Minute Impurities Contribute Significantly to Olfactory Receptor Ligand Studies: Tales from Testing the Vibration Theory.

Several studies have attempted to test the vibrational hypothesis of odorant receptor activation in behavioral and physiological studies using deuterated compounds as odorants. The results have been mixed. Here, we attempted to test how deuterated compounds activate odorant receptors using calcium imaging of the fruit fly antennal lobe. We found specific activation of one area of the antennal lobe corresponding to inputs from a specific receptor. However, upon more detailed analysis, we discovered that an impurity of 0.0006% ethyl acetate in a chemical sample of benzaldehyde-d5 was entirely responsible for a sizable odorant-evoked response in Drosophila melanogaster olfactory receptor cells expressing dOr42b. Without gas chromatographic purification within the experimental setup, this impurity would have created a difference in the responses of deuterated and nondeuterated benzaldehyde, suggesting that dOr42b be a vibration sensitive receptor, which we show here not to be the case. Our results point to a broad problem in the literature on use of non-GC-pure compounds to test receptor selectivity, and we suggest how the limitations can be overcome in future studies.

[Breast augmentation with autologous fat - experience of 96 procedures with the BEAULI-technique]. / Brustaugmentation mit autologem Fett - Erfahrungen aus 96 Operationen mit der BEAULITM-Methode.

Over the past 30 years, interest in the use of autologous fat for aesthetic body contouring, especially for breast augmentation has been continuously on the rise. The benefits of an autologous fat transplant include the absence of any inflammatory reaction to a foreign body, its harmonious appearance and a natural feeling. In earlier years, complications such as necrosis, infections or the formation of cysts, poor resorption rates as well as the difficulty of harvesting large amounts of fat within a reasonable amount of time provided grounds for criticism of the methodology of autologous fat transplantation. With the advent of the so-called BEAULI method, since 2007 a procedure is available for the efficient harvesting and processing of larger quantities of transplantable fat. The aim of the study is to describe the technique in detail and reproducibly and to present a detailed overview of autologous fat transfer due to the basis of our own clinical experience. Between 1 September 2010 and 30 June 2012 the author performed 96 fat transfer procedures on 84 patients. Patients aged 18-60 with a desire for a moderate augmentation of volume and shape of the breasts were selected for the procedure. The fat was harvested using water jet-assisted liposuction (Bodyjet) to flush out the fat cells and subsequent separation of the fat components with the Lipo-Collector. There were no occurrences of post-operative complications in any of the cases. The results were evaluated in the context of a check-up, a photographic comparison and with the completion of a questionnaire. With regard to the increase in size and/or shape enhancement of the breasts, 31% of the patients were very happy with the results, 45% indicated they were satisfied, 23% would have liked more volume, while 1% were dissatisfied. This study indicates that the autologous fat transplant into the female breast using the BEAULI method represents an excellent, safe method for the achievement of a moderate and harmonious breast size augmentation as well as sustainable and natural-looking contour improvements in selected patients. Additional studies with a larger number of cases and longer observation periods over several years as well as guidelines from the professional associations could contribute to the further perfection of the autologous fat transplant method in terms of resorption rate, efficiency and safety.

Role of autophagy in disease resistance and hypersensitive response-associated cell death.

Ancient autophagy pathways are emerging as key defense modules in host eukaryotic cells against microbial pathogens. Apart from actively eliminating intracellular intruders, autophagy is also responsible for cell survival, for example by reducing the deleterious effects of endoplasmic reticulum stress. At the same time, autophagy can contribute to cellular suicide. The concurrent engagement of autophagy in these processes during infection may sometimes mask its contribution to differing pro-survival and pro-death decisions. The importance of autophagy in innate immunity in mammals is well documented, but how autophagy contributes to plant innate immunity and cell death is not that clear. A few research reports have appeared recently to shed light on the roles of autophagy in plant-pathogen interactions and in disease-associated host cell death. We present a first attempt to reconcile the results of this research.

Structural and proteomic analyses reveal regional brain differences during honeybee aging.

