Development of a Staphylococcus aureus reporter strain with click beetle red luciferase for enhanced in vivo imaging of experimental bacteremia and mixed infections.

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 104 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.

Experimental and Theoretical Studies on the Aqueous Solvation and Reactivity of SmCl2 and Comparison with SmBr2 and SmI2.

Water addition to Sm(II) has been shown to increase reactivity for both SmI2 and SmBr2. Previous work in our groups has demonstrated that this increase in reactivity can be attributed to coordination induced bond weakening enabling substrate reduction through proton-coupled electron transfer. The present work examines the interaction of water with samarium dichloride (SmCl2) and illustrates the importance of the Sm-X interaction and bond distance upon water addition critical for the reactivity of the reagent system. Born-Oppenheimer molecular dynamics simulations identify substantial variations among the reductants created in solution upon water addition to SmI2, SmBr2, and SmCl2 with the latter showing the least halide dissociation. This results in a lower water coordination number for SmCl2, creating a more powerful reducing system. As previously shown with the other SmX2-water systems, coordination-induced bond-weakening of the O-H bond of water bound to Sm(II) results in significant bond weakening. In the case of SmCl2, the bond weakening is estimated to be in the range of 83 to 88.5 kcal/mol.

Interplay between Substrate and Proton Donor Coordination in Reductions of Carbonyls by SmI2-Water Through Proton-Coupled Electron-Transfer.

The reduction of a carbonyl by SmI2-water is the first step in a range of reactions of synthetic importance. Although the reduction is often proposed to proceed through an initial stepwise electron-transfer-proton-transfer (ET-PT), recent work has shown that carbonyls and related functional groups are likely reduced though proton-coupled electron-transfer (PCET). In the present work, the reduction of an activated ester, aldehyde, a linear and cyclic ketone, and related sterically demanding carbonyls by SmI2-H2O was examined through a series of mechanistic experiments. Kinetic studies demonstrate that all substrates exhibit significant increases in the rate of reduction by SmI2 as [H2O] is increased. Under identical conditions, ketones and an aldehyde containing a methyl adjacent to the carbonyl are reduced slower than an unsubstituted variant by an order of magnitude, demonstrating the importance of substrate coordination. In the case of unactivated substrates, rates of reduction show excellent correlation with the calculated bond dissociation free energy of the O-H bond of the intermediate ketyl and the calculated free energy of intermediate ketyl radical anions derived from unhindered substrates: findings consistent with concerted PCET. Activated esters derived from methylbenzoate are likely reduced through stepwise or asynchronous PCET. Overall, this work demonstrates that the combination of the coordination of substrate and water to Sm(II) provides a configuration uniquely suited to a coupled electron- and proton-transfer process.

Experimental and Theoretical Studies on the Implications of Halide-Dependent Aqueous Solvation of Sm(II).

The addition of water to samarium(II) has been demonstrated to have a significant impact on the reduction of organic substrates, with the majority of research dedicated to the most widely used reagent, samarium diiodide (SmI2). The work presented herein focuses on the reducing capabilities of samarium dibromide (SmBr2) and demonstrates how the modest change in halide ligand results in observable mechanistic differences between the SmBr2-water and the SmI2-water systems that have considerable implications in terms of reactivity between the two reagents. Quantum chemical results from Born-Oppenheimer molecular dynamics simulations show significant differences between SmI2-water and SmBr2-water, with the latter displaying less dissociation of the halide, which results in a lower coordination number for water. Experimental results are consistent with computational results and demonstrate that the coordination sphere of SmBr2 is saturated at lower concentrations of water. In addition, coordination-induced bond-weakening of the O-H bond is demonstrably different for water bound to SmBr2, leading to an estimated O-H bond-weakening of at least 83 kcal/mol, nearly 10 kcal/mol larger than the bond-weakening observed in SmI2-H2O. Experimental results also demonstrate that the use of alcohols in place of water with SmBr2 leads to substrate reduction, albeit several orders of magnitude slower than for SmBr2-water. The difference in rates resulting from the change in proton donor is attributed to a rate-limiting proton-coupled electron transfer in SmBr2-water and a sequential electron transfer then proton transfer in SmBr2-alcohol systems, where electron transfer is rate-limiting.

