In vitro induction of the acrosome reaction in spermatozoa from endangered Spanish bulls: Effect of breed, culture media and incubation time

Current conservation programs for endangered species have been widely benefited from the use of modern assisted reproductive techniques. However, at the present time, there is little information available regarding the use of locally adapted cattle breeds on IVF programs, and even less about their sperm behavior. The aims of this study were to (1) evaluate standard acrosome reaction (AR) protocols in cryopreserved semen doses of Andalusian endangered cattle breeds, and (2) investigate the effect of breed, culture medium supplementation and incubation time on AR test results. To this end, 80 frozen semen doses from 16 bulls (5 semen samples per bull) of five different cattle breeds (3 Berrendo en Colorado, 6 Berrendo en Negro, 2 Cardena Andaluza, 1 Pajuna and 4 Retinta) were in vitro incubated in three test media (Heparin, BSA or their combina-tion), using Sp-TALP as a control, to induce in vitro AR. The percentage of acrosome-reacted spermatozoa was evaluated using fluorescence techniques (FITC-PNA and Propidium Iodide) at four time-points (0, 30, 60 or 120 min.). Throughout the whole experiment, spermatozoa incubated with a combination of both supplements (Heparin and BSA) showed significantly (Po0.05) higher results than those processed with Heparin or BSA alone. In contrast, the control media (without supplementation) showed the lowest results. We also found a significant (Po0.05) influence of cattle breed on the dynamics of the in vitro induced AR using different protocols. Based on our results, we can conclude that the response pattern of in vitro induced AR depends not only on culture media and incubation time but also on the cattle breed. Therefore, all these factors must be taken in consideration to assess the rate of AR sperm in bulls

Detoxification of azo dyes by a novel pH-versatile, salt-resistant laccase from Streptomyces ipomoea

A newly identified extracellular laccase produced by Streptomyces ipomoea CECT 3341 (SilA) was cloned and overexpressed, and its physicochemical characteristics assessed together with its capability to decolorize and detoxify an azotype dye. Molecular analysis of the deduced sequence revealed that SilA contains a TAT-type signal peptide at the N-terminus and only two cupredoxine domains; this is consistent with reports describing two other Streptomyces laccases but contrasts with most laccases, which contain three cupredoxine domains. The heterologous expression and purification of SilA revealed that the homodimer is the only active form of the enzyme. Its stability at high pH and temperature, together with its resistance to high concentrations of NaCl and to typical laccase inhibitors such as sodium azide confirmed the unique properties of this novel laccase. The range of substrates that SilA is able to oxidize was found to be pH-dependent; at alkaline pH, SilA oxidized a wide range of phenolic compounds, including the syringyl and guayacil moieties derived from lignin. The oxidative potential of this enzyme to use phenolic compounds as natural redox mediators was shown through the coordinated action of SilA and acetosyringone (as mediator), which resulted in the complete detoxification of the azo-type dye Orange II.This work was financially supported by the Spanish Ministry of Science (CICYT Project CTQ2004-0344/PPQ to MEA and BFU 2006-00972/BMC to JMD). We thank the Autonomous Government of Madrid and the University of Alcalá for the fellowship awarded to R.M

Copper-iron mixed oxide supported onto cordierite honeycomb as a heterogeneous catalyst in the Kharasch-Sosnovsky oxidation of cyclohexene

A copper-iron mixed oxide was deposited by the washcoating procedure over cordierite honeycomb monoliths for its use as a heterogeneous catalyst in organic synthesis processes. In particular, the prepared catalyst, characterized by techniques such as X-ray fluorescence, X-ray diffraction, SEM-EDS, laser granulometry, adherence tests, Temperature-Programmed Oxidation and Temperature-Programmed Reduction, showed an excellent yield and stability in the selective production of the allylic ester derived from the Kharasch-Sosnovsky oxidation of cyclohexene with benzoic acid. The use of a structured catalyst here proposed opens up an interesting alternative to homogeneous catalysis in the field of synthetic chemistry. © 2021 The AuthorsThe authors thank the Ministry of Economy and Competitiveness of Spain (Projects MAT2017-85-719-R , and AGL2017-88083-R ), the Junta de Andalucía ( FQM-110 and FQM-169 groups), and the Institute of Electron Microscopy and Materials (IMEYMAT) of Cadiz University (UCA) (Projects HOMOGREEN and NUPRECAT) for their financial support. They also acknowledge the SC-ICYT of the UCA for using its XRD NMR, and electron microscopy division facilities

