Free-Standing, Water-Resistant, and Conductivity-Enhanced PEDOT:PSS Films from In Situ Polymerization of 3-Hydroxymethyl-3-Methyl-Oxetane.

Free-standing films based on conducting polymers, such as poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) (PEDOT:PSS), offer many benefits over traditional metal electrodes for applications in flexible electronics. However, to ensure structural integrity when contacting aqueous environments and high levels of electrical conductivity, solution-processed polymers require additives that act as crosslinking agents and conductivity enhancers. In this work, a new approach is presented to fabricate water-resistant free-standing films of PEDOT:PSS and simultaneously increase their conductivity, using an oxetane compound as an additive. It is shown that at moderate temperatures, oxetane polymerizes within the PEDOT:PSS acidic medium, forming hydroxymethyl-substituted polyether compounds that form a network upon crosslinking with PSS. The polymer composite films show self-sustainability, structural stability in aqueous environments, and enhanced conductivity. Finally, the potential of the free-standing films as health-monitoring electrodes, specifically for human electrocardiography, is explored.

Advancing cortisol measurements in zebrafish: Analytical validation of a commercial ELISA kit for skin mucus cortisol analysis.

Cortisol is the main stress biomarker used for zebrafish. However, zebrafish small size made it challenging to extract cortisol without harming or killing the fish. Thus, researchers adopted a terminal method, the trunk cortisol, as standard practice. Here, we developed and validated an alternative and minimally invasive technique for measuring cortisol in the skin mucus of adult zebrafish, using a commercial enzyme-linked immunosorbent assay (ELISA). For this, AB zebrafish were randomly assigned to a precision, accuracy, and specificity test. Each sample contained the skin mucus of five to ten fish or one fish trunk. The cortisol was extracted using methanol as organic solvent. The results obtained showed an adequate precision (intra-assay coefficient of variation (CV) <15%; inter-assay CV = 26%), accuracy (CV <120%), and specificity (r2 =0.96-0.98) for skin mucus cortisol levels, as well as for trunk cortisol.•A commercial ELISA was analytically validated to measure cortisol in the skin mucus of zebrafish.•Skin mucus cortisol is a non-terminal method that reduce the number of animals used and allows longitudinal studies.

Housing Conditions Affect Adult Zebrafish (Danio rerio) Behavior but Not Their Physiological Status.

Zebrafish is a valuable model for neuroscience research, but the housing conditions to which it is exposed daily may be impairing its welfare status. The use of environmental enrichment and the refinement of methodology for cortisol measurement could reduce stress, improving its welfare and its suitability as an animal model used in stress research. Thus, this study aimed to evaluate (I) the influence of different housing conditions on zebrafish physiology and behavior, and (II) skin mucus potential for cortisol measurement in adult zebrafish. For this, AB zebrafish were raised under barren or enriched (PVC pipes and gravel image) environmental conditions. After 6 months, their behavior was assessed by different behavioral paradigms (shoaling, white-black box test, and novel tank). The physiological response was also evaluated through cortisol levels (whole-body homogenates and skin mucus) and brain oxidative stress markers. The results revealed that enriched-housed fish had an increased nearest neighbors' distance and reduced activity. However, no effect on body length or stress biomarkers was observed; whole-body and skin mucus cortisol levels had the same profile between groups. In conclusion, this study highlights the skin mucus potential as a matrix for cortisol quantification, and how housing conditions could influence the data in future studies.

Behavioural Aversion and Cortisol Level Assessment When Adult Zebrafish Are Exposed to Different Anaesthetics.

The use of zebrafish (Danio rerio) as an animal model is growing and occurs in a wide range of scientific areas. Therefore, researchers need better and more appropriate anaesthetics for stressful and/or painful procedures to prevent unpleasant experiences. Thus, we aimed to study if adult zebrafish displayed aversion-associated behaviours (conditioned place aversion) and alterations in cortisol levels when exposed to equipotent concentrations of MS222, propofol/lidocaine, clove oil, or etomidate. Adult AB zebrafish (mixed-sex, N = 177) were randomly assigned to MS222 (150 mg/L), Propofol/Lidocaine (5 mg/L propofol + 150 mg/L lidocaine), Clove Oil (45 mg/L), or Etomidate (2 mg/L) groups. The conditioned place aversion test was used to assess behavioural aversion. Only etomidate resulted in a similar aversion to the positive control group (HCl; pH = 3). Cortisol levels were measured 5 and 15 min after loss of equilibrium. Etomidate induced low levels of cortisol by impairing its synthesis, whereas all the other groups had similar cortisol levels. Based on our data, etomidate was ruled out as an alternative to MS222, as it showed an aversive profile. The remaining protocols were not innocuous, displaying a weak aversive profile when compared to the positive control. In conclusion, a combination of propofol with lidocaine, clove oil, and MS222 were valid candidates for use as anaesthetic protocols.

Anesthesia Overdose Versus Rapid Cooling for Euthanasia of Adult Zebrafish.

