Individual and combined impacts of carbon dioxide enrichment, heatwaves, flow velocity variability, and fine sediment deposition on stream invertebrate communities.

Climate change and land-use change are widely altering freshwater ecosystem functioning and there is an urgent need to understand how these broad stressor categories may interact in future. While much research has focused on mean temperature increases, climate change also involves increasing variability of both water temperature and flow regimes and increasing concentrations of atmospheric CO2, all with potential to alter stream invertebrate communities. Deposited fine sediment is a pervasive land-use stressor with widespread impacts on stream invertebrates. Sedimentation may be managed at the catchment scale; thus, uncovering interactions with these three key climate stressors may assist mitigation of future threats. This is the first experiment to investigate the individual and combined effects of enriched CO2, heatwaves, flow velocity variability, and fine sediment on realistic stream invertebrate communities. Using 128 mesocosms simulating small stony-bottomed streams in a 7-week experiment, we manipulated dissolved CO2 (ambient; enriched), fine sediment (no sediment; 300 g dry sediment), temperature (ambient; two 7-day heatwaves), and flow velocity (constant; variable). All treatments changed community composition. CO2 enrichment reduced abundances of Orthocladiinae and Chironominae and increased Copepoda abundance. Variable flow velocity had only positive effects on invertebrate abundances (7 of 13 common taxa and total abundance), in contrast to previous experiments showing negative impacts of reduced velocity. CO2 was implicated in most stressor interactions found, with CO2 × sediment interactions being most common. Communities forming under enriched CO2 conditions in sediment-impacted mesocosms had ~20% fewer total invertebrates than those with either treatment alone. Copepoda abundances doubled in CO2-enriched mesocosms without sediment, whereas no CO2 effect occurred in mesocosms with sediment. Our findings provide new insights into potential future impacts of climate change and land use in running freshwaters, in particular highlighting the potential for elevated CO2 to interact with fine sediment deposition in unpredictable ways.

Expanding ecological assessment by integrating microorganisms into routine freshwater biomonitoring.

Bioindication has become an indispensable part of water quality monitoring in most countries of the world, with the presence and abundance of bioindicator taxa, mostly multicellular eukaryotes, used for biotic indices. In contrast, microbes (bacteria, archaea and protists) are seldom used as bioindicators in routine assessments, although they have been recognized for their importance in environmental processes. Recently, the use of molecular methods has revealed unexpected diversity within known functional groups and novel metabolic pathways that are particularly important in energy and nutrient cycling. In various habitats, microbial communities respond to eutrophication, metals, and natural or anthropogenic organic pollutants through changes in diversity and function. In this review, we evaluated the common trends in these changes, documenting that they have value as bioindicators and can be used not only for monitoring but also for improving our understanding of the major processes in lotic and lentic environments. Current knowledge provides a solid foundation for exploiting microbial taxa, community structures and diversity, as well as functional genes, in novel monitoring programs. These microbial community measures can also be combined into biotic indices, improving the resolution of individual bioindicators. Here, we assess particular molecular approaches complemented by advanced bioinformatic analysis, as these are the most promising with respect to detailed bioindication value. We conclude that microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems.

High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research.

Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities.

Hyaluronic acid used for the correction of nasal deviation in an 18-year-old Middle Eastern man.

The use of fillers for nonsurgical rhinoplasty has advanced in both materials and methods, and continues to gain popularity in North America. This technique is most often used for secondary revisions, although reports of fillers used in primary rhinoplasty in selected patients have been recently described. The present report details the use of a hyaluronic acid dermal filler in a young Middle Eastern man for a post-traumatic crooked nose deformity. Primary correction of the patient's right-sided nasal bone deviation using hyaluronic acid as a soft tissue filler was achieved with excellent results and patient satisfaction. The current use of fillers in nasal contouring is reviewed.

Bilateral pulmonary emboli after bilateral mastectomy in a 15-year-old boy with hypogonadism: A case report.

Pulmonary emboli are rare, yet serious, complications of body contouring surgery. When they occur, they more often follow as complications of long, invasive procedures in adults. The present report details a case of bilateral pulmonary emboli in an obese 15-year-old boy with hypogonadism undergoing bilateral mastectomy for gynecomastia. The diagnosis of bilateral pulmonary emboli was made on the basis of clinical presentation and positive ventilation/perfusion scan. The patient responded well to heparin anticoagulation treatment. The relevance of pediatric obesity, pediatric body contouring surgery and the risk of thromboembolic events in pediatric patients are discussed.

Chemometric modeling to relate antioxidants, neutral lipid fatty acids, and flavor components in chicken breasts.