Acyloxylation of 1,4-Dioxanes and 1,4-Dithianes Catalyzed by a Copper−Iron Mixed Oxide

The use of a copper−iron mixed oxide as a heterogeneous catalyst for the efficient synthesis of α-acyloxy-1,4-dioxanes and 1,4-dithianes employing t-butyl peroxyesters is reported. The preparation and characterization of the catalyst are described. The effect of the heteroatoms and a plausible mechanism are discussed. The method is operationally simple and involves low-cost starting materials affording products in good to excellent yields

DoE (Design of Experiments) Assisted Allylic Hydroxylation of Enones Catalysed by a Copper–Aluminium Mixed Oxide

The allylic hydroxylation of enones using dioxygen as the oxidant has been studied. The reaction was first examined in the absence of any catalyst, using β-ionone as a model substrate. Then a new copper–aluminium mixed oxide, Cu–Al Ox, was prepared and characterized in order to be used as a catalyst. This oxide showed good activity, and provided the corresponding γ- or ε-hydroxylated enones, starting from different α,β- or α,β,γ,δ-unsaturated ketones. In all cases, the yields were significantly improved compared to experiments run in the absence of the catalyst. The reaction was selective, and the formation of epoxides or other overoxidation products was detected only to a minor extent. The described procedure is a technically straightforward synthetic alternative to those methods described to date involving many reaction steps or toxic reagents. The reactions were optimized using design of experiments techniques (DoE)

The Myxococcus xanthus Two-Component System CorSR Regulates Expression of a Gene Cluster Involved in Maintaining Copper Tolerance during Growth and Development

Myxococcus xanthus is a soil-dwelling member of the δ–Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains.This work has been funded by the Spanish Government (grants CSD2009-00006 and BFU2012-33248, 70% funded by FEDER). This work was also supported by the National Institute of General Medical Science of the National Institutes of Health under award number R01GM095826 to LJS, and by the National Science Foundation under award number MCB0742976 to LJS. JMD and JP received a fellowship from Junta de Andalucía to do some work at University of Georgia

CorE from Myxococcus xanthus Is a Copper-Dependent RNA Polymerase Sigma Factor

The dual toxicity/essentiality of copper forces cells to maintain a tightly regulated homeostasis for this metal in all living organisms, from bacteria to humans. Consequently, many genes have previously been reported to participate in copper detoxification in bacteria. Myxococcus xanthus, a prokaryote, encodes many proteins involved in copper homeostasis that are differentially regulated by this metal. A σ factor of the ECF (extracytoplasmic function) family, CorE, has been found to regulate the expression of the multicopper oxidase cuoB, the P1B-type ATPases copA and copB, and a gene encoding a protein with a heavy-metal-associated domain. Characterization of CorE has revealed that it requires copper to bind DNA in vitro. Genes regulated by CorE exhibit a characteristic expression profile, with a peak at 2 h after copper addition. Expression rapidly decreases thereafter to basal levels, although the metal is still present in the medium, indicating that the activity of CorE is modulated by a process of activation and inactivation. The use of monovalent and divalent metals to mimic Cu(I) and Cu(II), respectively, and of additives that favor the formation of the two redox states of this metal, has revealed that CorE is activated by Cu(II) and inactivated by Cu(I). The activation/inactivation properties of CorE reside in a Cys-rich domain located at the C terminus of the protein. Point mutations at these residues have allowed the identification of several Cys involved in the activation and inactivation of CorE. Based on these data, along with comparative genomic studies, a new group of ECF σ factors is proposed, which not only clearly differs mechanistically from the other σ factors so far characterized, but also from other metal regulators

