The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele.

Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.

Molecularly imprinted polymer based microtiter chemiluminescence array for determination of phenothiazines and benzodiazepines in pork.

In this study, a molecularly imprinted polymer based chemiluminescence array capable of simultaneous determining phenothiazines and benzodiazepines was first reported. Two polymers were coated in different wells of the conventional 96-well microtiter plate as the recognition reagents, and the added analytes competed with a horseradish peroxidase-labeled bi-hapten conjugate to bind the recognition reagents. The light signal was induced by using a highly effective luminol-H2O2-IMP system. The assay procedure consisted of only one sample-loading step prior to data acquisition. Then, the array was used to determine 4 phenothiazines and 5 benzodiazepines in pork simultaneously. The limits of detection for the 9 drugs were in a range of 0.001-0.01 ng/mL, and the recoveries from the fortified blank pork were in a range of 63.5%-94.1%. Furthermore, the array could be reused for 8 times. The detection results for some real pork samples were consistent with an ultra performance liquid chromatography method.

Spiders have rich pigmentary and structural colour palettes.

Elucidating the mechanisms of colour production in organisms is important for understanding how selection acts upon a variety of behaviours. Spiders provide many spectacular examples of colours used in courtship, predation, defence and thermoregulation, but are thought to lack many types of pigments common in other animals. Ommochromes, bilins and eumelanin have been identified in spiders, but not carotenoids or melanosomes. Here, we combined optical microscopy, refractive index matching, confocal Raman microspectroscopy and electron microscopy to investigate the basis of several types of colourful patches in spiders. We obtained four major results. First, we show that spiders use carotenoids to produce yellow, suggesting that such colours may be used for condition-dependent courtship signalling. Second, we established the Raman signature spectrum for ommochromes, facilitating the identification of ommochromes in a variety of organisms in the future. Third, we describe a potential new pigmentary-structural colour interaction that is unusual because of the use of long wavelength structural colour in combination with a slightly shorter wavelength pigment in the production of red. Finally, we present the first evidence for the presence of melanosomes in arthropods, using both scanning and transmission electron microscopy, overturning the assumption that melanosomes are a synapomorphy of vertebrates. Our research shows that spiders have a much richer colour production palette than previously thought, and this has implications for colour diversification and function in spiders and other arthropods.

Simple extraction method for quantification of phenothiazine residues in pork muscle using liquid chromatography-triple quadrupole tandem mass spectrometry.

In this study, an analytical method was developed for quantification of residues of the anthelmintic drug phenothiazine (PTZ) in pork muscle using liquid chromatography-tandem mass spectrometry. Muscles were extracted using 0.2% formic acid and 10 mm ammonium formate in acetonitrile, defatted and purified using n-hexane. The drug was well separated on a Waters XBridge™ C18 analytical column using a binary solvent system consisting of 0.2% formic acid and 10 mm ammonium formate in ultrapure water (A) and acetonitrile (B). Good linearity was achieved over a six-point concentration range in matrix-matched calibration with determination coefficient =0.9846. Fortified pork muscle having concentrations equivalent to and double the limit of quantification (1 ng/g) yielded recovery ranges between 100.82 and 104.03% and relative standard deviations <12%. Samples (n = 5) collected from large markets located in Seoul City tested negative for PTZ residue. In conclusion, 0.2% formic acid and ammonium formate in acetonitrile can effectively extract PTZ from pork muscle without solid-phase extraction, a step normally required for cleanup before analysis and the validated method can be used for routine analysis to ensure the quality of animal products.

Development of an enzyme linked immunosorbent assay for the determination of phenothiazine drugs in meat and animal feeds.

In this study, 2-chlorophenothiazine was used to synthesize a hapten for production of monoclonal antibody. The obtained monoclonal antibody showed high crossreactivities to chlorpromazine, promethazine and perphenazine, and showed low crossreactivities to acepromazine and fluphenazine. After evaluation of three coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the five phenothiazines in animal feeds and the residues of chlorpromazine, promethazine and perphenazine in meat. The crossreactivities to the five analytes were in a range of 2.4%-98%. The limits of detection for the five drugs in feeds were in a range of 0.1-3.0 µg g-1, and that for chlorpromazine, promethazine and perphenazine in meat were in a range of 0.5-0.8 ng g-1. Their recoveries from standards fortified blank samples (chicken, pork and feeds) were in a range of 74.1%-96.5% with coefficients of variation of 6.4%-15.1%. Therefore, this method could be used as a rapid screen tool to determine phenothiazine drugs in animal feeds and animal derived foods.