Among insects, learning is particularly well studied in the fruit fly Drosophila melanogaster and the honeybee Apis mellifera. A senescence-dependent decline in classic pavlovian conditioning is demonstrated for both species. To understand how aging affects learning, genetic approaches used with Drosophila can benefit from complementary studies in Apis. Specifically, honeybees have a larger brain size allowing for compartment-specific approaches, and a unique life-history plasticity. They usually perform within-nest tasks early in life (nest bees) and later they collect food (foragers). Senescence of learning performance is a function of the bees' foraging duration but underlying causes are poorly understood. As cognitive aging is commonly associated with structural and biochemical changes in the brain, we hypothesized that brain areas implicated in learning change in synaptic and biochemical composition with increased foraging duration. First, we used synapse-specific immunohistochemistry and proteomics to screen for alterations in the calyx region of the mushroom body, a key structure for memory formation. Using proteomics, we next profiled the central brain, which comprises all higher-order integration centers. We show that, with increased foraging duration, levels of kinases, synaptic- and neuronal growth-related proteins decline in the central brain while the calyx region remains intact both in structure and biochemistry. We suggest that proteome-level changes within major anatomical sites of memory formation other than the calyx region could be central to learning dysfunction. These include large compartments of the central brain, such as the mushroom body's output regions and the antennal lobes. Our data provide novel information toward heterogeneity in the aging insect brain, and demonstrate advantages of the honeybee for invertebrate neurogerontological research.

Ageing in a eusocial insect: molecular and physiological characteristics of life span plasticity in the honey bee.

Commonly held views assume that ageing, or senescence, represents an inevitable, passive, and random decline in function that is strongly linked to chronological age. In recent years, genetic intervention of life span regulating pathways, for example, in Drosophila as well as case studies in non-classical animal models, have provided compelling evidence to challenge these views.Rather than comprehensively revisiting studies on the established genetic model systems of ageing, we here focus on an alternative model organism with a wild type (unselected genotype) characterized by a unique diversity in longevity - the honey bee.Honey bee (Apis mellifera) life span varies from a few weeks to more than 2 years. This plasticity is largely controlled by environmental factors. Thereby, although individuals are closely related genetically, distinct life histories can emerge as a function of social environmental change.Another remarkable feature of the honey bee is the occurrence of reverted behavioural ontogeny in the worker (female helper) caste. This behavioural peculiarity is associated with alterations in somatic maintenance functions that are indicative of reverted senescence. Thus, although intraspecific variation in organismal life span is not uncommon, the honey bee holds great promise for gaining insights into regulatory pathways that can shape the time-course of ageing by delaying, halting or even reversing processes of senescence. These aspects provide the setting of our review.We will highlight comparative findings from Drosophila melanogaster and Caenorhabditis elegans in particular, and focus on knowledge spanning from molecular- to behavioural-senescence to elucidate how the honey bee can contribute to novel insights into regulatory mechanisms that underlie plasticity and robustness or irreversibility in ageing.

Selective method for the analysis of perchlorate in drinking waters at nanogram per liter levels, using two-dimensional ion chromatography with suppressed conductivity detection.

The United States Environmental Protection Agency (EPA) collected drinking water occurrence data for perchlorate in the Unregulated Contaminant Monitoring Regulation (UCMR 1; 2001-2005) using EPA Method 314.0. To address the interest in increasing sensitivity and selectivity for the analysis of perchlorate, three new methods, EPA Methods 314.1, 331.0 and 332.0, were subsequently published by EPA for the analysis of perchlorate in drinking water. In 2006, an automated two-dimensional ion chromatography (2D-IC) method for measuring perchlorate with suppressed conductivity detection was developed. Two-dimensional IC is essentially an automated "heart-cutting", column concentration and matrix elimination technique. In the first dimension, a large sample volume is injected onto a first separation column and the separated matrix ions are diverted to waste while the analyte(s) of interest are selectively cut, trapped and concentrated in a concentrator column. In the second dimension, the contents from the concentrator column are eluted onto a second analytical column for separation and quantitation of the analyte(s) of interest. Incorporation of two columns with different affinities for the analyte(s) in a single analysis can provide comparable selectivity and superior sensitivity to a method using second column confirmation in a second separate analysis step. Use of this approach led to the development of a new, highly sensitive and selective 2D-IC, suppressed conductivity method with a Lowest Concentration Minimum Reporting Level (LCMRL) of 55 ng/L for perchlorate in drinking water samples. This new method has comparable sensitivity and selectivity and is simpler and more economical than IC-mass spectrometric (MS) or IC-MS-MS techniques. The method is now being prepared for publication as EPA Method 314.2.

