Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation.

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.

Experimentally Induced Dieback Conditions Limit Phragmites australis Growth.

Phragmites australis is a cosmopolitan grass species common in wetland ecosystems across the world. In much of North America, the non-native subspecies of Phragmites threatens wetland biodiversity, hinders recreation, and is a persistent problem for natural resource managers. In other parts of the world, populations are in decline, as Reed Die-Back Syndrome (RDBS) plagues some Phragmites stands in its native range. RDBS is defined by a clumped growth form, stunted root and shoot growth, premature senescence, and shoot death. RDBS has been associated with a build-up of short-chain fatty acids (SCFAs) and altered bacterial and oomycete communities in soils, but the exact causes are unknown. To control invasive Phragmites populations, we sought to develop treatments that mimic the conditions of RDBS. We applied various SCFA treatments at various concentrations to mesocosm soils growing either Phragmites or native wetland plants. We found that the high-concentration SCFA treatments applied weekly induced strong significant declines in above- and belowground biomass of Phragmites. Declines were significant but slightly weaker in native species. In addition, soil bacterial abundance increased, diversity decreased, and bacterial community composition significantly differed following treatments, such that treated pots maintained a higher relative abundance of Pseudomonadaceae and fewer Acidobacteriaceae than untreated pots. Our results suggest that application of SCFAs to Phragmites can lead to stunted plants and altered soil bacterial communities similar to populations affected by RDBS. However, the lack of species-specificity and intensive application rate may not make this treatment ideal as a widespread management tool.

The COVID-19 pandemic: resilient organisational response to a low-chance, high-impact event.

The global healthcare sector is currently in the midst of the COVID-19 pandemic, a 'low-chance, high-impact' event which will require healthcare systems, and the organisations within them, to maintain organisational resilience in order to respond effectively. However, contrary to the instinctive reaction to tighten control, the quality of response depends on healthcare systems' capacity to loosen control and, subsequently, enhance improvisation. Three factors critical to enhancing an organisation's capacity for improvisation are highlighted; increasing autonomy, maintaining structure and creating a shared understanding. By drawing on the case of Christchurch Hospital's response to a major earthquake, this paper demonstrates the vital role that improvisation can play within a clinical setting, when responding to a low-chance, high-impact event.

Synthesis, transfer, and characterization of core-shell gold-coated magnetic nanoparticles.

Magnetic separation has gained new popularity as a versatile partitioning method with the recent growth in nanotechnology and related biotechnology applications. In this study, iron oxide magnetic nanoparticles were synthesized via solvothermal methods and directly coated with gold to form core-shell gold-coated magnetic nanoparticles (Fe3O4-AuNPs). High-resolution transmission electron microscopy with Energy dispersive X-ray spectroscopy results suggests that temperature and reaction time play an important role in the formation of small, monodisperse Fe3O4-AuNPs. We also demonstrate that increased 4- dimethyl(amino)pyridine (DMAP) concentrations and vigorous stirring were required to successfully transfer Fe3O4-AuNPs into aqueous solution. The structure and morphology of the synthesized and transferred Fe3O4-AuNPs was further confirmed by UV-vis absorption spectroscopy and solubility experiments. •Direct coating of Fe3O4 with Au: Slowly heating by (10 °C/ min) until 180-190 °C without exceeding this reaction temperature and increasing the reaction time to 3 h from 1.5 h•High yield transfer of Fe3O4-AuNPs was achieved using 4- dimethyl(amino)pyridine (DMAP) as phase transfer catalyst.

Gas Chromatography-Mass Spectrometry Microbial Metabolomics for Applications in Strain Optimization.

Metabolomics is the systems-scale measurement of biochemical intermediates in biological systems; by virtue of its deep and broad study of metabolism, it has great potential for applications in metabolic engineering. While a number of the analytical techniques used widely in metabolomics are familiar to metabolic engineers performing post hoc analyses of product titers, the requirements for accurately capturing metabolism at a systems scale rather than just measuring a single secreted product are much more complicated. Nonetheless, metabolomics (which is still not widely available as an affordable consumer service like many molecular biology services) is within reach of many properly equipped metabolic engineering groups. To this end, we present a detailed metabolomics protocol with application to strain optimization. Specifically, we focus on characterizing metabolism in the yeast Saccharomyces cerevisiae using gas chromatography coupled to mass spectrometry. The measurement of metabolic intermediates that results from such approaches has the potential to enable more informed and rational efforts towards pathway engineering and strain optimization.

