Considerations for experimental animal ethics in the research planning and evaluation process

Research using experimental animals has substantially contributed to advances in science and medicine. Animal experiments are nearly essential for biomedical research and development efforts. Because many animals are sacrificed, researchers should consider the welfare of experimental animals and related ethical issues, along with the successful results of their experiments. This review introduces the criteria that should be considered in terms of experimental animal ethics, based on the principles of the 3 R’s: replacement, representing careful consideration of the need for animal experiments; reduction, representing the use of the minimal number of animals to obtain meaningful experimental results; and refinement, representing continuous effects to find alternative methods to reduce pain and distress in experimental animals. Based on these principles, the following points should be considered when planning experiments: the necessity of animal experiments; alternatives to animal experiments; the relevance of the species and numbers of experimental animals; appropriate assessment and management of pain; the proper usage of sedatives, painkillers, and anesthesia; and valid timing for humane endpoints and euthanasia. These criteria are beneficial for both experimental animals and researchers because careful handling to ensure experimental animal welfare guarantees that scientific research will yield convincing, repeatable, and accurate results

The full repertoire of Drosophila gustatory receptors for detecting an aversive compound.

The ability to detect toxic compounds in foods is essential for animal survival. However, the minimal subunit composition of gustatory receptors required for sensing aversive chemicals in Drosophila is unknown. Here we report that three gustatory receptors, GR8a, GR66a and GR98b function together in the detection of L-canavanine, a plant-derived insecticide. Ectopic co-expression of Gr8a and Gr98b in Gr66a-expressing, bitter-sensing gustatory receptor neurons (GRNs) confers responsiveness to L-canavanine. Furthermore, misexpression of all three Grs enables salt- or sweet-sensing GRNs to respond to L-canavanine. Introduction of these Grs in sweet-sensing GRNs switches L-canavanine from an aversive to an attractive compound. Co-expression of GR8a, GR66a and GR98b in Drosophila S2 cells induces an L-canavanine-activated nonselective cation conductance. We conclude that three GRs collaborate to produce a functional L-canavanine receptor. Thus, our results clarify the full set of GRs underlying the detection of a toxic tastant that drives avoidance behaviour in an insect

Clinical and Echocardiographic Findings of Newly Diagnosed Acute Decompensated Heart Failure in Elderly Patients

PURPOSE: Elderly patients (pts) (EPs; ≥ 65 years old) with newly diagnosed-acute decompensated heart failure (ND-ADHF) have not yet been studied. The aim of the present study was to investigate clinical characteristics, including echocardiographic findings and prognosis, for EPs with ND-ADHF and to compare those with non-elderly pts (NEPs). MATERIALS AND METHODS: We retrospectively investigated 256 pts (144 males, 63.0 ± 14.8 years old) who were admitted to our hospital between January 2005 and March 2009 with ND-ADHF. Clinical characteristics and echocardiographic parameters were analyzed in EPs (n = 135, 58 males) and NEPs (n = 121, 86 males). RESULTS: In intergroup comparison, female gender, diabetes mellitus, previous stroke and hypertension were more common in EPs. Body mass index (22.3 ± 4.5 vs. 24.0 ± 4.4 kg/m(2)), estimated glomerular filtration rate (54.8 ± 24.3 vs. 69.2 ± 30.7 mL/min/m(2)), C-reactive protein (28.5 ± 46.9 vs. 7.6 ± 11.6 mg/dL), hemoglobin (12.3 ± 2.1 vs. 13.6 ± 2.3 g/dL) and N-terminal pro-brain natriuretic peptide level (10,538.2 ± 10,942.3 vs. 6,771.0 ± 8,964.7 pg/mL) were significantly different (p < 0.05 for all). Early mitral inflow velocity to early diastolic mitral annular velocity (E/E') was significantly higher in EPs than in NEPs (21.2 ± 9.4 vs. 18.0 ± 8.9, p < 0.05). During follow-up (44.7 ± 14.5 months), there were no significant differences in in-hospital mortality, re-hospitalization and cardiovascular mortality between EPs and NEPs (p = NS for all). CONCLUSION: EPs with ND-ADHF have different clinical characteristics and higher LV filling pressure when compared with NEPs. However, the clinical outcomes for NEPs with ND-ADHF are not necessarily more favorable than those for EPs.ope

Down-Regulation of NF-κB Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in Drosophila

The activation of several transcription factors is required for the elimination of infectious pathogens via the innate immune response. The transcription factors NF-κB, AP-1, and STAT play major roles in the synthesis of immune effector molecules during innate immune responses. However, the fact that these immune responses can have cytotoxic effects requires their tight regulation to achieve restricted and transient activation, and mis-regulation of the damping process has pathological consequences. Here we show that AP-1 and STAT are themselves the major inhibitors responsible for damping NF-κB–mediated transcriptional activation during the innate immune response in Drosophila. As the levels of dAP-1 and Stat92E increase due to continuous immune signaling, they play a repressive role by forming a repressosome complex with the Drosophila HMG protein, Dsp1. The dAP-1–, Stat92E-, and Dsp1-containing complexes replace Relish at the promoters of diverse immune effector genes by binding to evolutionarily conserved cis-elements, and they recruit histone deacetylase to inhibit transcription. Reduction by mutation of dAP-1, Stat92E, or Dsp1 results in hyperactivation of Relish target genes and reduces the viability of bacterially infected flies despite more efficient pathogen clearance. These defects are rescued by reducing the Relish copy number, thus confirming that mis-regulation of Relish, not inadequate activation of dAP-1, Stat92E, or Dsp1 target genes, is responsible for the reduced survival of the mutants. We conclude that an inhibitory effect of AP-1 and STAT on NF-κB is required for properly balanced immune responses and appears to be evolutionarily conserved