Relationships among quality factors in retailed free-range, corn-fed, organic, and conventional chicken breasts (9) were modeled using chemometric approaches. Use of principal component analysis (PCA) to neutral lipid composition data explained the majority (93%) of variability (variance) in fatty acid contents in 2 significant multivariate factors. PCA explained 88 and 75% variance in 3 factors for, respectively, flame ionization detection (FID) and nitrogen phosphorus (NPD) components in chromatographic flavor data from cooked chicken after simultaneous distillation extraction. Relationships to tissue antioxidant contents were modeled. Partial least square regression (PLS2), interrelating total data matrices, provided no useful models. By using single antioxidants as Y variables in PLS (1), good models (r2 values > 0.9) were obtained for alpha-tocopherol, glutathione, catalase, glutathione peroxidase, and reductase and FID flavor components and among the variables total mono and polyunsaturated fatty acids and subsets of FID, and saturated fatty acid and NPD components. Alpha-tocopherol had a modest (r2 = 0.63) relationship with neutral lipid n-3 fatty acid content. Such factors thus relate to flavor development and quality in chicken breast meat.

The development of novel flexible electrode arrays for the electrochemotherapy of solid tumour tissue. (Potential for endoscopic treatment of inaccessible cancers).

Therapeutic "electroporation" involves application of electric fields to target cells/tissues, thereby rendering their cell membranes transiently porous, thus making feasible the cellular uptake and efficacy of previously impermeant and ineffective therapeutic agents. The objectives of this research are a) the development of flexible electrode arrays for incorporation into microsystem endoscopic devices, and b) the assessment of their efficacy in delivering selected genetic and pharmaceutical anticancer therapies. Gold electrodes were fabricated on flexible polyimide substrates following predictive modeling and simulation of electric fields using FEMLAB software. Subsequent assessment of electroporation efficiency in-vitro involved 1) enumeration of viable tumour cells after delivery of electric pulses and exposure to low concentrations of bleomycin, otherwise known as electrochemotherapy 2) Efficacy of gene delivery by detection of emitted green fluorescence by cells after electroporation with the pEGFP plasmid and 3) In-vivo efficacy of electrochemotherapy in a variety of human solid tumour masses in nude mouse models (xenografts). The flexible electrode system was found to be successful for electrical delivery of plasmids and drugs in-vitro and in-vivo. We found in-vivo complete regression of prostate, colon, oesophageal, and renal cancers with reduced growth rates for fibrosarcoma and breast cell lines. These flexible electrodes are suitable for electrochemotherapy or gene therapy to solid tumours masses and may be fabricated for application to the treatment of some cancers in humans by transcutaneous or endoscopic delivery systems.

Release cells, breath analysis and in-mouth analysis in flavour research.

The flavour of a food or beverage is not perceived in a single event, but rather as a series of events experienced as the food is consumed. Recent methods in flavour research have taken account of this, and techniques have been developed to study flavour release in model systems (release cells or simulated mouths) and from the mouth or nose of assessors, while consuming foods. However, while there is agreement on the need in some cases for hydration or artificial saliva in simulated mouths, other parameters must be optimised on a case-by-case basis. Individual variability may still be a problem in breath analysis, and further work is required to determine the extent to which there are real differences in volatile profiles. The techniques of release cells and breath analysis must now be applied to provide data, which will allow flavour release to be modelled.

Responses of two genotypes of chicken to the diets and stocking densities of conventional UK and Label Rouge production systems-II. Sensory attributes.

French Label Rouge quality chickens are reputed to possess improved sensory characteristics compared with birds reared under intensive conditions. The effects of genotype, diet, stocking density and age on eating quality were evaluated in a 2 × 2 × 2 experiment. The results suggest that genotype, diet, age and, to a lesser extent, stocking density can influence eating quality. The most pronounced sensory differences between the two genotypes were in the appearance and texture of the cooked meat. In particular, scores for toughness were higher for breast meat from Ross than ISA birds, though the opposite effect was observed for the thigh meat. Diet and age also affected texture, with the breast meat from chickens on the Label Rouge diet, or from older birds, having lower scores for toughness. Effects on odour and flavour were generally small and subject to genotype × diet interactions. The odour and flavour intensity of breast meat increased with age, while the odour intensity of thigh meat was higher in birds reared at low stocking density.

ZG-1494 alpha, a novel platelet-activating factor acetyltransferase inhibitor from Penicillium rubrum, isolation, structure elucidation andbiological activity.

A novel inhibitor of platelet-activating factor (PAF) acetyltransferase, an essential enzyme in the remodeling pathway of platelet-activating factor synthesis, was identified by a high throughout screen of natural product extracts of microbial origin. The compound, ZG-1494 alpha, was isolated from an ethyl acetate extract of a culture broth of Penicillium rubrum through bioassay guided fractionation. The structure of ZG-1494 alpha was determined by spectroscopic methods. A key feature of the structure, which is relatively rare among natural products, is the 5-hydroxy-3-pyrrolin-2-one moiety. A 13C-13C INADEQUATE was utilized to unambiguously determine the regiochemistry of this molecule.

Analysis of inositol metabolites produced by Saccharomyces cerevisiae in response to glucose stimulation.