Perchlorate in water via US Environmental Protection Agency Method 331 Determination of method uncertainties, lowest concentration minimum reporting levels, and Hubaux-Vos detection limits in reagent water and simulated drinking water.

US Environmental Protection Agency (EPA) Method 331 determines perchlorate in drinking water using non-suppressed ion chromatography with tandem mass spectrometry. This study reports the results of calibration and recovery studies in reagent water, as well as of a recovery study in simulated drinking water (i.e., total dissolved solids are 500 mg/mL each of chloride, sulfate, and bicarbonate). The perchlorate concentrations in the study ranged from 0.05 to 64 ng/mL. At 95% confidence, the Hubaux-Vos detection limit (H-V DL) was 0.04 ng/mL for the calibration study and the simulated-drinking-water recovery study, and 0.03 ng/mL for the reagent-water recovery study. The lowest concentration minimum reporting level was 0.03 ng/mL for reagent water and 0.0 7 ng/mL for simulated drinking water, again at 95% confidence.

US Environmental Protection Agency Method 314.1, an automated sample preconcentration/matrix elimination suppressed conductivity method for the analysis of trace levels (0.50 microg/L) of perchlorate in drinking water.

Since 1997 there has been increasing interest in the development of analytical methods for the analysis of perchlorate. The US Environmental Protection Agency (EPA) Method 314.0, which was used during the first Unregulated Contaminant Monitoring Regulation (UCMR) cycle, supports a method reporting limit (MRL) of 4.0 microg/L. The non-selective nature of conductivity detection, combined with very high ionic strength matrices, can create conditions that make the determination of perchlorate difficult. The objective of this work was to develop an automated, suppressed conductivity method with improved sensitivity for use in the second UCMR cycle. The new method, EPA Method 314.1, uses a 35 mm x 4 mm cryptand concentrator column in the sample loop position to concentrate perchlorate from a 2 mL sample volume, which is subsequently rinsed with 10 mM NaOH to remove interfering anions. The cryptand concentrator column is combined with a primary AS16 analytical column and a confirmation AS20 analytical column. Unique characteristics of the cryptand column allow perchlorate to be desorbed from the cryptand trap and refocused on the head of the guard column for subsequent separation and analysis. EPA Method 314.1 has a perchlorate lowest concentration minimum reporting level (LCMRL) of 0.13 microg/L in both drinking water and laboratory synthetic sample matrices (LSSM) containing up to 1,000 microg/L each of chloride, bicarbonate and sulfate.

Microbial inhibitors for U.S. EPA drinking water methods for the determination of organic compounds.

Preservation of chemical analytes in drinking water samples is necessary to obtain accurate information concerning contaminant occurrence. Sample preservation to prevent biodegradation is important for most samples and analytes. With the unique demands of environmental methods, it is not always possible to kill all microorganisms without having undesirable effects. To find a suitable preservative, the sample, analysis, and preservation needs should be considered. During method development of U.S. Environmental Protection Agency (EPA) Methods 526 (for unstable semivolatile compounds) and 532 (for phenylurea pesticides), a number of studies were conducted to identify compatible microbial inhibitors. Copper sulfate was successfully used in Method 532 and is an excellent first-choice antimicrobial agent for many applications. Copper sulfate can catalyze hydrolysis reactions for some pesticides such as those analyzed in Method 526. Under these conditions, a nonmetal compound of antimicrobial activity must be considered. During the development of Method 526, a survey of alternate organic based antimicrobial compounds found that diazolidinyl urea worked well in the method. Several other candidate microbial inhibitors were identified that could have application to other environmental methods. A general approach to selecting antimicrobial compounds in future environmental methods in water matrixes is discussed.

Review of the methods of the US Environmental Protection Agency for bromate determination and validation of method 317.0 for disinfection by-product anions and low-level bromate.

