The objective of this prospective study was a blinded comparison of three methods for the identification of bacteria isolated on Columbia blood agar from milk samples of dairy cows. Basic biochemical ...testing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and 16S rRNA partial genome sequence analysis were compared for bacterial identification to the genus or species level. Milk samples submitted from a commercial dairy farm from recently calved cows or clinical mastitis cases were cultured, and 181 isolates were identified by biochemical testing, MALDI-TOF MS, and 16S rRNA sequence analysis (179 isolates; 2 isolates could not be recovered from storage). For
and
, agreement was determined at the species level. For other microbes, agreement was determined at the genus level or at the group level for streptococcus-like organisms. The positive agreement among all 3 diagnostic methods was 94%, with 95% to 98% between each pair of methods. The overall (including negative agreement) agreement among all 3 methods ranged from 97% to 100%. MALDI-TOF MS is becoming more commonplace for the genus- and/or species-level identification of bacteria isolated from milk samples and, in some laboratories, has replaced conventional biochemical methods. The results of the present study suggest that when identifying pathogens at the genus or group level, conventional culture followed up with either secondary biochemical testing or MALDI-TOF MS is of practical value. For purposes of milk quality and udder health monitoring or research, any of the 3 methods is a valuable tool for genus-level identification of bacteria isolated from dairy cow milk.
A series of substituted xanthenes was synthesized and screened for activity using DU-145, MCF-7, and HeLa cancer cell growth inhibition assays. The most potent compound, 9g ...(N,N-diethyl-9-hydroxy-9-(3-methoxyphenyl)-9H-xanthene-3-carboxamide), was found to inhibit cancer cell growth with IC50 values ranging from 36 to 50μM across all three cancer cell lines. Structure–activity relationship (SAR) data is presented that indicates additional gains in potency may be realized through further derivatization of the compounds (e.g., the incorporation of a 7-fluoro substituent to 9g). Results are also presented that suggest the compounds function through a unique mechanism of action as compared to that of related acridine and xanthone anticancer agents (which have been shown to intercalate into DNA and inhibit topoisomerase II activity). A structural comparison of these compounds suggests the differences in function may be due to the structure of the xanthene heterocycle which adopts a nonplanar conformation about the pyran ring.
We have developed an automated photochemical microfluidics platform that integrates a 1 kW high-pressure Hg vapor lamp and allows for analytical pulse flow or preparative continuous flow reactions. ...Herein, we will discuss the use of this platform toward the discovery of new chemotypes through multidimensional reaction screening. We will highlight the ability to discretely control wavelengths with optical filters, allowing for control of reaction outcomes.
An automated, silicon-based microreactor system has been developed for rapid, low-volume, multidimensional reaction screening. Use of the microfluidic platform to identify transformations of densely ...functionalized bicyclo3.2.1octanoid scaffolds will be described.
Silyloxypyrone-based (5+2) cycloadditions were facilitated by amides that allowed for increased reactivity and a pathway for cleaving the tether to afford net intermolecular cycloadducts. Various ...amides underwent facile cycloaddition, and several experiments revealed steric and electronic factors that accelerate the reaction. tert-Butyl amides reacted faster than less hindered variants in multiple cases. In the case of dearomative oxidopyrylium-indole (5+2) cycloadditions, an amine-based tether was ineffective, whereas amides enabled this powerful transformation. Theoretical calculations evidenced a concerted asynchronous reaction in which the amide facilitates a conformational driving force enabling cycloaddition. Finally, a one-pot acylation/(5+2) cycloaddition/nucleophilic lactam opening and other examples of tosyl lactam opening of a modified cycloadduct were demonstrated.
The reaction pathway of silyloxypyrone-based (5 + 2) cycloadditions was determined to be extremely dependent on the nature of the dipolarophile. Neutral alkenes were the least reactive, whereas both ...electron-deficient and electron-rich dipolarophiles were more reactive, thus providing evidence for ambident oxidopyrylium intermediates. Qualitative rate studies, Hammett linear free energy relationships, and theoretical calculations combined to provide evidence for a spectrum of reactivity that passes through the borderlands of concerted and stepwise.
