In vivo structure probing of RNA in Archaea: novel insights into the ribosome structure of Methanosarcina acetivorans.

Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date, such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.

Lactate oxidation is linked to energy conservation and to oxygen detoxification via a putative terminal cytochrome oxidase in Methanosarcina acetivorans.

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.

Methods for Culturing Anaerobic Microorganisms.

Anaerobic microorganisms (anaerobes) proliferate in diverse oxygen-free environments. They inhabit Earth's soils and aquatic sediments, the rumen and gut of mammals, and the gut of insects among many other oxygen-free environments. Anaerobes impact biotechnological, biomedical, ecological, and astrobiological fields. Sensitivity to oxygen is of prime consideration for successful culturing which is essential to understand function. Although cultivated for many years, the protocols and media components have been modified and adapted to the special needs of species, as well as conditions and variables for experimental evaluations. Here we describe a revised method used in our laboratories for the growth of methane-producing anaerobes (methanogenic archaea) which are among the most oxygen sensitive. The method is an example for the preparation of more specific media to cultivate a wide diversity of anaerobes.

Transcriptomic Responses of Rhizobium phaseoli to Root Exudates Reflect Its Capacity to Colonize Maize and Common Bean in an Intercropping System.

Corn and common bean have been cultivated together in Mesoamerica for thousands of years in an intercropping system called "milpa," where the roots are intermingled, favoring the exchange of their microbiota, including symbionts such as rhizobia. In this work, we studied the genomic expression of Rhizobium phaseoli Ch24-10 (by RNA-seq) after a 2-h treatment in the presence of root exudates of maize and bean grown in monoculture and milpa system under hydroponic conditions. In bean exudates, rhizobial genes for nodulation and degradation of aromatic compounds were induced; while in maize, a response of genes for degradation of mucilage and ferulic acid was observed, as well as those for the transport of sugars, dicarboxylic acids and iron. Ch24-10 transcriptomes in milpa resembled those of beans because they both showed high expression of nodulation genes; some genes that were expressed in corn exudates were also induced by the intercropping system, especially those for the degradation of ferulic acid and pectin. Beans grown in milpa system formed nitrogen-fixing nodules similar to monocultured beans; therefore, the presence of maize did not interfere with Rhizobium-bean symbiosis. Genes for the metabolism of sugars and amino acids, flavonoid and phytoalexin tolerance, and a T3SS were expressed in both monocultures and milpa system, which reveals the adaptive capacity of rhizobia to colonize both legumes and cereals. Transcriptional fusions of the putA gene, which participates in proline metabolism, and of a gene encoding a polygalacturonase were used to validate their participation in plant-microbe interactions. We determined the enzymatic activity of carbonic anhydrase whose gene was also overexpressed in response to root exudates.

Patient-reported taste change assessment questionnaires used in the oncology setting: A narrative review.

PURPOSE: Patient-reported questionnaires to assess taste changes (TC) among patients with cancer and the common domains or aspects assessed by those questionnaires are identified in this narrative review. Taste changes are a prevalent symptom experienced by patients with cancer that impact food choice and enjoyment, reduce food intake, and diminish quality of life. Appropriate assessment is essential to detect and manage this symptom. METHOD: A systematic search of relevant databases between 1999 and 2018 yielded 1959 articles; 38 articles were included in the review. RESULTS: Seventeen questionnaires designed specifically to assess patient-reported taste changes among patients with cancer are described in the review. Seven domains were identified among the questionnaires; the most frequently assessed domain was the description of the taste change in 14 questionnaires). Timeframe, scoring, number of items and domains, and item phrasing varied greatly among questionnaires and the approach to domain and item evaluation was inconsistent. Comprehensive questionnaires (n = 7) assessed five or more domains to characterize the taste change experience. The majority of questionnaires have been cited only once or twice. CONCLUSION: Patient-reported taste change assessment in oncology has been achieved by a large number of diverse questionnaires; no standard tool or approach is used. Development of a question bank of validated or standardized taste change modules or items may strengthen the consistency and applicability of research in this area.

Structure and function of an unusual flavodoxin from the domain Archaea.