In recent years several methods have been published by the United States Environmental Protection Agency (EPA) which specify bromate as a target analyte. The first of these was EPA Method 300.0. As technological improvements in ion chromatographic hardware have evolved and new detection techniques have been designed, method detection limits for bromate have been reduced and additional procedures have been written, including EPA Method 300.1, 321.8 and, most recently, EPA Method 317.0. An overview of the evolution of these bromate methods since 1989 is presented. The focus is specific to each of these respective procedures, highlighting method strengths, weaknesses, and addressing how these methods fit into EPA's regulatory agenda. In addition, performance data are presented detailing the joint EPA/American Society for Testing and Materials multilaboratory validation of EPA Method 317.0 for disinfection by-product anions and low-level bromate.

Collaborative study of EPA Method 317.0 for the determination of inorganic oxyhalide disinfection by-products in drinking water using ion chromatography with the addition of a postcolumn reagent for trace bromate analysis.

The development of the U.S. Environmental Protection Agency (EPA) Method 317.0 is initiated to provide a sufficiently sensitive and fundamental technique for the compliance monitoring of trace levels of bromate in drinking water. After a comparative evaluation of Method 317.0 and elimination of a chlorite interference, this method is tested by a collaborative study in order to determine the precision and bias of the method and evaluate its potential role as a future compliance-monitoring method for inorganic disinfection by-products (DBPs) and trace bromate. This technique provides a practical method for future compliance monitoring for all of the inorganic oxyhalide DBPs including trace concentrations of bromate.

Considerations necessary in gathering occurrence data for selected unstable compounds in the USEPA Unregulated Contaminant Candidate List in USEPA Method 526.

U.S. Environmental Protection Agency (EPA) Method 526 was developed for the analysis of target analytes that are subject to degradation by hydrolysis. Two technical hurdles that had to be overcome were preservation of the target analytes and selection of a suitable solid-phase extraction material. The target analytes were diazinon, disulfoton, fonofos, terbufos, prometon, 1,2-diphenylhydrazine, nitrobenzene, acetochlor, 2,4,6-trichlorophenol, 2,4-dichlorophenol, and cyanazine. Diazolidinyl urea was used for the first time as a microbial inhibitor in an EPA drinking water method. Experiment confirmed antimicrobial agents containing copper or mercury salts increased hydrolysis degradation rates. Trisodium ethylenediaminetetraacetic acid salt was added to chelate metal ions that may increase hydrolysis rates. A pH 7 buffer of tris(hydroxymethyl)aminomethane (Tris) and Tris hydrochloride was used to minimize rates of hydrolysis. The use of ascorbic acid prevented degradation of 2,4-dichlorophenol, terbufos, fonofos, diazinon, and disulfoton due to residual chlorine. Samples were extracted using a styrene divinylbenzene solid-phase material and analyzed by capillary column gas chromatography/mass spectrometry. A 21-day storage stability study, together with precision and accuracy studies, showed that this method has suitable sensitivity, accuracy, precision, and ruggedness for use in the EPA's Unregulated Contaminant Monitoring Rule drinking water occurrence survey.

[Initial manifestation of thromboangiitis obliterans as necrotizing colitis]. / Erstmanifestation einer Thrombangiitis obliterans als nekrotisierende Kolitis.

Necrotizing colitis as primary manifestation of Buerger's disease. We report the disease process of a 41 year old woman, who was referred to our clinic with intermittent claudication of the leg. She has been an excessive smoker since early youth. Three years ago a hemicolectomy was carried out because of a necrotizing colitis. The clinical, angiographic and histologic findings are presented. Finally the frequency of intestinal Buerger's disease and the types of clinical course are discussed.

Performance evaluation of a method for the determination of bromate in drinking water by ion chromatography (EPA method 317.0) and validation of EPA method 324.0.

The potential carcinogenic nature of bromate has prompted global regulatory agencies, and industrial and academic institutions to publish several methods for the analysis of bromate in both drinking and bottled waters. The United States Environmental Protection Agency (EPA) has reported two methods capable of detecting bromate at or below the promulgated maximum contaminant level of 10.0 microg/l. These methods are EPA Method 300.1 and 317.0. Method 300.1 has been promulgated by EPA for compliance monitoring of bromate under Stage 1 of the Disinfectants/Disinfection By-Products Rule. Due to its sensitivity, selectivity and simplicity, Method 317.0 has been drafted and evaluated for potential use as a future compliance monitoring method. This manuscript describes the performance evaluation work with Method 317.0 and efforts completed at EPA's Technical Support Center that improved the sensitivity of Method 317.0, leading to the development of EPA Method 324.0

Eliminating the chlorite interference in US Environmental Protection Agency Method 317.0 permits analysis of trace bromate levels in all drinking water matrices.

