-beta1 followed by exposure of cells to IGF-I in three-dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSC's remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF-beta1 without sequential IGF-I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF-I supplemented cultures, particularly when used in concert with TGF-beta1 pretreatment.

‘Foreign breads slice white sales’ appeared as a headline of an article by Joanna Bale, in The Times on 13 August 1996. The article stated that ‘While croissants and others increase in popularity the humble loaf of bread continues to fall out of favour. Consumers are becoming more adventurous and acquiring a taste for the new products available’.

Cartilage function after resurfacing with cell-based transplantation procedures or during the early stages of arthritic disease may be bolstered by the addition of growth factor genes to the transplanted tissue. Insulinlike growth factor-I maintains chondrocyte metabolism in normal cartilage homeostasis and has been shown to improve cartilage healing in vivo. Given the relatively short half-life of insulinlike growth factor-I in biologic systems, however, maintenance of effective concentrations of this peptide has necessitated either very high initial doses or repeated treatment. Delivery of the insulinlike growth factor-I gene, using a deleted adenovirus vector, specifically targeting graftable articular chondrocytes, bone marrow-derived chondroprogenitor cells, or synovial lining cells, may provide more durable insulinlike growth factor-I fluxes to articular tissues. Cultured equine articular chondrocytes, mesenchymal stem cells, synovial explants, and synovial intimal cells were readily transfected with an E1-deleted adenoviral vector containing equine insulinlike growth factor-I coding sequence. Optimal viral concentrations for effective transduction were 100 multiplicities of infection in synoviocytes, 500 multiplicities of infection in chondrocytes, and 1000 multiplicities of infection in mesenchymal stem cells. Production of insulinlike growth factor-I ligand varied from 65 ng/mL to 246 ng/mL in medium from chondrocytes and synovial explants, respectively. For chondrocytes, these concentrations were sufficient to produce significant stimulation of cartilage matrix gene expression and subsequent proteoglycan production. Moreover, cells in infected cultures maintained a chondrocytic phenotype and continued to express elevated insulinlike growth factor-I levels during 28 days of monolayer culture. Minimal synthetic activity, other than insulinlike growth factor-I ligand synthesis, was evident in synovial cultures. These experiments suggest several avenues for insulinlike growth factor-I supplementation of articular cartilage, including preimplantation adenoviral-insulinlike growth factor gene transfer to chondrocytes or chondroprogenitor cells, and direct injection of adenoviral-insulinlike growth factor to transfect the synovial structures in situ.

Spices are aromatic vegetable products which are used in powder form to flavour various confections and, more generally, as a condiment for seasoning food. These powders consist of the vegetable tissue of certain plants which have been dried and ground or pulverized by heavy machinery. The aromas and strong pungent flavours of spices are due to the presence of particular essential oils and glucosides. Owing to their strong odour and flavour, they are comparatively easy to adulterate with worthless material, such as the shells and husks of various nuts. The adulterant is seldom harmful in itself, but it reduces considerably the flavouring value of the spices. Another form of adulteration consists in extraction of the essential oils with alcohol to obtain the essence; then the residue is ground to a powder and sold as spice.

Chemically aerated goods are those morning goods which are lightened by the use of chemicals instead of yeast or eggs. Most of these chemicals are baking powders, which consist of an acid substance together with bicarbonate of soda. The main differences between the various powders on the market is the nature of the acid material which is used to liberate carbon dioxide gas from bicarbonate of soda, and so effect the necessary aeration. The various chemical actions which take place have been fully discussed in the chapter dealing with chemical aeration. Cream of tartar is still regarded as the classic acid component, and for this reason most of the others used are known under the general name of cream powders. They are marketed under proprietary names and are so compounded that by the addition of an inert filler they can be used in approximately the same quantities as cream of tartar when making baking powder.

Undoubtedly the replacement of sugar and fat in any formulation presents a challenge if the quality of the product is to remain high. Recent work at the Flour Milling and Baking Research Association has shown that substantial sugar and fat reductions can be made in traditional formulations using a combination of a high-intensity sweetener, Sunett (which is approximately 200 times sweeter than sucrose in diluted aqueous solutions and is Hoechst’s trademark for the sweetener acesulfame K), and a low-calorie bulking agent, Litesse. Litesse is described by the manufacturer as a superior quality polydextrose. It is a randomly bonded polymer of dextrose containing minor amounts of bound sorbitol and citric acid. It is a water-soluble, low-calorie functional ingredient that allows the creation of light baked goods by replacing all or part of the sugars and some of the fats. Fats supply both a sensorial and functional benefit to foods, in addition to calories. Generally, the higher the level of fat, the harder it is to replace the organoleptic and functional qualities of fat with any one ingredient. Similarly, when sugar is replaced in a formulation the major considerations include the impact on the general organoleptic qualities of the baked product but more specifically the sweetness level of the finished product. Replacing the sugar also impacts upon the shelf-life of the product.

Gums have been used in bakery for very many years, but in more recent years an increasing number have found their way into flour confectionery, either in the manufacture of prepared materials or used directly in the baked product.

The fat from cow’s milk, milkfat, is normally available to the baker or bakery technologist as cream, butter and anhydrous milkfat (AMF) (or butteroil). Since the early 1970s, fractionated butter/butteroil products have also become available for bakery applications. Several other milk-based products contain milkfat, in the range 5–90% fat, and they have all found applications in bakery products (Figure 5.1).