Secondary Amides as Hydrogen Atom Transfer Promoters for Reactions of Samarium Diiodide.

Two secondary amides (N-methylacetamide and 2-pyrrolidinone) were used as additives with SmI2 in THF to estimate the extent of N-H bond weakening upon coordination. Mechanistic and synthetic studies demonstrate significant bond-weakening, providing a reagent system capable of reducing a range of substrates through formal hydrogen atom transfer.

Proton-Coupled Electron Transfer in the Reduction of Carbonyls by Samarium Diiodide-Water Complexes.

Reduction of carbonyls by SmI2 is significantly impacted by the presence of water, but the fundamental step(s) of initial transfer of a formal hydrogen atom from the SmI2-water reagent system to produce an intermediate radical is not fully understood. In this work, we provide evidence consistent with the reduction of carbonyls by SmI2-water proceeding through proton-coupled electron transfer (PCET). Combined rate and computational studies show that a model aldehyde and ketone are likely reduced through an asynchronous PCET, whereas reduction of a representative lactone occurs through a concerted PCET. In the latter case, concerted PCET is likely a consequence of significantly endergonic initial electron transfer.

High-Affinity Proton Donors Promote Proton-Coupled Electron Transfer by Samarium Diiodide.

The relationship between proton-donor affinity for Sm(II) ions and the reduction of two substrates (anthracene and benzyl chloride) was examined. A combination of spectroscopic, thermochemical, and kinetic studies show that only those proton donors that coordinate or chelate strongly to Sm(II) promote anthracene reduction through a PCET process. These studies demonstrate that the combination of Sm(II) ions and water does not provide a unique reagent system for formal hydrogen atom transfer to substrates.

Detection of monofluoroacetate in Palicourea and Amorimia species.

Numerous plant species worldwide including Palicourea marcgravii and Tanaecium bilabiatum in Brazil cause sudden death and are known to contain monofluoroacetate (MFA). Other species in Brazil including some species traditionally assigned to Mascagnia but now properly called Amorimia species and other Palicourea species are reported to cause sudden death in livestock and are suspected to contain MFA due to the similarity of clinical signs. In this study, an HPLC-APCI-MS method to detect and quantify MFA was developed and was used to investigate plant material from field collections and/or herbarium specimens of Mascagnia, Amorimia, and Palicourea species suspected of causing sudden death. MFA was detected in Amorimia amazonica, Amorimia camporum, Amorimia exotropica, Amorimia pubiflora, Amorimia rigida, and Amorimia septentrionalis as well as Palicourea aeneofusca. MFA concentrations differ greatly between Palicourea species and Amorimia species, which may explain the incidence of poisoning and the amount of plant material required to cause sudden death between these taxa.

A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology.

PREMISE OF THE STUDY: The Malpighiaceae include ∼1300 tropical flowering plant species in which generic definitions and intergeneric relationships have long been problematic. The goals of our study were to resolve relationships among the 11 generic segregates from the New World genus Mascagnia, test the monophyly of the largest remaining Malpighiaceae genera, and clarify the placement of Old World Malpighiaceae. • METHODS: We combined DNA sequence data for four genes (plastid ndhF, matK, and rbcL and nuclear PHYC) from 338 ingroup accessions that represented all 77 currently recognized genera with morphological data from 144 ingroup species to produce a complete generic phylogeny of the family. • KEY RESULTS AND CONCLUSIONS: The genera are distributed among 14 mostly well-supported clades. The interrelationships of these major subclades have strong support, except for the clade comprising the wing-fruited genera (i.e., the malpighioid+Amorimia, Ectopopterys, hiraeoid, stigmaphylloid, and tetrapteroid clades). These results resolve numerous systematic problems, while others have emerged and constitute opportunities for future study. Malpighiaceae migrated from the New to Old World nine times, with two of those migrants being very recent arrivals from the New World. The seven other Old World clades dispersed much earlier, likely during the Tertiary. Comparison of floral morphology in Old World Malpighiaceae with their closest New World relatives suggests that morphological stasis in the New World likely results from selection by neotropical oil-bee pollinators and that the morphological diversity found in Old World flowers has evolved following their release from selection by those bees.