Pharmacotherapy negative outcomes resulting in Primary Care Emergency visits

Objetivo Analizar la prevalencia de los resultados negativos asociados a la medicación (RNM) que son causa de consulta en un servicio de urgencias de atención primaria (SUAP) en un entorno rural. Determinar la evitabilidad y la gravedad de los mismos. Diseño Estudio observacional descriptivo transversal. Emplazamiento SUAP de Mula. Murcia. Participantes Un total de 330 pacientes, en un periodo de 33 semanas. Mediciones principales Número y tipo de RNM: el farmacéutico, a través de los datos obtenidos de un cuestionario validado y la historia clínica, evaluó si existía relación entre los medicamentos que toma el paciente y el motivo de acudir a urgencias. En caso de sospecha de RNM se reevaluaba con el médico y se confirmaban o no los RNM identificados. Resultados De los 330 pacientes fueron evaluables 317. La media de edad de los pacientes era de 39,63 años y el 51,42% eran mujeres. La media de medicamentos que utilizaban fue de 1,38. Se detectaron un 26,50% (IC 95% 21,94-31,62) de pacientes con RNM como causa de visita a urgencias. El 53,57% de los RNM detectados fue de la categoría de efectividad y el 40,48% de necesidad. El 77,41% (IC-95% 67,35-85,01) de las visitas causadas por RNM fueron evitables. En cuanto a la gravedad, el 92,986% de los RNM eran leves. Conclusiones Una de cada 4 visitas al SUAP de Mula está causada por un RNM y, de ellas, el 77,41% son evitables.Objective Our aim was to estimate the prevalence of Pharmacotherapy negative outcomes in Primary Care Emergency visits in a rural environment, and to determine their preventability and severity. Design Descriptive study with an analytical component. Site Primary Care Emergency Service (SUAP), Mula. Murcia. Patients The study consisted of 330 patients over a 33 week period. Method Number and type of Pharmacotherapy negative outcomes: Pharmacist through the data, a validated questionnaire and medical history, assessing whether there was a relationship between the medications and the patient, and the reason for going to the Primary Care Emergency. In case of suspicion of Pharmacotherapy negative outcomes the patient is reassessed by the doctor, and the Pharmacotherapy negative outcomes confirmed or not identified. Results Of the 330 patients, 317 were evaluable. The mean age of patients was 39.63 years and 51.42% were women. The mean number of drugs used was 1.38, and 26.50% (95% CI, 21.94% -31.62%) patients were detected with Pharmacotherapy negative outcomes as a cause of visiting the Primary Care Emergency. 53.57% of the detected Pharmacotherapy negative outcomes detected as regards efficacy was 53.75%, 40.48% as regards need. More than three-quarters (77.41%; 95% CI, 67.35% -85.01%) of emergency visits caused by Pharmacotherapy negative outcomes were avoidable. In terms of severity, 92.86% of the Pharmacotherapy negative outcomes were mild. Conclusions One in four Mula SUAP visits are due to a Pharmacotherapy negative outcomes, and 77.41% of them are preventable

Bacterial laccases: some recent advances and applications

Laccases belong to the large family of multi-copper oxidases (MCOs) that couple the one-electron oxidation of substrates with the four-electron reduction of molecular oxygen to water. Because of their high relative non-specific oxidation capacity particularly on phenols and aromatic amines as well as the lack of requirement for expensive organic cofactors, they have found application in a large number of biotechnological fields. The vast majority of studies and applications were performed using fungal laccases, but bacterial laccases show interesting properties such as optimal temperature above 50 °C, optimal pH at the neutral to alkaline range, thermal and chemical stability and increased salt tolerance. Additionally, bacterial systems benefit from a wide range of molecular biology tools that facilitates their engineering and achievement of high yields of protein production and set-up of cost-effective bioprocesses. In this review we will provide up-to-date information on the distribution and putative physiological role of bacterial laccases and highlight their distinctive structural and biochemical properties, discuss the key role of copper in the biochemical properties, discuss thermostability determinants and, finally, review biotechnological applications with a focus on catalytic mechanisms on phenolics and aromatic amines.info:eu-repo/semantics/publishedVersio