When cultures of Saccharomyces cerevisiae are grown to stationary phase in medium containing [3H] inositol, significant amounts of radioactivity can be detected in phosphatidylinositol, phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. Addition of glucose to such cultures results in the generation of [3H]glycerophosphoinositol, [3H]glycerophosphoinositol 4-phosphate, and [3H]glycerophosphoinositol 4,5-bisphosphate in the extracellular medium. We found no evidence, however, for the stimulated formation of other inositol polyphosphates. This result suggests that glucose does not stimulate the "phospholipase C" signalling pathway established in higher eukaryotic cells but, in contrast, stimulates specific phospholipases A or B. A variety of cell division cycle (cdc) mutants have been studied to investigate the relationship between cell cycle progression and inositol metabolism in S. cerevisiae. Mutants which are defective for completion of cell cycle "START" (i.e. commitment to mitosis) show reduced formation of glycerophosphoinositol 4-phosphate and glycerophosphoinositol 4,5-bisphosphate in response to glucose. In contrast, cdc mutants which are defective in post-"START" processes show a larger glucose response than wild type cells. These results suggest that deacylation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate may be coordinated with cell cycle control in S. cerevisiae.

The expression of canine cardiac phospholamban in heterologous systems.

A synthetic phospholamban gene has been cloned and expressed in Escherichia coli, producing both native phospholamban and a fusion protein with 81 amino acids of the influenza virus NS1 protein. Both the native phospholamban and fusion proteins produced extensive cell lysis upon induction of gene expression, but only the native protein underwent spontaneous pentamer formation in E. coli. Translation in vitro of mRNA produced by transcription in vitro of phospholamban cDNA was used to demonstrate the spontaneous aggregation of phospholamban to form pentamers in this system also, both in the presence and absence of exogenous microsomes from canine pancreas or heart. Phospholamban produced by translation in vitro was apparently susceptible to proteolysis by enzymes present in the particulate fractions in these experiments.

Phospholamban is a good substrate for cyclic GMP-dependent protein kinase in vitro, but not in intact cardiac or smooth muscle.

1. Cyclic GMP-dependent protein kinase phosphorylates purified phospholamban. It also phosphorylates phospholamban present in vesicles of cardiac sarcoplasmic reticulum and smooth muscle microsomal fractions, and in transformants of Escherichia coli which contain a plasmid into which a gene encoding phospholamban has been inserted. 2. In vitro the phospholamban present in cardiac sarcoplasmic reticulum membranes is a better substrate for cyclic GMP-dependent protein kinase than for cyclic AMP-dependent protein kinase. 3. Studies using [32P]Pi to label the cellular ATP in intact cardiac or smooth muscle failed to demonstrate that phosphorylation of phospholamban occurs in response to stimuli which increase intracellular cyclic GMP. Possible reasons for this functional separation between increased cyclic GMP and phosphorylation of phospholamban are discussed.

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae.

A 2 micron circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in "high-copy" and "low-copy" number cells was determined. "High-copy" number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.

The secretion and post translational modification of interferons from Saccharomyces cerevisiae.

Studies with three interferon molecules, IFN-alpha 2, IFN-beta 1, and a "hybrid" interferon, IFNX-430 are described which illustrate that both the expression and secretion characteristics of heterologous proteins in yeast cells reflect properties of the proteins themselves. Recombinant DNA techniques have also been used to demonstrate that the efficient processing of mature heterologous proteins from the yeast alpha factor secretion leader can be affected by sequences on the carboxyl side of the initial cleavage site. Secretion studies with heterologous proteins in S. cerevisiae are aimed at maximising yield, the percentage of extracellular product and correct amino terminus sequence. The results presented here show that all three factors are susceptible to currently unpredictable properties of the foreign sequence. This situation, in turn, means that heterologous proteins can be used as tools in the biochemical dissection of the yeast secretion process.

Secretion of mammalian polypeptides from yeast.

Yeast cells are capable of expressing and secreting foreign polypeptides into the medium. Mammalian glycoproteins are glycosylated when secreted from yeast although the exact oligosaccharide sequence is not reproduced.

A bifunctional gene product involved in two phases of the yeast cell cycle.

The cell cycle in Saccharomyces cerevisiae is divided into two distinct phases. Unbudded, mononucleate cells in the G1 phase can react to relevant environmental changes by mating, sporulating, or by entering stationary phase. DNA synthesis and bud initiation occur almost simultaneously and mark 'commitment' to the completion of mitosis. Temperature-sensitive mutations at the cdc28 locus are known to cause arrest in the G1 phase of the cell cycle at the restrictive temperature. Here we show that the cdc28 gene product is also active in post-G1 cell cycle functions, and that a different property of the gene product may be required for each phase of the cycle in which it acts.

Food preferences of some United Kingdom residents.

A food preference questionnaire was completed by 1304 people. There were considerable differences in food preferences related to age, sex, and area of residence of the respondents. In spite of these differences, five distinct clusters of foods showing similar preference patterns were indentified. These clusters were of sweet fruits, meats, vegetables, alcoholic beverages, and some high carbohydrate foods. The cause of the clustering was uncertain.