Spiders do have melanin after all.

Melanin pigments are broadly distributed in nature - from bacteria to fungi to plants and animals. However, many previous attempts to identify melanins in spiders were unsuccessful, suggesting that these otherwise ubiquitous pigments were lost during spider evolution. Yet, spiders exhibit many dark colours similar to those produced by melanins in other organisms, and the low solubility of melanins makes isolation and characterization difficult. Therefore, whether melanins are truly absent or have simply not yet been detected is an open question. Raman spectroscopy provides a reliable way to detect melanins in situ, without the need for isolation. In this study, we document the presence of eumelanin in diverse species of spiders using confocal Raman microspectroscopy. Comparisons of spectra with theoretically calculated data falsify the previous hypothesis that dark colours are produced solely by ommochromes in spiders. Our data indicate that melanins are present in spiders and further supporting that they are present in most living organisms.

Structural characterization of product ions by electrospray ionization and quadrupole time-of-flight mass spectrometry to support regulatory analysis of veterinary drug residues in foods. Part 2: Benzimidazoles, nitromidazoles, phenothiazines, and mectins.

RATIONALE: Analysis for identification and quantification of regulated veterinary drug residues in foods is usually achieved by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The instrumental method requires the selection of characteristic ions, but structural elucidation is seldom performed to help ensure accuracy. This study is a continuation of previous work to characterize selected product ions in support of regulatory monitoring programs. METHODS: The tandem mass spectra of 28 veterinary drugs from a previously published LC/MS/MS method were acquired with a high-resolution quadrupole time-of-flight (Q-TOF) mass spectrometer using electrospray ionization (ESI) in positive mode. The TOF analyzer was calibrated to achieve a mass accuracy error <5 ppm for the MS and MS/MS modes, and samples were infused for data acquisition. RESULTS: The high mass accuracy achieved in Q-TOF allowed elucidation of the formulae of the product ions previously selected for qualitative identification. Rational interpretation of results was made and compared with the published literature, and the structure for the MS/MS product ions of four classes of regulated drugs (mectins, benzimidazoles, nitroimidazoles, and phenothiazines), totaling 28 compounds, were examined leading to the report of new structures or confirmation of published structures using low-resolution MS. CONCLUSIONS: Structural characterization of the product ions selected for identification and quantification of veterinary drug residues is important information for regulatory monitoring programs in defense of regulatory enforcement actions. This study has allowed structural elucidation of 84 MS/MS product ions previously selected for the LC/MS/MS analysis of 28 drug analytes.

Broad specific enzyme-linked immunosorbent assay for determination of residual phenothiazine drugs in swine tissues.

In this study, a novel generic hapten of phenothiazine drugs was synthesized by derivatization of 2-chlorophenothiazine with sodium bromoacetate. Then the hapten was coupled to bovine serum albumin for production of the monoclonal antibody. Results showed that the obtained monoclonal antibody recognized five phenothiazine drugs simultaneously: chlorpromazine, promethazine, acepromazine, perphenazine, and fluphenazine. After evaluation of different coating antigens, a heterologous competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of the five phenothiazine drugs in swine tissues (muscle, liver, and kidney). The cross-reactivities to the five analytes were in the range of 71 to 98%, and the limits of detection were in the range of 0.2 to 0.4 ng/ml, depending on the drug. Their recoveries from the fortified blank samples were in the range of 73.8 to 96.2%, with coefficients of variation in the range of 4.1 to 14.3%. This is the first study reporting a broad specific immunoassay for multi-determination of the residues of five phenothiazine drugs in animal-derived foods.

Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma.

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green fluorescence. Relative fluorescence intensity of the products was measured at λ exc 398 nm and λ em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N-dealkylation or oxidation of the corresponding sulphoxides or sulphones.

Chemical wastes in the sea: new forms of marine pollution.

Drums containing chemical wastes have been found in and along the North Sea. The wastes were analyzed and were found to include lower chlorinated aliphatic compounds, vinyl esters, chlorinated aromatic amines and nitrocompounds, and the insecticide endosulfan. Because dropping these drums into the sea endangers the environment and results in damage to fishing operations, measures should be taken to stop this practice.