An emulsion is described as a dispersion of one immiscible substance within another. Often enough, both substances, known in this case as ‘phases’, will be liquids, e.g. oil and water, although other substances could be involved such as gas or air in the case of aerated foams, or solids in suspension, e.g. sugar in chocolate.

Experiments to determine melting points are conducted in which paired indium samples have been placed in the sample and reference cells in power compensated and differential temperature DSC instruments. In some cases, it has been found that the peak temperatures for the two cells differ within an instrument, contrary to general expectations.

Populus balsamifera var. virginiana (Sargent), balsam poplar, is capable of withstanding liquid nitrogen temperatures when cooling is slow (less than 6°C h −1 ) and the twigs are winter dormant. Cooling to −50°C at rates as high as 42°C h −1 is also tolerated. The mechanical behavior during warming of dormant twigs cooled at rates varying from 3°C h −1 to 600 °C h −1 has been investigated using resonance mode dynamic mechanical analysis (DMA). The results were correlated with those of freeze-etch electron microscopy investigations of the samples as well as differential scanning calorimetry (DSC) of model solutions. The results of these investigations were compared with mortality data on the twigs. This has led us to hypothesize that at critical cooling rates below 0° C plasma membranes tear, leading to the rapid growth of intracellular ice. This in turn prevents the intracellular liquid-liquid phase separations seen by DMA in samples cooled at sublethal rates. DSC analysis of salt-sugar-protein model solutions supports the idea that when liquid-liquid phase separation does occur, some domains are sugar-depleted but rich in protein, while others are sugar-rich but low in protein. This may have important implications regarding the organization of extreme freezing resistance mechanisms in this natural system.

SUMMARY: Soluble proteins extracted from mycelium of Penicillium griseofulvum were separated by acrylamide-gel electrophoresis. The pattern and overall intensity of the protein bands varied greatly with age of culture. A marked decrease in band intensity occurred at the time of exhaustion of the nitrogen source in shaken cultures, and at an earlier stage in static cultures; the total protein in whole or ultracentrifuged extracts did not decrease to the same extent. Changes in the pattern and intensity of protein bands during incubation occurred also in P. chrysogenum and P. frequentans. Mycelium of P. griseofulvum which was induced to sporulate in shaken culture yielded little protein as shown by electrophoresis at early stages of culture, in comparison with non-sporing mycelium. Each of these three Penicillium species could be distinguished by the protein pattern, which was reproducible and characteristic of the fungus at any particular stage of culture. The results indicate the need to determine the effects of age and conditions of culture when gel electrophoresis of mycelial proteins is used for taxonomic purposes.

Novel experimental techniques have been used to show that in thermofix dyeing disperse dyes are transferred from cotton to polyester fibres—in a blend or union fabric—solely by a vapour-phase mechanism. Other possible modes of transfer were examined, but no evidence was found to support them. The heat fastness of paniculate disperse dyes on cotton was correlated with their transfer properties to polyester fibres. The various types of behaviour of disperse dyes in bulk application are explained. Functions of padding assistants and, in particular, a migration inhibitor and the importance of time and temperature in thermofixation procedures are dealt with in practical terms in Part II of the paper.

SUMMARY Certain fungicides prevented the growth of powdery mildews at distances extending several millimetres beyond the edge of localized deposits on leaves. Most of the materials also acted at a distance when separated from the leaf by glass cover-slips, indicating that the effect was due to the emission of vapour. In addition to sulphur and lime-sulphur, whose vapour action is already known, fungicides based on drazoxolon, oxythioquinox, binap-acryl, dinitro-octyl phenyl crotonates and a phthalimido-phosphonothionate were found to act in this way. Two systemic fungicides, griseofulvin and triamiphos, did not give a detectable vapour action. Tests were conducted mostly on open glasshouse benches. A draught from a fan shifted the zones of inhibition to one side of the deposits, but did not reduce their areas. Vapour effects were similar in extent on plants maintained at 18–25° C and at 27–32° C. Variation in the areas protected was studied in relation to the size and fungicide content of the deposits, to different powdery mildews, to time of incubation and to different types of formulation. Deposits applied to leaves by high- or low-volume sprays at concentrations used in the field gave significant protection at a distance. Vapour effects were demonstrated also on mildew conidia incubated on glass slides bearing a spot of fungicide, and on infected plants placed in beakers coated on the bottom with fungicide. Movement of fungicides in the gaseous state is discussed in relation to the control of foliage diseases in the field.

Sequential changes in albumin, transferrin, alpha 1-acid glycoprotein, C reactive protein, fibrinogen, copper, iron, and zinc in plasma up to 24 h after hysterectomy were measured. No increases in the concentrations of the acute phase proteins alpha 1-acid glycoprotein, C reactive protein, and fibrinogen were observed until 6 h after the skin incision. These increases were preceded by significant falls at 2-4 h, and this was shown also by albumin, transferrin, iron, zinc, and copper. The ratios of iron and zinc to their binding proteins, transferrin and albumin, did not decrease until 4-6 h and their concentrations remained low for at least 24 h. These patterns suggest that at least two mechanisms operate after trauma. The early fall in the concentrations of the proteins in plasma is consistent with a prompt increase in microvascular permeability. The later decrease in binding of the metals iron and zinc to their transport proteins and the increase in concentrations of the acute phase proteins could be initiated by a common mediator.