Gene transfer from a parasitic flowering plant to a fern.

The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts.

Accuracy of Nanoscale Pitch Standards Fabricated by Laser-Focused Atomic Deposition.

The pitch accuracy of a grating formed by laser-focused atomic deposition is evaluated from the point of view of fabricating nanoscale pitch standard artifacts. The average pitch obtained by the process, nominally half the laser wavelength, is simply traceable with small uncertainty to an atomic frequency and hence can be known with very high accuracy. An error budget is presented for a Cr on sapphire sample, showing that a combined standard uncertainty of 0.0049 nm, or a relative uncertainty of 2.3 × 10(-5), is readily obtained, provided the substrate temperature does not change. Precision measurements of the diffraction of the 351.1 nm argon ion laser line from such an artifact are also presented. These yield an average pitch of (212.7777 ± 0.0069) nm, which agrees well with the expected value, as corrected for thermal contraction, of (212.7705 ± 0.0049) nm.

Epirubicin and meglumine gamma-linolenic acid: a logical choice of combination therapy for patients with superficial bladder carcinoma.

BACKGROUND: Anthracyclines have been established as first-line drugs for intravesical use in the treatment of patients with superficial bladder carcinoma, although they result only in a modest reduction in tumor recurrence rates. The essential fatty acid gamma-linolenic acid (GLA) also is an effective cytotoxic agent against superficial bladder carcinoma when it is applied topically. The objective of this study was to assess the efficacy of combined epirubicin and GLA with the purpose of developing a suitable model for modification of existing intravesical regimens. METHODS: The human urothelial carcinoma cell lines MGH-U1 and RT112 were used in standard cytotoxicity assays and were exposed to meglumine GLA (MeGLA) and epirubicin in two-dimensional concentration matrices. A thiozolyl blue (methyl-thiazoldiphenyl tetrazolium) assay was used to determine residual cell biomass. Drug interaction was quantified by median-effect analysis software (CalcuSyn), and the evaluation of drug uptake utilized fluorescence confocal microscopy (FCM) and flow cytometry. RESULTS: MeGLA caused a significant enhancement of anthracycline uptake, viewed by FCM, from 92 fluorescence units to 222 fluorescence units (P < 0.001). Flow cytometry confirmed the increased drug uptake and showed that the mean epirubicin content per cell increased from 23 to 57 units and from 8 to 24 units for MGH-U1 and RT112 cells, respectively (99% confidence interval < 0.3). This resulted in improved cytotoxicity, and it was shown that the drugs acted synergistically with all but the highest MeGLA concentrations. CONCLUSIONS: The efficacy of epirubicin was enhanced significantly when it was used in combination with most concentrations of MeGLA (< 300 microg/mL), and the two agents acted synergistically. There was a corresponding increase in epirubicin uptake by cells under these conditions. At high MeGLA concentrations, however, anthracycline solubility was compromised, and drug synergy was lost.

Occurrence and phylogenetic significance of latex in the Malpighiaceae.

Latex and laticifers are reported for the first time in the genera Galphimia and Verrucularia (Malpighiaceae), with description and illustration of the leaf and stem anatomy of both genera. Those genera and the other two in which latex is known (Lophanthera and Spachea) constitute a single tribe, Galphimieae, that is at or near the base of the family's phylogeny, which suggests that latex in the Malpighiaceae may indicate an ancestor shared with the Euphorbiaceae.