Characterisation of white and yellow eye colour mutant strains of house cricket, Acheta domesticus.

Two eye-colour mutant strains, white (W) and yellow (Y) of house cricket Acheta domesticus were established in our laboratory. We phenotyped and genotyped the mutants, performed genetic crossings and studied the eye structure and pigment composition using light and electron microscopy and biochemical analysis. We show that W and Y phenotypes are controlled by a single autosomal recessive allele, as both traits are metabolically independent. The analysis of the mutants`eye structure showed a reduced number of dark pigment granules while simultaneously, and an increased amount of light vacuoles in white eye mutants was observed. Significant differences in eye pigment composition between strains were also found. The Y mutant had a lower number of ommochromes, while the W mutant had a lower number of ommochromes and pteridines. This indicates that mutated genes are involved in two different, independent metabolic pathways regulating tryptophan metabolism enzymes, pigment transporter granules or pigment granule formation.

Pitfalls and prevention strategies for liquid chromatography-tandem mass spectrometry in the selected reaction-monitoring mode for drug analysis.

BACKGROUND: We observed cases of false-positive results with the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Different LC-MS/MS techniques that use the selected reaction-monitoring mode, routinely employed for the analysis and quantification of drugs and toxic compounds in biological matrices, were involved in the false-positive and potentially false-positive results obtained. We sought to analyze the causes of and solutions to this problem. METHODS: We used a previously reported LC-MS/MS general unknown screening method, as well as manual spectral investigation in 1 case, to perform verification and identification of interfering compounds. RESULTS: We observed that false-positive results involved: a metabolite of zolpidem that might have been mistaken for lysergic acid diethylamide, benzoylecgonine mistaken for atropine, and clomipramine and 3 phenothiazines that share several common ion transitions. CONCLUSIONS: To prevent problems such as those we experienced, we recommend the use of stable-isotope internal standards when possible, relative retention times, 2 transitions or more per compound when possible, and acceptable relative abundance ratios between transitions, with an experience-based tolerance of +/-15% for transitions with a relative abundance >10% and with an extension to +/-25% for transitions <10% when the concentration is at the limit of quantification. A powerful general unknown screening procedure can help to confirm suspected interferences. Our results indicate that the specificity of screening procedures is questionable for LC-MS/MS analyses performed in the selected reaction-monitoring mode and involving a large number of compounds with only 1 transition per compound.

Application of chaotropic effect in reversed-phase liquid chromatography of structurally related phenothiazine and thioxanthene derivatives.

Chromatographic behavior of two main classes of neuroleptics, derivatives of phenothiazine and thioxanthene in RP systems modified by anionic additives: sodium perchlorate and sodium hexafluorophosphate possessing chaotropic properties, was examined. Influence of the method of pH lowering (by addition of acids: trifluoroacetic or perchloric or by adding the appropriate concentration of phosphate buffer) and the kind of organic modifier in the mobile phase (methanol, acetonitrile) were estimated. Stability of complexes created between protonated drugs and anions of added salts was evaluated by comparison of their desolvation parameters (K), limiting retention factors for unsolved molecules calculated on the basis of chromatographic data. Experimentally obtained parameters were used in QSAR studies. It appeared that chosen parameters reflect not only physico-chemical properties of analytes but also contain information about the strength of their antipsychotic activity. Multidimensional cluster analysis has been performed. On the basis of the results obtained, it could be concluded that chaotropic systems can generate useful parameters for further QSAR studies.

Different ommochrome pigment mixtures enable sexually dimorphic Batesian mimicry in disjunct populations of the common palmfly butterfly, Elymnias hypermnestra.

