Synthesis and Biological Evaluation of Novel Chromogenic Substrates for the Enhanced Detection of Pathogenic Bacteria

The present work investigated the preparation of phenoxazinone derivatives and evaluated their performances for the detection of pathogenic bacteria. The first method investigated the condensation of nitroaminophenol with tetrahalogenobenzoquinones; the corresponding nitrohalogenophenoxazinones were all characterised and evaluated for the detection of nitroreductase activity in a range of clinically important microorganisms. The detection of nitroreductase activity has been previously suggested for the monitoring of bacterial growth; however, nitrohalogenophenoxazinones were proven to be less suitable for this purpose than other, previously reported, nitroreductase substrates. The second route investigated the synthesis of phenoxazinone derivatives via the oxidative cyclisation of diamino-dihydroxydiphenylethers and of diaminobenzoquinones. The reactive intermediates were trapped and characterised in order to rationalize the mechanism of formation of aminophenoxazinones via this route. 7- and 8-Aminophenoxazinones derivatives were prepared and further derivatised with b-alanine. Similarly, some nitrohalogenophenoxazinones were reduced to their corresponding aminophenoxazinones and derivatised with b-alanine. Thirteen new chromogenic substrates were prepared, characterised and evaluated for their sensitivity to detect b-alanine aminopeptidase on agar medium; this enzyme is expressed by only three types of bacteria, the most important being Pseudomonas aeruginosa, a pathogen commonly known to affect cystic fibrosis sufferers. Their performance for the detection of Pseudomonas aeruginosa were compared to the lead compound (7-N-(b-alanyl)amino-1-pentylphenoxazin-3-one), the substrate contained in a commercially available medium, chromIDTM ID Ps. aeruginosa. The substrates, if hydrolysed, resulted in a low colouration of the colonies when compared to the lead compound; however, 2-pentyl substituted aminophenoxazinones were found to be less toxic and had an excellent affinity for the bacterial colonies.

Synthesis and evaluation of halogenated nitrophenoxazinones as nitroreductase substrates for the detection of pathogenic bacteria

The synthesis and microbiological evaluation of 7-, 8- and 9-nitro-1,2,4-trihalogenophenoxazin-3-one substrates with potential in the detection of nitroreductase-expressing pathogenic microorganisms are described. The 7- and 9-nitrotrihalogenophenoxazinone substrates were reduced by most Gram negative microorganisms and were inhibitory to the growth of certain Gram positive bacteria; however, the majority of Gram positive strains that were not inhibited by these agents, along with the two yeast strains evaluated, did not reduce the substrates. These observations suggest there are differences in the active site structures and substrate requirements of the nitroreductase enzymes from different strains; such differences may be exploited in the future for differentiation between pathogenic microorganisms. The absence of reduction of the 8-nitrotrihalogenophenoxazinone substrates is rationalized according to their electronic properties and correlates well with previous findings

Phosphonopeptides Revisited, in an Era of Increasing Antimicrobial Resistance

Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in the context of widespread resistance to conventional agents. In the 1970s, much work was performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin. We investigated the activity of alafosfalin, di-alanyl fosfalin and β-chloro-L-alanyl-β-chloro-L-alanine against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128), methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci (GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE. Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis (MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active against MRSA (MIC90; 8 mg/L), whereas β-chloro-L-alanyl-β-chloro-L-alanine was slightly more active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have a therapeutic role to play in an era of increasing antibacterial resistance

Synthesis and evaluation of novel 7- and 8-aminophenoxazinones for the detection of β-alanine aminopeptidase activity and the rapid identification of Pseudomonas aeruginosa in clinical samples

A series of novel 8-aminophenoxazin-3-one and 7-aminophenoxazin-3-one chromogens and their corresponding β-alanine derivatives were synthesized and evaluated for their ability to detect β-alanyl aminopeptidase activity in bacteria known to hydrolyse β-alanine derivatized substrates. The results provided insight into the structural requirements for effective visualization of enzymatic activity and the mechanism of formation of phenoxazinon-3-ones. 8-Aminophenoxazin-3-one substrates 23c, 23d and 23e were prepared in good to high overall yield and were selective for β-alanyl aminopeptidase activity in bacteria, producing a lighter agar background coloration facilitating visualization of colored colonies, with variable localization to the colonies, but had lower sensitivities for the detection of Pseudomonas aeruginosa in comparison to the analogous 7-aminophenoxazin-3-one substrates. The synthetic methodology employed here allows the preparation of a range of substrates for evaluation and the establishment of structure-activity relationships. For example, the 2-pentyl substituted aminophenoxazin-3-one 22b performed with analogous sensitivity to the corresponding 1-pentyl-7-aminophenoxazin-3-one substrate 1 used commercially, highlighting that the position of the pentyl substituent can be varied while maintaining detection sensitivity