Flavodoxins, electron transfer proteins essential for diverse metabolisms in microbes from the domain Bacteria, are extensively characterized. Remarkably, although genomic annotations of flavodoxins are widespread in microbes from the domain Archaea, none have been isolated and characterized. Herein is described the structural, biochemical, and physiological characterization of an unusual flavodoxin (FldA) from Methanosarcina acetivorans, an acetate-utilizing methane-producing microbe of the domain Archaea In contrast to all flavodoxins, FldA is homodimeric, markedly less acidic, and stabilizes an anionic semiquinone. The crystal structure reveals an flavin mononucleotide (FMN) binding site unique from all other flavodoxins that provides a rationale for stabilization of the anionic semiquinone and a remarkably low reduction potentials for both the oxidized/semiquinone (-301 mV) and semiquinone/hydroquinone couples (-464 mV). FldA is up-regulated in acetate-grown versus methanol-grown cells and shown here to substitute for ferredoxin in mediating the transfer of low potential electrons from the carbonyl of acetate to the membrane-bound electron transport chain that generates ion gradients driving ATP synthesis. FldA offers potential advantages over ferredoxin by (i) sparing iron for abundant iron-sulfur proteins essential for acetotrophic growth and (ii) resilience to oxidative damage.

Preclinical Assessment of a New Polyvalent Antivenom (Inoserp Europe) against Several Species of the Subfamily Viperinae.

The European continent is inhabited by medically important venomous Viperinae snakes. Vipera ammodytes, Vipera berus, and Vipera aspis cause the greatest public health problems in Europe, but there are other equally significant snakes in specific regions of the continent. Immunotherapy is indicated for patients with systemic envenoming, of which there are approximately 4000 annual cases in Europe, and was suggested as an indication for young children and pregnant women, even if they do not have systemic symptoms. In the present study, the safety and venom-neutralizing efficacy of Inoserp Europe-a new F(ab')2 polyvalent antivenom, designed to treat envenoming by snakes in the Eurasian region-were evaluated. In accordance with World Health Organization recommendations, several quality control parameters were applied to evaluate the safety of this antivenom. The venom-neutralizing efficacy of the antivenom was evaluated in mice and the results showed it had appropriate neutralizing potency against the venoms of several species of Vipera, Montivipera, and Macrovipera. Paraspecificity of the antivenom was demonstrated as well, since it neutralized venoms of species not included in the immunization schemes and contains satisfactory levels of total proteins and F(ab')2 fragment concentration. Therefore, this new polyvalent antivenom could be effective in the treatment of snake envenoming in Europe, including Western Russia and Turkey.

Physical symptom burden of post-treatment head and neck cancer patients influences their characterization of food: Findings of a repertory grid study.

BACKGROUND AND AIM: Dietary advice for post treatment head and neck cancer (HNC) patients emphasizes food characteristics of nutritional value and texture, and not patients' characterization of food. The aim of this study was to determine patients' characterization of food. METHODS: Repertory grid interviews were conducted with 19 orally-fed HNC patients between 4 and 10 months post-treatment to characterize foods commonly eaten, avoided and eaten sometimes. Patients compared and rated 12 foods using their own descriptors. Data were analyzed by General Procrustes Analysis (GPA). Socio-demographic status, taste and smell alterations, appetite and food intake data were also collected. Patient physical symptom burden was defined by University of Washington-Quality of Life Physical Function domain scores and used to stratify patients with "less physical symptom burden" (n = 11, score ≥ 61.7) or "greater physical symptom burden" (n = 8, score < 61.7). RESULTS: All patients used descriptors of taste, ease of eating, convenience, texture, potential to worsen symptoms and liking to characterize foods. Overall, avoided foods were characterized as having dry texture, while foods commonly eaten were characterized by their ease of eating and low potential to worsen symptoms. Descriptors of nutrition and smell were significant only for patients with greater physical symptom burden. CONCLUSIONS: Physical symptom burden influenced the characterization of foods among post-treatment HNC patients. Nutrition counseling must consider patients' physical symptom burden and the subsequent characterization of food that drive food selection or avoidance to facilitate dietary advice for adequate, appropriate and enjoyable food intake.

The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes.

Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental advantage for methanosarcinales when carbon sources are scarce. Also, the understanding of the central carbohydrate metabolism in methanosarcinales may help to optimize methane production.

A review of chemosensory perceptions, food preferences and food-related behaviours in subjects with Prader-Willi Syndrome.