With varied, brightly patterned wings, butterflies have been the focus of much work on the evolution and development of phenotypic novelty. However, the chemical structures of wing pigments from few butterfly species have been identified. We characterized the orange wing pigments of female Elymnias hypermnestra butterflies (Lepidoptera: Nymphalidae: Satyrinae) from two Southeast Asian populations. This species is a sexually dimorphic Batesian mimic of several model species. Females are polymorphic: in some populations, females are dark, resemble conspecific males, and mimic Euploea spp. In other populations, females differ from males and mimic orange Danaus spp. Using LC-MS/MS, we identified nine ommochrome pigments: six from a population in Chiang Mai, Thailand, and five compounds from a population in Bali, Indonesia. Two ommochromes were found in both populations, and only two of the nine compounds have been previously reported. The sexually dimorphic Thai and Balinese populations are separated spatially by monomorphic populations in peninsular Malaysia, Singapore, and Sumatra, suggesting independent evolution of mimetic female wing pigments in these disjunct populations. These results indicate that other butterfly wing pigments remain to be discovered.

Lipophilicity of New Anticancer 1,6- and 3,6-diazaphenothiazines by of Use RP TLC and Different Computational Methods.

The lipophilicity of new two series of anticancer active 10-substituted 1,6- and 3,6-diazaphenothiazines has been investigated using reversed-phase thin-layer chromatography. Their lipophilicity (RM0 and log PTLC) was determined with mixtures of acetone and Tris buffer as mobile phases. The relative lipophilicity parameter RM0 and specific hydrophobic surface area b were significantly intercorrelated showing congeneric classes of diazaphenothiazines. The parameter RM0 was transformed into parameter log PTLC by use of the calibration curve. The parameter log PTLC was compared with computationally calculated lipophilic parameters log Pcalcd. The lipophilicity was discussed with the structure elements and was correlated with molecular descriptors, ADME properties and in vitro anticancer activities.

Repeated attempted homicide by administration of drugs documented by hair analysis.

Attempted murder by repeated poisoning is quite rare. The authors describe the case of a 62-year-old man who was admitted to an intensive care unit (ICU) for neurological disturbances complicated by inhalation pneumopathy. He presented a loss of consciousness while his wife was visiting him at the ICU (H0). Forty-eight hours later (H48), police officers apprehended the patient's wife pouring a liquid into his fruit salad at the hospital. Toxicological analyses of a blood sample and the infusion equipment (H0), as well as the fruit salad and its container (H48), confirmed the attempted poisoning with cyamemazine (H0) and hydrochloric acid (H48). In order to evaluate the anteriority of poisonings, hair analysis was requested and the medical records of the 6 previous months were also examined. Two 6-cm brown hair strands were sampled and the victim's medical record was seized in order to determine the treatments he had been given during the previous six months. Segmental hair testing on two 6-cm brown hair was conducted by GC-MS, LC-DAD and LC-MS/MS (0-2/2-4/4-6 cm; pg/mg). Haloperidol (9200/1391/227), amitriptyline (7450/1850/3260), venlafaxine (332/560/260), that had never been part of the victim's treatment were detected, as well as some benzodiazepines (alprazolam, bromazepam, nordazepam); cyamemazine was also detected in all the segments (9960/1610/2367) though only a single dose administration was reported in the medical records. The toxicological analyses performed at H0 and H48 confirmed the homicide attempts in the ICU. In addition, comparison of the results in hair analysis with the medical records confirmed repeated poisoning attempts over the previous six months, and thus explain the origin of the disorders presented by the victim. This case serves to remind us that repeated attempted murder can be difficult to diagnose and that hair analysis can be an effective way to detect such attempts.

Oxidation of Selected Phenothiazine Drugs During Sample Preparation: Effects of Varying Extraction Conditions on the Extent of Oxidation.

Characterization of degradation products formed from selected phenothiazine drugs during standard solid-phase extraction (SPE) approaches is described. An analytical method for promethazine (PMZ), chlorpromazine (CPZ) and their respective N-desmethyl and sulfoxide metabolites in biological samples (bone tissue extract and blood) by ultra performance liquid chromatography-photodiode array detection, using mixed-mode SPE for basic drugs was developed. When ethyl acetate:isopropanol:ammonium hydroxide (80:17:3) was used as the elution solvent during method development, extraneous peaks were observed that were absent in the negative controls. Analysis of extracts of PMZ and CPZ individually showed extraneous peaks, including peaks with retention time and UV spectra suggesting the formation of the sulfoxide metabolites, amongst others. Analytes were then extracted individually and analyzed by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The results confirmed the oxidation of PMZ to its sulfoxide and N-oxide metabolites and oxidation of CPZ to its sulfoxide metabolite. Oxidation was also observed in analysis of whole blood, and thus was not specific to bone tissue extract. To determine if extraction with minimal oxidation was possible, extractions using SPE with a different elution solvent system (dichloromethane:isopropanol:ammonium hydroxide) and filtration/pass through extraction (FPTE) with and without evaporation were evaluated. The results demonstrated that the sample preparation method highly influenced the extent of oxidation. FPTE without an evaporation step was the only method that did not measurably induce analyte oxidation.