Efficacy and safety of a 30-day methylprednisolone treatment protocol for subacute thyroiditis: a prospective study

Objective: The optimal corticosteroid treatment regimen for subacute thyroiditis has not yet been established. To avoid side effects, tapering of the initial dose of corticosteroid is recommended. With reducing dose, the symptoms can recur. Design: In a prospective clinical study, a 30-day methylprednisolone (MPSL) treatment protocol with a starting dose of 24 mg/day and tapered by 4 mg every 5 days was assessed for effectiveness and safety regarding possible adrenal insufficiency. Methods: Fifty-nine patients with subacute thyroiditis were included. At visit 1, after establishing the diagnosis, a short stimulation adrenocorticotrophic hormone (ACTH) test was performed and methylprednisolone treatment was prescribed. At visit 2 (40 ± 5 days after visit 1), clinical, laboratory (including short stimulation ACTH test), and ultrasound evaluation were repeated. Results: Forty-eight patients (81.4%) were cured by the prescribed protocol, having significantly lower cortisol levels after stimulation at visit 1 than patients who were not cured (mean, 674.9 nmol/L and 764.0 nmol/L, respectively, P = 0.012). Seven patients (12.3%) developed adrenal insufficiency; this group had significantly lower cortisol levels after stimulation at visit 1 than patients without adrenal insufficiency development (mean, 561.5 nmol/L and 704.7 nmol/L, respectively, P = 0.005). Using stimulated cortisol level at visit 1 as the explanatory variable, logistic models were optimized to determine treatment efficacy (AUC = 0.745, optimal threshold 729 nmol/L, specificity 71%, sensitivity 73%) and adrenal function (AUC = 0.861, optimal threshold 629 nmol/L, specificity 73%, sensitivity 100%). Conclusions: The described protocol was efficient for more than 80% of patients. Using this protocol, the corticosteroid treatment interval is shorter than proposed in current guidelines. Significance statement: A short but effective protocol for treatment of subacute thyroiditis with methylprednisolone is presented in this article. Using this protocol, the treatment interval is shorter than proposed in current guidelines. Its safety regarding possible adrenal insufficiency is assessed

Discovery of sisunatovir (RV521), an inhibitor of respiratory syncytial virus fusion

RV521 is an orally bioavailable inhibitor of respiratory syncytial virus (RSV) fusion that was identified after a lead optimization process based upon hits that originated from a physical property directed hit profiling exercise at Reviral. This exercise encompassed collaborations with a number of contract organizations with collaborative medicinal chemistry and virology during the optimization phase in addition to those utilized as the compound proceeded through preclinical and clinical evaluation. RV521 exhibited a mean IC50 of 1.2 nM against a panel of RSV A and B laboratory strains and clinical isolates with antiviral efficacy in the Balb/C mouse model of RSV infection. Oral bioavailability in preclinical species ranged from 42 to >100% with evidence of highly efficient penetration into lung tissue. In healthy adult human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a significant reduction in viral load and symptoms compared to placebo