The rapid increase in zebrafish use needs to be accompanied by research into the refinement of procedures. The European (EU) Directive lists three possible euthanasia methods for fish: anesthetic overdose, electrical stunning, and concussion. However, for small fish such as zebrafish, concussion and electrical stunning are difficult to perform, leaving anesthetic overdose as the most used method. Our aim was to test the efficacy and side effects of anesthesia overdose using different anesthetics and the rapid cooling method to euthanize adult zebrafish. Adult mixed-sex AB zebrafish were randomly assigned to: 250 mg/L MS222; 20 mg/L propofol +100 mg/L lidocaine; 6 mg/L etomidate; 50 mg/L clove oil; and rapid cooling (water at 2°C-4°C). Two minutes after opercular movement ceased, animals were transferred into clean water for 20 min and recovery assessed, or decapitated and used for biochemical analysis of the gills, muscle, liver, and brain; for the histological analysis of the gills and muscle; or for the assessment of cortisol levels. No animal recovered; rapid cooling was the quickest and etomidate overdose was the slowest method to cease the opercular movements. There were no major differences between euthanasia methods regarding the biochemical or histological data. Cortisol levels were higher in the rapid cooling group, but only when compared with the propofol/lidocaine group. The use of a physical method of euthanasia, such as rapid cooling, is essential when chemicals, such as anesthetics, may interfere with postmortem analyses. Although anesthetic overdose can be used without major effects on the analyses conducted in this work, rapid cooling can be another option with the advantage of being simple to administer, easily available, affordable, and very quick; this decreases the potential duration of suffering, being more humane. Therefore, a change in EU legislation should be considered to include additional humane options for euthanasia, such as rapid cooling, for zebrafish and other small tropical fish.

Adult Zebrafish Anesthesia: A Study of Efficacy and Behavioral Recovery of Different Anesthetics.

The use of proper anesthesia in zebrafish research is essential to ensure fish welfare and data reliability. However, anesthesia long-term side effects remain poorly understood. The purpose of this study was to assess anesthesia quality and recovery in adult zebrafish using different anesthetic protocols and to determine possible long-term effects on the fish activity and anxiety-like behaviors after anesthesia. Mixed-sex adult AB zebrafish were randomly assigned to five different groups (Control, 175 mg/L of tricaine methanesulfonate [MS222], 45 mg/L of clove oil, 2 mg/L of etomidate, and 5 mg/L of propofol combined with 150 mg/L of lidocaine) and placed in the respective anesthetic bath. Time to lose the equilibrium, response to touch and to caudal fin pinch stimuli, and recovery after anesthesia administration were evaluated. In addition, after stopping anesthesia, respiratory rate, activity, and anxiety-like behaviors in the novel tank test were studied. Overall, all protocols proved to be adequate for zebrafish anesthesia research as they showed full recovery at 1 h, and only etomidate had minor effects on fish behavior in the novel tank, a validated test for anxiety.

Framework and implementation of a fuzzy logic filter-an optimization strategy for the BWMS based on stakeholders' perspectives.

The International Convention for the Control and Management of Ships' Ballast Water and Sediments (IMO 2018 Edition) was adopted in 2004 and came into force on 8 September 2017, aiming to introduce global regulations to control the transfer of potentially invasive species. Large efforts have been made by the maritime industry in creating reliable strategies for the installation of systems on board (Register 2014). Environmental considerations (INTERTANKO 2018) and optimization for the management systems (IMO 2017 Report) are factors broadly considered to tackle this matter. A consistent implementation strategy must be stated before the ballast water management system (BWMS) installation project starts-the management of stakeholders (e.g. ship owners, classification societies, administration, shipyards) is an important aspect of this process. This relies on their expertise, which in turn results in a high level of engagement and supports the implementation plan into the organizations. The creation of a framework for the optimization process, considering the implementation project of a BWMS on board of oil tankers, is the first part of this research. The use of fuzzy logic principles in the second part-as an evaluation instrument from a ranking obtained by multicriteria principles-sums up the aim of this paper, where the peculiarities about oil tankers' modelling will be discussed throughout the analysis of 2 optimization cases (Suezmax and Aframax).

A survey on the BWM Convention: analysing the stakeholders' perceptions with emphasis on the acquisition process and operational expenditure.

The International Convention for the Control and Management of Ships' Ballast Water and Sediments (IMO 2018 Edition) was adopted in 2004 and entered into force on 8 September 2017, aiming to introduce global regulations to control the transfer of potentially invasive species Resolution (IMO MEPC.207(62) 2011). These efforts are translated into a wide range of solutions for retrofitting and new building services, which is evidenced through the substantial amount of data disclosed and analysed by different parties showing that optimization processes will have an important role for the existent management systems. Analysing a ballast water management installation project for oil tankers, categorizing the stakeholders involved and assessing properly the outcomes from an engineering perspective are important matters. The experience considered at the BWM Convention and its amendments set an important call to the industry in terms of milestones. Avoiding any delay in terms of operation is not the only answer to this call but also setting up an optimized and sustainable operation after the implementation process. The project management, on the subfield of stakeholders' management (Bourne 2016), brings a myriad of individuals connected to ballast water management matters with different levels of knowledge, experience and commitment from an engineering perspective. This article aims to measure these factors and the stakeholders' experience based on qualitative research into the ballast water management convention, with emphasis on the installation process on board. The data herewith analysed was obtained from a series of interviews conducted from January 2019 to August 2019.

Electronic circular dichroism of chiral alkenes: B3LYP and CAM-B3LYP calculations.

Time-dependent density functional theory (TDDFT) calculations of electronic circular dichroism (ECD) are widely used to determine absolute configurations (ACs) of chiral molecules. Two very popular DFT exchange-correlation functionals, one hybrid (B3LYP) and one long-range corrected (CAM-B3LYP), along with a hierarchical sequence of basis sets were investigated, and the ECD spectra predicted for eight alkenes and compared to gas-phase experimental spectra. Little variation in predicted ECD spectra was found with the basis set size enlargement, but the sensitivity to the functional is greater. Good agreement was obtained only with the CAM-B3LYP functional, leading to the conclusion that TDDFT calculations of ECD spectra can routinely provide reliable ACs if and only if an appropriate functional is used. For camphene, twistene, syn-(E)-bisfenchylidene, and phyllocladene, solvent effects were estimated.