Hyperphagia and obsessive preoccupation with food are hallmark characteristics of Prader-Willi Syndrome (PWS). Although hyperphagia in PWS is linked to hypothalamic dysfunction, the underlying mechanisms behind this problem are poorly understood. Moreover, our understanding of how chemosensory perceptions and food choice/preferences relate to hyperphagia in individuals with PWS is very limited. This narrative review synthesizes studies that assessed chemosensory perceptions, food choices and food-related behaviours in PWS individuals and highlights knowledge gaps in research for further exploration. Twenty seven publications from relevant databases met inclusion criteria and were organized thematically by study technique in the review. Results suggested that PWS individuals have consistent preferences for sweet tastes and in most studies have exhibited a preference for calorie-dense foods over lower calorie foods. No firm conclusions were drawn concerning the chemosensory perceptions of PWS individuals and their influence on food preferences or choices; chemosensation among PWS individuals is an understudied topic. Current evidence suggests that eating behaviour in PWS is a complex phenomenon that involves a dysfunctional satiation and not excessive hunger. Food preferences, choices, and related behaviours and the impact of these on obesity management in those with PWS remain poorly understood and require further study using validated tools and methodologies.

Fauna europaea: Diptera - brachycera.

Fauna Europaea provides a public web-service with an index of scientific names (including important synonyms) of all extant multicellular European terrestrial and freshwater animals and their geographical distribution at the level of countries and major islands (east of the Urals and excluding the Caucasus region). The Fauna Europaea project comprises about 230,000 taxonomic names, including 130,000 accepted species and 14,000 accepted subspecies, which is much more than the originally projected number of 100,000 species. Fauna Europaea represents a huge effort by more than 400 contributing taxonomic specialists throughout Europe and is a unique (standard) reference suitable for many user communities in science, government, industry, nature conservation and education. The Diptera-Brachycera is one of the 58 Fauna Europaea major taxonomic groups, and data have been compiled by a network of 55 specialists. Within the two-winged insects (Diptera), the Brachycera constitute a monophyletic group, which is generally given rank of suborder. The Brachycera may be classified into the probably paraphyletic 'lower brachyceran grade' and the monophyletic Eremoneura. The latter contains the Empidoidea, the Apystomyioidea with a single Nearctic species, and the Cyclorrhapha, which in turn is divided into the paraphyletic 'aschizan grade' and the monophyletic Schizophora. The latter is traditionally divided into the paraphyletic 'acalyptrate grade' and the monophyletic Calyptratae. Our knowledge of the European fauna of Diptera-Brachycera varies tremendously among families, from the reasonably well known hoverflies (Syrphidae) to the extremely poorly known scuttle flies (Phoridae). There has been a steady growth in our knowledge of European Diptera for the last two centuries, with no apparent slow down, but there is a shift towards a larger fraction of the new species being found among the families of the nematoceran grade (lower Diptera), which due to a larger number of small-sized species may be considered as taxonomically more challenging. Most of Europe is highly industrialised and has a high human population density, and the more fertile habitats are extensively cultivated. This has undoubtedly increased the extinction risk for numerous species of brachyceran flies, yet with the recent re-discovery of Thyreophoracynophila (Panzer), there are no known cases of extinction at a European level. However, few national Red Lists have extensive information on Diptera. For the Diptera-Brachycera, data from 96 families containing 11,751 species are included in this paper.

Aplicación del reactivo de Neesler en la cuantificación de amonio para las fermentaciones de productos biotecnológicos / Application of Neesler reagent in the ammonium quantification used in the fermentations of biotechnology product

Las sales de amonio se utilizan en las fermentaciones para suplementar las cantidades deficitarias de nitrógeno y estabilizar el pH del medio de cultivo. El ion amonio en exceso, ejerce un efecto perjudicial en el proceso fermentativo ya que inhibe el crecimiento microbiano. Con el objetivo de monitorear y controlar la concentración de amonio durante el proceso de fermentación, se desarrolló un método con el reactivo de Neesler, para la cuantificación de dicho analito. El ensayo se estandarizó mediante: selección del equipo de medición; tiempo de reacción del ensayo y comparación de las sales estándares de amonio. El método se caracterizó con la evaluación de los parámetros: especificidad; linealidad y rango del sistema, límite de cuantificación, exactitud y precisión. El método demostró ser específico. Se establecieron dos sistemas con curvas que fueron lineales en los rangos de cloruro de amonio (2 a 20 µg/mL) y sulfato de amonio (5 a 30 µg/mL). Los límites de cuantificación fueron los puntos inferiores de cada rango de trabajo. El método resultó ser preciso y exacto. Este ensayo se aplicó a muestras de cultivos de levadura y bacterias del género Saccharomyces y Escherichia. coli respectivamente. Se desarrolló un método novedoso en microplaca para la cuantificación y control analítico del amonio. Mediante este método se controla este componente químico fundamental en las fermentaciones, para optimizar el medio de cultivo. Se logra una adecuada expresión de proteínas recombinantes y la obtención de candidatos vacunales para uso clínico(AU)