Die Lehrerreflexion des Unterrichts

Diplomsko delo obravnava učiteljevo refleksijo pouka in njeno razvitost oziroma razširjenost v praksi. V teoretičnem delu so predstavljena temeljna teoretična pojmovanja, opredeljen je tudi pojem refleksija ter so opisani njena vloga, pomen, vrste in pristopi, ki se pojavljajo v izobraževalnem procesu. Predstavljene so tudi ugotovitve empiričnega raziskovanja refleksije pouka v neposredni vzgojno-izobraževalni praksi, ki osvetljujejo pogostost, način, korist in vsebino te dejavnosti učiteljev, razlike glede na delovno dobo učiteljev in razlike glede zaposlitev učiteljev v osnovni ali srednji šoli.Meine Diplomarbeit behandelt die Lehrerreflexion des Unterrichts und wie sie in der Praxis entwickelt bzw. verbreitet ist. In dem theoretischen Teil meiner Diplomarbeit sind die grundlegenden theoretischen Auffassungen und die Bestimmung des Begriffs Reflexion, ihre Rolle und Bedeutung, verschiedene Arten und Ansätze der Reflexion, die im Bildungsprozess vorkommen, vorgestellt. In der Diplomarbeit sind auch die Feststellungen der empirischen Forschung der Reflexion des Unterrichts in dem unmittelbaren Erziehungs-und-Unterrichtswesen dargestellt. Sie erhellen die Häufigkeit, die Art und Weise, den Nutzen und den Inhalt dieser Lehrertätigkeit, die Unterschiede in Hinsicht auf die Lebensarbeitszeit der Lehrer und die Unterschiede in Hinsicht auf die Beschäftigung der Lehrer in der Grundschule oder Mittelschule

AUSWIRKUNGEN DIDAKTISCHER SPIELE AUF DAS GRAMMATIKLERNEN IM DAF-UNTERRICHT

Moja diplomska naloga obravnava vplive didaktičnih iger na učenje slovnice pri nemškem jeziku kot tujem jeziku in primerjavo učnih ur z oziroma brez uporabe didaktičnih iger. V teoretičnem delu so osvetljena temeljna teoretična spoznanja, definicija termina didaktična igra, njeni elementi, funkcije ter igre, ki so primerne za poučevanje slovnice. Na kratko sem pojasnila tudi termin trpnika, ker sem to slovnično kategorijo poučevala v razredu. V diplomski nalogi so zapisana tudi spoznanja, pridobljena na podlagi praktične raziskave. Vsebujejo ugotovitve o uporabnosti didaktičnih iger pri poučevanju slovnice in rezultate primerjav s klasičnim poukom. Tako najdete odgovore na vprašanja glede uporabe, priljubljenosti in uspešnosti didaktičnih iger pri poučevanju slovnice.Meine Diplomarbeit behandelt die Auswirkungen didaktischer Spiele auf das Grammatiklernen im Daf-Unterricht und eine Gegenüberstellung des Unterrichts mit und ohne didaktische Spiele anhand einer praktischen Durchführung. Im theoretischen Teil meiner Diplomarbeit sind die grundlegenden theoretischen Auffassungen und die Bestimmung des Begriffs didaktische Spiele, ihre Elemente, Funktionen, Spiele, die für den Grammatikunterricht geeignet sind, und die kurze Behandlung des Passivs, den ich für den praktischen Teil meiner Diplomarbeit unterrichtet habe, angeführt. In meiner Diplomarbeit sind auch die Feststellungen der praktischen Forschung über die didaktischen Spiele im Unterricht dargestellt. Sie erhellen den Gebrauch der didaktischen Spiele beim Grammatikunterricht und die Unterschiede gegenüber dem klassischen Unterricht basierend auf einem Test und Fragebogen. So werden die Fragen über den Gebrauch, die Beliebtheit und den Erfolg der didaktischen Spiele beim Unterricht beantwortet

SUCCESSIVE INFECTIONS WITH DIFFERENT AGENTS IN 76-YEARS OLD PATIENT

Background. A case comprising six successive infections in period lasting from September 12, 1999 to November 23, 1999 in 76-years old female is presented. The etiological agents were confirmed in/with the first four infections but with the last two the purulent meningitis and septic shock infection, blood and liquor cultures were negative. We presume that this was caused by antibiotic therapy before her second admission in the hospital and because of intensive antibiotic therapy of purulent meningitis. The first five infections were successfully treated: enteritis caused by Clostridium difficile, sepsis caused by Salmonella enteritidis, candidiasis that appeared during the therapy of both sepsis, Campylobacter jejuni infection and purulent meningitis. The number of leukocytes and granulocytes fell under the normal values in all infections and during the recovery their increase could be observed. The patient died in septic shock, with acute renal failure, with bleedings in the lung, in the skin, in the urinary tract and in the brain.Conclusions. The leukopenia and the granulocytopenia are themselves the signs of serious infections. They accelerate the appearance of new infections and they reduce the success of therapy. The expected mortality total rate for this six infections is very high. The massive bleedings in the brain are lethal.</p