[Determination of bufothionine in skin of Bufo bufo gargarizans and Huachansu injection].

OBJECTIVE: To establish a HPLC method for bufothionine in the skin of Bufo bufo gargarizans and Huachansu injection. METHOD: The samples were separated using a Lichrosob C18 column with CH3CN-H2O (10:90) as mobile phase. Flow rate was at 1.0 mL x min (-1) and the detection wavelength was at 225 nm. RESULT: The calibration curve of bufothionine was linear over the range of 0.0772-0.4632 microg and the average recovery was 99. 2%. The contents of bufothionine were fluctuated from 36.4-641.8 microg x g(-1) in the skin of Bufo bufo gargarizans and 22.47-33.16 microg x mL(-1) in Huachansu injection, respectively. CONCLUSION: The contents of bufothionine were greatly different between cultured and wild species. The method was suitable for the quality control of the skin of Bufo bufo gargarizans and its preparation.

Fenton process on single and mixture components of phenothiazine pharmaceuticals: Assessment of intermediaries, fate, and preliminary ecotoxicity.

Pharmaceuticals do not occur isolated in the environment but in multi-component mixtures and may exhibit antagonist, synergistic or additive behavior. Knowledge on this is still scarce. The situation is even more complicated if effluents or potable water is treated by oxidative processes or such transformations occur in the environment. Thus, determining the fate and effects of parent compounds, metabolites and transformation products (TPs) formed by transformation and degradation processes in the environment is needed. This study investigated the fate and preliminary ecotoxicity of the phenothiazine pharmaceuticals, Promazine (PRO), Promethazine (PRM), Chlorpromazine (CPR), and Thioridazine (THI) as single and as components of the resulting mixtures obtained from their treatment by Fenton process. The Fenton process was carried out at pH7 and by using 0.5-2mgL-1 of [Fe2+]0 and 1-12.5mgL-1 of [H2O2]0 at the fixed ratio [Fe2+]0:[H2O2]0 of 1:10 (w:w). No complete mineralization was achieved. Constitutional isomers and some metabolite-like TPs formed were suggested based on their UHPLC-HRMSn data. A degradation pathway was proposed considering interconnected mechanisms such as sulfoxidation, hydroxylation, N-dealkylation, and dechlorination steps. Aerobic biodegradation tests (OECD 301 D and OECD 301 F) were applied to the parent compounds separately, to the mixture of parent compounds, and for the cocktail of TPs present after the treatment by Fenton process. The samples were not readily biodegradable. However, LC-MS analysis revealed that abiotic transformations, such hydrolysis, and autocatalytic transformations occurred. The initial ecotoxicity tested towards Vibrio fischeri as individual compounds featured a reduction in toxicity of PRM and CPR by the treatment process, whereas PRO showed an increase in acute luminescence inhibition and THI a stable luminescence inhibition. Concerning effects of the mixture components, reduction in toxicity by the Fenton process was predicted by concentration addition and independent action models.

Preparation of a generic monoclonal antibody and development of a highly sensitive indirect competitive ELISA for the detection of phenothiazines in animal feed.

In this study, a broadly specific monoclonal antibody was prepared and a sensitive monoclonal-based indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was subsequently developed to determine the phenothiazines in animal feed with a simple sample preparation procedure for the first time. The obtained antibody 3A5 was of the immunoglobulin G1 (IgG1) isotype possessing a kappa light chain, which broadly cross-reacted to nine phenothiazines. The limit of detections of the method ranged from 1.1µgkg-1 to 15.3µgkg-1 in the swine feed and the fish feed. The recoveries of the phenothiazines were in the range of 78.2-116.6%. The coefficient of variations were less than 16.7%. A positive correlation (r>0.9249) between the results of the ic-ELISA and the high-performance liquid chromatography were also observed, which indicated that the developed ic-ELISA is reliable and can be used to monitor phenothiazines in animal feed.