The ammonium salts are used in fermentations to supplement the deficient amounts of nitrogen and stabilize the pH of the culture medium. The excess ammonium ion exerts a detrimental effect on the fermentation process inhibiting microbial growth. An analytical method based on Neesler reagent was developed for monitoring and controlling the concentration of ammonium during the fermentation process. The test was standardized, by means of the selection of measuring equipment, and the reaction time as well as comparing standards of ammonium salts. The method was characterized with the evaluation of the next parameters: Specificity, Linearity and Range, Quantification Limit, Accuracy and Precision. The method proved to be specific. Two linear curves were defined in the ranges of concentrations of ammonium chloride salt (2-20 µg/ml) and ammonium sulfate salt (5-30 µg/ml). The limits of quantification were the lowest points of each one. The method proved to be accurate and precise. This assay was applied to samples of the yeast culture and bacteria of the genus Saccharomyces and E. coli respectively . A novel method in micro plate for quantification and analytical control of ammonia was developed. T his method is used to control this fundamental chemical component in the fermentations , to optimize the culture medium. Thus, an appropriate expression of recombinant proteins and proper vaccine candidates for clinical use are achieved(AU)

Tratamiento no quirúrgico de colección pancreática en pancreatitis aguda grave: presentación de caso / Non surgical treatment of pancreatic collection in severe acute pancreatitis: case presentation

La pancreatitis aguda es un gran drama abdominal, de gravedad variable, seleccionar el tratamiento apropiado requiere identificación de pacientes conriesgo de complicaciones y muerte, ningún método detecta todos los pacientesque van a desarrollar complicación. La atención varía desde conducta quirúrgicatemprana a tratamiento médico en las Unidades de Cuidados Intensivos cuyoprincipal objetivo es corregir la hipovolemia, y detención o regresión del Síndrome de Respuesta Inflamatoria Sistémica. La atención debe ser por unequipo multidisciplinario. Los antimicrobianos profilácticos han sidocontroversiales; en países en vías de desarrollo con recursos limitados, estaterapéutica puede ser beneficiosa. Se presenta una mujer de 36 años que sufre de pancreatitis aguda grave con colección pancreática, que se le indicó tratamiento médico no intervencionista y antibioprofilaxis teniendo una evolución favorable, con resolución total, la cual estuvo ingresada en la Unidad de Cuidados Intensivas del Hospital General Docente de Placetas en octubre del 2012(AU)

Evaluación de los métodos de la normatividad mexicana para la determinación de nitritos en alimentos infantiles

Se evaluaron los dos métodos para la determinación de nitritos, que solicita la normatividad mexicana, en alimentos infantiles cárnicos con verduras. Se determinó el contenido de nitritos a los alimentos infantiles, materias primas y productos intermedios del proceso de elaboración; en cada corrida analítica se incluyeron un blanco de reactivos y una muestra testigo; además se determinó la sensibilidad, porcentaje de recuperación y precisión de las metodologías. Los resultados en los alimentos infantiles indicaron una importante diferencia en los contenidos de nitritos obtenidos entre las metodologías, debido a la persistente presencia de turbidez en los extractos. Se propusieron diferentes tratamientos físicos para eliminarla, pero únicamente la redujeron; tal turbidez se atribuyó a los hidratos de carbono; las concentraciones de nitritos informadas presentaron una dispersión grande y estuvieron por debajo del límite de cuantificación de ambos métodos, por lo que no es recomendable la aplicación de estas técnicas para alimentos que se sospechan contienen trazas de nitritos.

Evaluation of the methods for the determination of nitrites in baby foods according Mexican legislation. We evaluated the two methods accepted by the Mexican norm for the determination of nitritesin infant meatbased food with vegetables. We determined the content of nitrites in the infant food, raw materials as well as products from the intermediate stages of production. A reagent blank and a reference sample were included at each analytical run. In addition, we determined the sensitivity, recovery percentage and accuracy of each methodology. Infant food results indicated an important difference in the nitrite content determined under each methodology, due to the persistent presence of turbidity in the extracts. Different treatments were proposed to eliminate the turbidity, but these only managed to reduce it. The turbidity was attributed to carbohydrates which disclosed concentration exhibit a wide dispersion and were below the quantifiable limit under both methodologies; therefore it is not recommended to apply these techniques with food suspected to contain traces of nitrites.