A post-column reagent (PCR) method for bromate analysis in drinking water with a method detection limit (MDL) and method reporting limit (MRL) of 0.1 and 0.5 microg/l, respectively, has been developed by the United States Environmental Protection Agency (EPA) for future publication as EPA Method 317.0. The PCR method provides comparable results to the EPA's Selective Anion Concentration (SAC) method used to support the laboratory analysis of Information Collection Rule (ICR) low-level bromate samples and offers a simple, rugged, direct injection method with potential to be utilized as a compliance monitoring technique for all inorganic Disinfectants/Disinfection By-Products (D/DBPs). It has superior sensitivity for bromate compared to EPA Method 300.1, which was promulgated as the compliance monitoring method for bromate under Stage 1 of the D/DBP rule. This paper addresses elimination of the chlorite interference that was previously reported in finished waters from public water systems (PWSs) that employ chlorine dioxide as the disinfectant. An evaluation of Method 317.0 for the analysis of bromate in commercial bottled waters is also reported.

Analysis of 500-ng/l levels of bromate in drinking water by direct-injection suppressed ion chromatography coupled with a single, pneumatically delivered post-column reagent.

In July 1997, the US Environmental Protection Agency (EPA) began sampling and analyzing drinking water matrices from US municipalities serving populations greater than 100,000 for low-level bromate (> 0.20 microgram/l) in support of the Information Collection Rule (ICR) using the selective anion concentration (SAC) method. In September 1997, EPA published Method 300.1 which lowered the Method 300.0 bromate method detection limit (MDL) from 20.0 to 1.4 micrograms/l. This paper describes the research conducted at the EPA's Technical Support Center laboratory investigating a single post-column reagent, o-dianisidine (ODA), which has been successfully coupled to EPA Method 300.1 to extend the MDL for bromate. Initial studies indicate that this method offers a MDL which approaches the EPA's SAC method with the added benefit of increased specificity, shortened analysis time and reduced sample preparation. The method provides excellent ruggedness and acceptable precision and accuracy with a bromate MDL in reagent water of 0.1 microgram/l, and a method reporting limit of 0.50 microgram/l.

Contribution to Zn-speciation in human breast milk: fractionation of organic compounds by HPLC and subsequent Zn-determination by DCP-AES.

Human milk was collected between the 2nd and 7th day after delivery from different women, pooled and separated into fat, proteins and low molecular weight (LMW) substances by centrifugation. The fatty share was rejected, the remaining two fractions were further separated by size exclusion chromatography (SEC) as described in (1), and analyzed for zinc (Zn). The HPLC-method was checked for stability of organo-metal complexes. Only bi-distilled water served as mobile phase during HPLC in order to maintain the Zn/organic molecule-complex intact and to avoid unnecessary contamination sources. Among milk proteins, zinc was associated with casein, albumin, lactoferrin, and metallothionein, whereas among LMW substances a zinc peak could be observed exclusively with citrate. The identity of citrate and proteins was verified with comparable HPLC runs of standard solutions, by citrate-specific examination of HPLC fractions and by isoelectric focusing (IEF) of collected HPLC fractions. Furthermore, native human milk, as well as fractions of proteins and LMW substances (with and without HPLC separation), were quantified with regard to total content of zinc, protein and citrate. No loss of substances was found. In human milk, zinc is primarily bound to citrate (approximately 3200 micrograms/L of Zn in pooled human milk = 95%), and only about 5% of the total amount of zinc is attached to proteins (approximately 150 micrograms/L of Zn in pooled human milk). Determination of citrate content in human milk used in this study yielded values approximately twice as high as data cited in the literature (325-655 mg/L compared with 95-270 mg/L, (2,3)).(ABSTRACT TRUNCATED AT 250 WORDS)