Current Status and Future Prospects for Aptamer-Based Mycotoxin Detection.

Aptamers are single-stranded oligonucleotides with the ability to bind tightly and selectively to a target analyte. High-affinity and specific aptamers for a variety of mycotoxins have been reported over the past decade. Increasingly, these molecular recognition elements are finding applications in biosensors and assays for the detection of mycotoxins in a variety of complex matrixes. This review article highlights the mycotoxin aptamers that are available for mycotoxin detection and the array of biosensing platforms into which they have been incorporated. Key advantages that aptamers have over analogous technology, and areas in which these advantages may be applied for the benefit of practical mycotoxin detection, are also discussed.

Comparison of In-Solution Biorecognition Properties of Aptamers against Ochratoxin A.

Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by several species of Aspergillus and Penicillium and frequently found as a natural contaminant in a wide range of food commodities. Novel and robust biorecognition agents for detecting this molecule are required. Aptamers are artificial nucleic acid ligands able to bind with high affinity and specificity to a given target molecule. In the last few years, three separate research groups have selected aptamers for ochratoxin A. While each of these three families of aptamers have been incorporated into various methods for detecting OTA, it is unclear if each aptamer candidate is better suited for a particular application. Here, we perform the first head-to-head comparison of solution-based binding parameters for these groups of aptamers. Based on our results, we provide recommendations for the appropriate choice of aptamer for incorporation into solution-based biorecognition assays and applications.

Differential metabolite levels in response to spawning-induced inappetence in Atlantic salmon Salmo salar.

Atlantic salmon Salmo salar undergo months-long inappetence during spawning, but it is not known whether this inappetence is a pathological state or one for which the fish are adapted. Recent work has shown that inappetent whale sharks can exhibit circulating metabolite profiles similar to ketosis known to occur in humans during starvation. In this work, metabolite profiling was used to explore differences in analyte profiles between a cohort of inappetent spawning run Atlantic salmon and captively reared animals that were fed up to and through the time of sampling. The two classes of animals were easily distinguished by their metabolite profiles. The sea-run fish had elevated É·-9 fatty acids relative to the domestic feeding animals, while other fatty acid concentrations were reduced. Sugar alcohols were generally elevated in inappetent animals, suggesting potentially novel metabolic responses or pathways in fish that feature these compounds. Compounds expected to indicate a pathological catabolic state were not more abundant in the sea-run fish, suggesting that the animals, while inappetent, were not stressed in an unnatural way. These findings demonstrate the power of discovery-based metabolomics for exploring biochemistry in poorly understood animal models.

Protostadienol biosynthesis and metabolism in the pathogenic fungus Aspergillus fumigatus.

Details of the fungal biosynthetic pathway to helvolic acid and other fusidane antibiotics remain obscure. During product characterization of oxidosqualene cyclases in Aspergillus fumigatus, we found the long-sought cyclase that makes (17Z)-protosta-17(20),24-dien-3beta-ol, the precursor of helvolic acid. We then identified a gene cluster encoding the pathway to helvolic acid, which is controlled by a transcription regulator (LaeA) associated with fungal virulence. Evidence regarding the evolutionary origin and taxonomic distribution of fusidane biosynthesis is also presented.

Conceptual and methodological issues in research on age and aging.

Studies of HIV/AIDS in people older than age 50 typically must address a series of distinct, age-related methodological concerns if they are to develop and implement age-sensitive research designs and measures. In this article, the authors identify and discuss seven methodological challenges that researchers confront when planning and conducting studies of how HIV/AIDS affects older adults. These challenges involve the following: defining who qualifies as an older adult in studies of HIV/AIDS and aging; determining which subset of older adults to study; selecting an appropriate level of analysis; measuring age and aging as research constructs; confronting the practical problems of studying people whose ages are nearing the end of the human life course; resolving the ethical dilemmas of HIV/AIDS research that targets older adults; and successfully going from research to practice in disseminating study findings.