Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).     

A unique automation platform for measuring low level radioactivity in metabolite identification studies

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using (14)C or (3)H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector.     

Predicting the epidemic sizes of influenza A/H1N1, A/H3N2, and B: a statistical method

Background

The epidemic sizes of influenza A/H3N2, A/H1N1, and B infections vary from year to year in the United States. We use publicly available US Centers for Disease Control (CDC) influenza surveillance data between 1997 and 2009 to study the temporal dynamics of influenza over this period.

Methods and Findings

Regional outpatient surveillance data on influenza-like illness (ILI) and virologic surveillance data were combined to define a weekly proxy for the incidence of each strain in the United States. All strains exhibited a negative association between their cumulative incidence proxy (CIP) for the whole season (from calendar week 40 of each year to calendar week 20 of the next year) and the CIP of the other two strains (the complementary CIP) from the start of the season up to calendar week 2 (or 3, 4, or 5) of the next year. We introduce a method to predict a particular strain''s CIP for the whole season by following the incidence of each strain from the start of the season until either the CIP of the chosen strain or its complementary CIP exceed certain thresholds. The method yielded accurate predictions, which generally occurred within a few weeks of the peak of incidence of the chosen strain, sometimes after that peak. For the largest seasons in the data, which were dominated by A/H3N2, prediction of A/H3N2 incidence always occurred at least several weeks in advance of the peak.

Conclusion

Early circulation of one influenza strain is associated with a reduced total incidence of the other strains, consistent with the presence of interference between subtypes. Routine ILI and virologic surveillance data can be combined using this new method to predict the relative size of each influenza strain''s epidemic by following the change in incidence of a given strain in the context of the incidence of cocirculating strains. Please see later in the article for the Editors'' Summary     

A Highlights from MBoC Selection: Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology

The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall.     

Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid beta-peptide cleaved from a recombinant fusion protein

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).     

Human proximity and habitat fragmentation are key drivers of the rangewide bonobo distribution

Habitat loss and hunting threaten bonobos (Pan paniscus), Endangered (IUCN) great apes endemic to lowland rainforests of the Democratic Republic of Congo. Conservation planning requires a current, data-driven, rangewide map of probable bonobo distribution and an understanding of key attributes of areas used by bonobos. We present a rangewide suitability model for bonobos based on a maximum entropy algorithm in which data associated with locations of bonobo nests helped predict suitable conditions across the species’ entire range. We systematically evaluated available biotic and abiotic factors, including a bonobo-specific forest fragmentation layer (forest edge density), and produced a final model revealing the importance of simple threat-based factors in a data poor environment. We confronted the issue of survey bias in presence-only models and devised a novel evaluation approach applicable to other taxa by comparing models built with data from geographically distinct sub-regions that had higher survey effort. The model’s classification accuracy was high (AUC = 0.82). Distance from agriculture and forest edge density best predicted bonobo occurrence with bonobo nests more likely to occur farther from agriculture and in areas of lower edge density. These results suggest that bonobos either avoid areas of higher human activity, fragmented forests, or both, and that humans reduce the effective habitat of bonobos. The model results contribute to an increased understanding of threats to bonobo populations, as well as help identify priority areas for future surveys and determine core bonobo protection areas.     

A silicone polymer as a Steroid Reservoir for enzyme-catalyzed Steroid reactions

Since steroids are only slightly soluble in the aqueous solutions in which enzymatic reactions take place, it is difficult to obtain high effective concentrations per unit reactor volume when enzymes are used to catalyze steroid reactions. In order to obtain high effective concentrations in the present work, we have used small particles of a hydrophobic polymer, poly (dimethyl siloxane), as a reservoir for the steroid substrate and product. The activity of a bacterial hydroxysteroid dehydrogenase in a buffer solution declines much more slowly in the presence of those polymer particles than in the presence of a comparable amount of butyl acetate or ethyl acetate, the organic solvents used as steroid reservoirs in previous work with steroid transforming enzymes. When another substrate of the hydroxysteroid dehydrogenase is loaded into the polymer particles and the particles are suspended in an aqueous solution containing the enzyme and its cofactor, more product is formed that when a similar solution is emulsified with butyl acetate.     

Heritability of morningness-eveningness and self-report sleep measures in a family-based sample of 521 hutterites

Individual variation in the phase and amplitude of human circadian rhythms is well known, but the impact of heritable factors on such variation is less clear. We estimated the narrow-sense heritability for selected circadian and sleep timing, quality, and duration measures among related members of the Hutterites, an endogamous, religious community (n=521 participants). “Morningness-eveningness” (M/E), a stable trait reflecting circadian phase, was evaluated using the Composite Scale (CS). Subjective sleep measures were assessed using the Sleep Timing Questionnaire. Initial analyses reconfirmed the impact of age on M/E. Previously reported correlations between M/E scores and the sleep measures were also noted, demonstrating the construct validity of the questionnaires among the participants. Following corrections for age, gender, and colony of residence, significant narrow-sense heritability was noted for M/E (23%). The heritability for subjective sleep measures (related to timing, duration, and quality) were statistically significant for all but one variable, and varied between 12.4% and 29.4%. Thus, significant heritable influences on human circadian phase and subjective sleep indices can be detected through family-based studies. In view of the impact of circadian malfunction on human health, it may be worthwhile to map genetic factors impacting circadian and sleep variation.