[Evaluation of the methods for the determination of nitrites in baby foods according Mexican legislation]. / Evaluación de los métodos de la normatividad mexicana para la determinación de nitritos en alimentos infantiles.

We evaluated the two methods accepted by the Mexican norm for the determination of nitritesin infant meat-based food with vegetables. We determined the content of nitrites in the infant food, raw materials as well as products from the intermediate stages of production. A reagent blank and a reference sample were included at each analytical run. In addition, we determined the sensitivity, recovery percentage and accuracy of each methodology. Infant food results indicated an important difference in the nitrite content determined under each methodology, due to the persistent presence of turbidity in the extracts. Different treatments were proposed to eliminate the turbidity, but these only managed to reduce it. The turbidity was attributed to carbohydrates which disclosed concentration exhibit a wide dispersion and were below the quantifiable limit under both methodologies; therefore it is not recommended to apply these techniques with food suspected to contain traces of nitrites.

Consumer preferences and willingness to pay for value-added chicken product attributes.

A growing demand for convenient and ready-to-eat products has increased poultry processors' interest in developing consumer-oriented value-added chicken products. In this study, a conjoint analysis survey of 276 chicken consumers in Edmonton was conducted during the summer of 2009 to assess the importance of the chicken part, production method, processing method, storage method, the presence of added flavor, and cooking method on consumer preferences for different value-added chicken product attributes. Estimates of consumer willingness to pay (WTP) premium prices for different combinations of value-added chicken attributes were also determined. Participants'"ideal" chicken product was a refrigerated product made with free-range chicken breast, produced with no additives or preservatives and no added flavor, which could be oven heated or pan heated. Half of all participants on average were willing to pay 30% more for a value-added chicken product over the price of a conventional product. Overall, young consumers, individuals who shop at Farmers' Markets and those who prefer free-range or organic products were more likely to pay a premium for value-added chicken products. As expected, consumers' WTP was affected negatively by product price. Combined knowledge of consumer product attribute preferences and consumer WTP for value-added chicken products can help the poultry industry design innovative value-added chicken products. Practical Application: An optimum combination of product attributes desired by consumers for the development of a new value-added chicken product, as well as the WTP for this product, have been identified in this study. This information is relevant to the poultry industry to enhance consumer satisfaction of future value-added chicken products and provide the tools for future profit growth.

New contributions to Pseudonapomyza (Diptera: Agromyzidae) from Spain: addition of three new species.

The genus Pseudonapomyza (Diptera: Agromyzidae) includes the main leafminer pests for monocots. Three new species are described that were captured using Malaise traps in "Tinença de Benifassà", "Font Roja" and "Lagunas de La Mata-Torrevieja" (Spain) Natural Parks: Pseudonapomyza curvata n. sp., P. longitata n. sp., and P. sicicornis n. sp. Systematics. Ecological data are discussed.

Acetylcholinesterase genes within the Diptera: takeover and loss in true flies.

It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by the ace-1 gene, distinct and divergent from the ace-2 gene, which performs this function in Drosophila. This is an unprecedented situation within the Diptera order because both ace genes derive from an old duplication and are present in most insects and arthropods. Nevertheless, Drosophila possesses only the ace-2 gene. Thus, a secondary loss occurred during the evolution of Diptera, implying a vital function switch from one gene (ace-1) to the other (ace-2). We sampled 78 species, representing 50 families (27% of the Dipteran families) spread over all major subdivisions of the Diptera, and looked for ace-1 and ace-2 by systematic PCR screening to determine which taxonomic groups within the Diptera have this gene change. We show that this loss probably extends to all true flies (or Cyclorrhapha), a large monophyletic group of the Diptera. We also show that ace-2 plays a non-detectable role in the synaptic AChE in a lower Diptera species, suggesting that it has non-synaptic functions. A relative molecular evolution rate test showed that the intensity of purifying selection on ace-2 sequences is constant across the Diptera, irrespective of the presence or absence of ace-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover of ace-2 and the loss of ace-1, taking into account our limited knowledge of non-synaptic functions of ace genes and some specific adaptations of true flies.