Antimicrobial and Antiinsectan Phenolic Metabolites of Dalea searlsiae

Continued interest in the chemistry of Dalea spp. led to investigation of Dalea searlsiae, a plant native to areas of the western United States. Methanol extractions of D. searlsiae roots and subsequent chromatographic fractionation afforded the new prenylated and geranylated flavanones malheurans A–D (1–4) and known flavanones (5 and 6). Known rotenoids (7 and 8) and isoflavones (9 and 10) were isolated from aerial portions. Structure determination of pure compounds was accomplished primarily by extensive 1D- and 2D-NMR spectroscopy. The absolute configurations of compounds 1–5, 7, and 8 were assigned using electronic circular dichroism spectroscopy. Antimicrobial bioassays revealed significant activity concentrated in the plant roots. Compounds 1–6 exhibited MICs of 2–8 μg/mL against Streptococcus mutans, Bacillus cereus, and oxacillin-sensitive and -resistant Staphylococcus aureus. Aerial metabolites 7–10 were inactive against these organisms, but the presence of 7 and 8 prompted investigation of the antiinsectan activity of D. searlsiae metabolites toward the major crop pest Spodoptera frugiperda (fall armyworm). While compounds 1–10 all caused significant reductions in larval growth rates, associated mortality (33–66%) was highest with flavanone 4 and rotenoids 7 and 8. These findings suggest a differential allocation of antimicrobial and antiinsectan plant resources to root and aerial portions of the plant, respectively

Desperately seeking niches: Grassroots innovations and niche development in the community currency field

The sustainability transitions literature seeks to explain the conditions under which technological innovations can diffuse and disrupt existing socio-technical systems through the successful scaling up of experimental ‘niches’; but recent research on ‘grassroots innovations’ argues that civil society is a promising but under-researched site of innovation for sustainability, albeit one with very different characteristics to the market-based innovation normally considered in the literature. This paper aims to address that research gap by exploring the relevance of niche development theories in a civil society context. To do this, we examine a growing grassroots innovation – the international field of community currencies – which comprises a range of new socio-technical configurations of systems of exchange which have emerged from civil society over the last 30 years, intended to provide more environmentally and socially sustainable forms of money and finance. We draw on new empirical research from an international study of these initiatives comprising primary and secondary data and documentary sources, elite interviews and participant observation in the field. We describe the global diffusion of community currencies, and then conduct a niche analysis to evaluate the utility of niche theories for explaining the development of the community currency movement. We find that some niche-building processes identified in the existing literature are relevant in a grassroots context: the importance of building networks, managing expectations and the significance of external ‘landscape’ pressures, particularly at the level of national-type. However, our findings suggest that existing theories do not fully capture the complexity of this type of innovation: we find a diverse field addressing a range of societal systems (money, welfare, education, health, consumerism), and showing increasing fragmentation (as opposed to consolidation and standardisation); furthermore, there is little evidence of formalised learning taking place but this has not hampered movement growth. We conclude that grassroots innovations develop and diffuse in quite different ways to conventional innovations, and that niche theories require adaptation to the civil society context

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Phthalate Esters Exacerbate Neurodegeneration in a Caenorhabditis elegans Parkinson’s Disease Model

Parkinson’s disease (PD) extracts a significant medical and financial toll on the developed world and is second only to Alzheimer’s disease in terms of prevalence as a neurodegenerative disease. Up to 90 percent of PD cases are idiopathic or environmental in origin. Benzyl Butyl Phthalate (BBzP) and Bis (2-ethylhexyl) Phthalate are plasticizers, compounds used in industry to improve the malleability and durability of plastic products, which were evaluated for their potential contribution to neurodegeneration of dopamine neurons in a Caenorhabditis elegans (roundworm) PD model. These phthalates were assessed for effect in two genetic worm strains of PD. The first genetic strain is a transgenic animal expressing the human alpha-synuclein gene that is known to contribute to PD in humans. The second strain was a transgenic animal that overexpressed tyrosine hydroxylase, leading to excess production of reactive oxygen species, another suspected cause of PD neurodegeneration in humans. It was discovered that different PD models were effected by different phthalate esters. DEHP induced neurodegeneration in non-genetically predisposed animals at ten days post-exposure and in the tyrosine hydroxylase model at seven and ten days post-exposure while BBzP only exacerbated neurodegeneration at ten days post-exposure. Further BBzP exacerbated neurodegeneration in the alpha-synuclein model at day seven and ten. Our results suggest that phthalate esters could be important in the development of PD

Development of an Assay to Detect Degenerative Dopaminergic Neurons in \u3ci\u3eCaenorhabditis\u3c/i\u3e \u3ci\u3eelegans\u3c/i\u3e

Parkinson’s disease (PK) is a disruption of motor function caused by loss of dopamine neurons. PK can be caused by environmental and genetic factors. One protein known to contribute to PK is the protein α-synuclein found in dopamine neurons. Over expression or mutations in α-synuclein can lead to PK. The soil nematode, Caenorhabditis elegans (C. elegans), has been developed as a model for PK by using a transgenically modified strain, which overexpresses the human α-synuclein protein. In this strain, the dopamine neurons, which have been labeled with a green fluorescent protein, were observed by fluorescent microscopy to degenerate after nine days of development. We have discovered that the transgenic strain expresses a locomotory behavioral defect that is indicative of deficient dopamine signaling at day three of development. When wild-type (normal) nematodes encounter their food, which is a bacterial lawn, they slow their locomotory speed. However, the transgenic strain does not exhibit the prototypical slowing behavior that stems from the excitation of the dopamine neurons. This defect was determined by utilizing an automated tracking system to quantify speed of locomotion on and off food. We can extrapolate that this behavior is correlated to nematodes that exhibit degenerative dopamine neurons, as this same behavioral defect is observed in cat-2 mutants that do not generate dopamine. In future studies, we will utilize this assay to examine the effects of environmental stressors on these neurons and their relation to PK

Bacteriological Testing of Novel Flavonoids Against Antibiotic Resistant Staphylococcus aureus

An urgent problem facing medicine is the growing number of bacterial species forming resistance to one or more common antibiotics. The need to find other treatments has lead to the isolation and characterization of natural compounds, including a family of biologically active chemicals known as flavonoids. These compounds are present in photosynthesizing organisms and have been shown to have anti-microbial, anti-oxidant, anti-cancer, and anti-inflammatory properties. In a collaborative project between the Chemistry and Biology Departments, we have isolated and characterized four novel flavonoids from Dalea searlsiae, a local prairie clover. From crude root extracts, we performed disk diffusion assays against Staphylococcus aureus to identify anti-bacterial fractions. These fractions were further separated using column chromatography and the disk diffusion assay was repeated until pure compounds were obtained. After isolation and characterization of these four flavonoids, we used a broth dilution method to determine minimum inhibitory concentrations (MICs) of the compounds against oxacillin sensitive S. aureus (OSSA) and oxacillin resistant S. aureus (ORSA). We found that the flavonoids have MICs of 5 μg/mL against both OSSA and ORSA compared to oxacillin MICs of 0.5 μg/mL and greater than 16 μg/mL, respectively. This indicates that our flavonoids function to inhibit bacterial growth through an independent mechanism compared to oxacillin. A novel antibiotic could possibly replace or augment compounds in the medical establishment’s antibiotic arsenal