Identification and Functional Analysis of Antifungal Immune Response Genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-κB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila

A numerical study of the ignition characteristics of nonpremixed methane/hydrogen versus air mixture within a rolled-up vortex

Department of Mechanical Engineeringclos

PhD

dissertationThe significant anti-tumor activity of 4-oxopyrido[2,3-d]-pyrimidine (1), 2,4-dioxopyrido[2,3-d]pryimidine (2) and 2-methylthio-4,5-dioxo-6carbethoxypyrido[2,3-d]pryimidine (3) prompted the development of new approaches to the synthesis of this ring system. The reagent selected for the conversion of a series of 6-aminouracil derivatives (57) to the corresponding pyrido[2,3-d]-pyrimidines was dimethyl acetylenedicarboxylate. The condensation was carried out in two different sets of reaction condition using aprotic media. The two sets of reaction condition gave, in fair yield, entirely different products. In aprotic media such as dimethyl formamide (DMF), 1, 1,2-dimethoxyethane and dioxane, the 6-aminouracil derivatives (57a-c) gave C-5 acylated compounds (58a-c) upon reaction with dimethyl acetylenedicarboxylate. The following three compounds, 1,3-dimethyl-6-aminouracil (57a), 1-methyl-6 aminouracil (57b) and 1, 3-dibenzyl-6-aminouracil (57c) in DMF gave, in fair yield, 1,3-dimethyl-6-amino-5-(3-carbomethoxy-wpropynoyl)uracil (58a), 1-methyl-6-amino-5-(3-carbomethoxy-2-propynoyl)uracil (58b( and 1,3-dibenzyl-6-amino-5-3-carbomethoxy-2-propynoy)uracil (58c) respectively. However, 6-aminouracil itself (57d) gave no such product. To resolve this apparent anomaly, acetylation of 6-aminouracil derivatives was evaluated as a model system. 1,3-Dimethyl-6-amino-uracil (57a) and 1-methyl-6-aminouracil (57b), both of which have N-1 substituents, gave 5-acetyl products (60a, 60b) whereas 6-aminouracil (57d) and 3-methyl-6-aminouracil (61), which have no N-1 substituents, yield N-6-acetyl derivatives (62am 62b). A plausible reaction mechanism for this difference in position of acylation is presented and discussed. Catalytic hydrogenation of 1,3-dimethyl-6-amino-5-(e-carbomethoxy-2-propynoyl)uracil (58a) gave the cis olefin 65. This olefin is very resistant to further reduction. The trans isomer 66 was prepared by C-t acylation of 1,3-dimethyl-6-aminouracil (57a) with methyl-3-choroformyl-tans-acrylate. The condensation of 6-aminouracil derivatives (57) with dimethyl acetylenedicarboxylate in protic media such as water and methanol produce the initially sought pyrido[2,3-d]pyrimidines. Treatment of 1,3-dimethyl-6-aminouracil (57a), 1-methyl-6-aminouracil (57a), 1-methyl-6-aminouracil (57b), 1,3-dibenzyl-6-amiouracil (57c) and 6-aminouracil (57d) with dimethyl acetylenedicarboxylate in refluxing methanol or water gave yields of substituted 2,4,5-troxo-7-carbomethoxypryido-[2,3-d]pyrimidines (67), regardless of presence of N-1 substituents. This was confirmed by establishing as identical the products (78) obtained by diazomethane methylation of both 1,3-dimethy1-2,4,5-trioxo-7-carbomethoxpyrido[2,3-d]pyrimidine (67a) and 2,4,5-troxo-7-carbomethoxyprido[2,3-d]pyrimidine (67d). 6-Aminouracil (57d) and 1, 3-dimethyl-6-aminouracil (57a) have also been shown to undergo the same type of condensation with diethyl ethoxymethylene-malonate to give 2,4,5,-trioxo-6-carbethoxypyrido [2,3-d]pyrimidine (71) and 1,3-dimethyl-2,4,5-trioxo-6-carbethoxyprido[2,3-d]-pyrimidine (72), respectively. A key intermediate, diethyl-N-(2,4-dioxo-6-pyrimidinyl)aminomethylenemalonate (70), was isolated from the reaction of 6-aminouracil (56d) with diethyl ethoxymethylene-malonate and subsequently cyclized thermally to compound 71. The structure assignment of the substituted 2,4,5-troxo-7-carbo-methoxypyrido[2, 3-d]pyrimidines (67 was established by preparing 1,3,-dimethyl-2,4,5-trioxopyrido[2,3-d]pyrimidine (80) from both of 1,3-dimethyl-2,4,5-trioxo-7-carbomethoxypyrido[2,3-d]pyrimidine (67a) and 1,3-dimethyl-2,4,5-troxo-6-carbethoxypyrido[2,3-d]-pyrimidine (72) by hydrolysis and decarboxylation. A relationship between product ratio (C-5 acylated vs. ring cyclized compound) and the effective proton concentration of reaction of reaction media in the reaction of 6-aminouracil derivatives (57) and dimethyl acetylenedicarboxylate, mechanistic considerations for the pyrido[2,3-d]pyrimidine ring cyclization and the pmr spectra of the new compounds synthesized were discussed in detai