Systematic investigation of intramolecular silyloxypyrone-based 5 + 2 cycloadditions revealed three significant factors impacting conversion to cycloadduct: (1) the silyl transfer group has a ...substantial influence on the rate of reaction, and the robust t-butyldiphenylsilyl group was found to be more effective overall than the conventional t-butyldimethylsilyl group; (2) α,β-unsaturated esters were generally more reactive than terminal olefins and afforded appreciable quantity of cycloadduct even at room temperature; and (3) the proximity of the tether to the silyl transfer group revealed a critical alignment trend between the pyrone and the alkene. Taken together, these investigations provided insight regarding the steric and electronic parameters that impact the scope and limitation of these reactions.
Oxidopyrylium-alkene 5 + 2 cycloaddition conjugate addition cascade (C3) sequences are described. Intramolecular cycloadditions involving terminal alkenes, enals, and enones were investigated. ...Substrates with tethers of varying lengths delivered five- and six-membered carbocycles and heterocycles thus demonstrating the scope and limitation of the cycloaddition–conjugate addition cascade. Several experiments and theoretical calculations provide evidence for the proposed mechanistic pathway.
A series of substituted 9-amino acridines inhibit topoisomerase II relaxation of DNA suggesting a mechanism similar to that of the anticancer drug aclarubicin.
The discovery of new non-nucleoside ...antiviral compounds is of significant and growing interest for treating herpes virus infections due to the emergence of nucleoside-resistant strains. Using a whole cell virus-induced cytopathogenic assay, we tested a series of substituted triaryl heterocyclic compounds including acridones, xanthones, and acridines. The compounds which showed activity against Herpes Simplex-1 and/or Herpes Simplex-2 were further assayed for inhibition of topoisomerase activity to gain insight into the mechanism of action. The results indicate that the acridine analogs bearing substituted carboxamides and bulky 9-amino functionalities are able to inhibit herpes infections as well as inhibit topoisomerase II relaxation of supercoiled DNA. Given the mechanism of action of amsacrine (a closely related, well-studied 9-amino substituted acridine), the compounds were further tested in a DNA topoisomerase II cleavage assay to determine if the compounds function as poisons. The results show that the acridines synthesized in this study function through a different mechanism to that of amsacrine, most likely by blocking topoisomerase binding to DNA (akin to that of aclarubicin). This not only suggests a unique mechanism of action in treating herpes virus infections, but also may be of great interest in the development of anticancer agents that target topoisomerase II activity.
The lack of antiviral compounds targeting flaviviruses represents a significant problem in the development of strategies for treating West Nile Virus (WNV), Dengue, and Yellow Fever infections. Using ...WNV high-throughput screening techniques developed in our laboratories, we report the identification of several small molecule anti-WNV compounds belonging to four different structural classes including pyrazolines, xanthanes, acridines, and quinolines. The initial set of “hits” was further refined using cell viability-cytotoxicity assays to two 1,3,5-triaryl pyrazoline compounds: 1-(4-chlorophenylacetyl)-5-(4-nitrophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole and 1-benzoyl-5-(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole. On the basis of their activity and favorable therapeutic indexes, these compounds were identified as viable leads and subjected to additional evaluation using an authentic viral titer reduction assay employing an epidemic strain of WNV. The compounds were further evaluated in a transient replicon reporting system to gain insight into the mechanism of action by identifying the step at which inhibition takes place during viral replication. The results indicate the pyrazolines inhibit RNA synthesis, pointing to viral RNA polymerase, RNA helicase, or other viral replication enzymes as potential targets. Progress was also made in understanding the structural requirements for activity by synthesizing a focused chemical library of substituted pyrazolines. Preliminary SAR data are presented that show the aryl-rings are required for activity against WNV. More importantly, the results indicate WNV activity is tolerant to aryl-substitutions paving the way for the design and development of much larger combinatorial libraries with varied physicochemical properties.
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