Techniques for Arbuscular Mycorrhiza Inoculum Reduction

It is well established that arbuscular mycorrhizal (AM) fungi can play a significant role in sustainable crop production and environmental conservation. With the increasing awareness of the ecological significance of mycorrhizas and their diversity, research needs to be directed away from simple records of their occurrence or casual speculation of their function (Smith and Read 1997). Rather, the need is for empirical studies and investigations of the quantitative aspects of the distribution of different types and their contribution to the function of ecosystems. There is no such thing as a fungal effect or a plant effect, but there is an interaction between both symbionts. This results from the AM fungi and plant community size and structure, soil and climatic conditions, and the interplay between all these factors (Kahiluoto et al. 2000). Consequently, it is readily understood that it is the problems associated with methodology that limit our understanding of the functioning and effects of AM fungi within field communities. Given the ubiquous presence of AM fungi, a major constraint to the evaluation of the activity of AM colonisation has been the need to account for the indigenous soil native inoculum. This has to be controlled (i.e. reduced or eliminated) if we are to obtain a true control treatment for analysis of arbuscular mycorrhizas in natural substrates. There are various procedures possible for achieving such an objective, and the purpose of this chapter is to provide details of a number of techniques and present some evaluation of their advantages and disadvantages. Although there have been a large number of experiments to investigated the effectiveness of different sterilization procedures for reducing pathogenic soil fungi, little information is available on their impact on beneficial organisms such as AM fungi. Furthermore, some of the techniques have been shown to affect physical and chemical soil characteristics as well as eliminate soil microorganisms that can interfere with the development of mycorrhizas, and this creates difficulties in the interpretation of results simply in terms of possible mycorrhizal activity. An important subject is the differentiation of methods that involve sterilization from those focussed on indigenous inoculum reduction. Soil sterilization aims to destroy or eliminate microbial cells while maintaining the existing chemical and physical characteristics of the soil (Wolf and Skipper 1994). Consequently, it is often used for experiments focussed on specific AM fungi, or to establish a negative control in some other types of study. In contrast, the purpose of inoculum reduction techniques is to create a perturbation that will interfere with mycorrhizal formation, although not necessarily eliminating any component group within the inoculum. Such an approach allows the establishment of different degrees of mycorrhizal formation between treatments and the study of relative effects. Frequently the basic techniques used to achieve complete sterilization or just an inoculum reduction may be similar but the desired outcome is accomplished by adjustments of the dosage or intensity of the treatment. The ultimate choice of methodology for establishing an adequate non-mycorrhizal control depends on the design of the particular experiments, the facilities available and the amount of soil requiring treatment

Impact of pulmonary exposure to gold core silver nanoparticles of different size and capping agents on cardiovascular injury

Background:The uses of engineered nanomaterials have expanded in biomedical technology and consumer manufacturing. Furthermore, pulmonary exposure to various engineered nanomaterials has, likewise, demonstrated the ability to exacerbate cardiac ischemia reperfusion (I/R) injury. However, the influence of particle size or capping agent remains unclear. In an effort to address these influences we explored response to 2 different size gold core nanosilver particles (AgNP) with two different capping agents at 2 different time points. We hypothesized that a pulmonary exposure to AgNP induces cardiovascular toxicity influenced by inflammation and vascular dysfunction resulting in expansion of cardiac I/R Injury that is sensitive to particle size and the capping agent. Methods: Male Sprague–Dawley rats were exposed to 200 μg of 20 or 110 nm polyvinylprryolidone (PVP) or citrate capped AgNP. One and 7 days following intratracheal instillation serum was analyzed for concentrations of selected cytokines; cardiac I/R injury and isolated coronary artery and aorta segment were assessed for constrictor responses and endothelial dependent relaxation and endothelial independent nitric oxide dependent relaxation. Results: AgNP instillation resulted in modest increase in selected serum cytokines with elevations in IL-2, IL-18, and IL-6. Instillation resulted in a derangement of vascular responses to constrictors serotonin or phenylephrine, as well as endothelial dependent relaxations with acetylcholine or endothelial independent relaxations by sodium nitroprusside in a capping and size dependent manner. Exposure to both 20 and 110 nm AgNP resulted in exacerbation cardiac I/R injury 1 day following IT instillation independent of capping agent with 20 nm AgNP inducing marginally greater injury. Seven days following IT instillation the expansion of I/R injury persisted but the greatest injury was associated with exposure to 110 nm PVP capped AgNP resulted in nearly a two-fold larger infarct size compared to naïve. Conclusions: Exposure to AgNP may result in vascular dysfunction, a potentially maladaptive sensitization of the immune system to respond to a secondary insult (e.g., cardiac I/R) which may drive expansion of I/R injury at 1 and 7 days following IT instillation where the extent of injury could be correlated with capping agents and AgNP size.This work was supported by the National Institute of Environmental Health Sciences U19ES019525, U01ES020127, U19ES019544 and East Carolina Universit

American Academy of Optometry Microbial Keratitis Think Tank

SIGNIFICANCE Think Tank 2019 affirmed that the rate of infection associated with contact lenses has not changed in several decades. Also, there is a trend toward more serious infections associated with Acanthamoeba and fungi. The growing use of contact lenses in children demands our attention with surveillance and case-control studies. PURPOSE The American Academy of Optometry (AAO) gathered researchers and key opinion leaders from around the world to discuss contact lens-associated microbial keratitis at the 2019 AAO Annual Meeting. METHODS Experts presented within four sessions. Session 1 covered the epidemiology of microbial keratitis, pathogenesis of Pseudomonas aeruginosa, and the role of lens care systems and storage cases in corneal disease. Session 2 covered nonbacterial forms of keratitis in contact lens wearers. Session 3 covered future needs, challenges, and research questions in relation to microbial keratitis in youth and myopia control, microbiome, antimicrobial surfaces, and genetic susceptibility. Session 4 covered compliance and communication imperatives. RESULTS The absolute rate of microbial keratitis has remained very consistent for three decades despite new technologies, and extended wear significantly increases the risk. Improved oxygen delivery afforded by silicone hydrogel lenses has not impacted the rates, and although the introduction of daily disposable lenses has minimized the risk of severe disease, there is no consistent evidence that they have altered the overall rate of microbial keratitis. Overnight orthokeratology lenses may increase the risk of microbial keratitis, especially secondary to Acanthamoeba, in children. Compliance remains a concern and a significant risk factor for disease. New insights into host microbiome and genetic susceptibility may uncover new theories. More studies such as case-control designs suited for rare diseases and registries are needed. CONCLUSIONS The first annual AAO Think Tank acknowledged that the risk of microbial keratitis has not decreased over decades, despite innovation. Important questions and research directions remain

The Pine River Statement: Human Health Consequences of DDT Use

Objectives: Dichlorodiphenyltrichloroethane (DDT) was used worldwide until the 1970s, when concerns about its toxic effects, its environmental persistence, and its concentration in the food supply led to use restrictions and prohibitions. In 2001, more than 100 countries signed the Stockholm Convention on Persistent Organic Pollutants (POPs), committing to eliminate the use of 12 POPs of greatest concern. However, DDT use was allowed for disease vector control. In 2006, the World Health Organization and the U.S. Agency for International Development endorsed indoor DDT spraying to control malaria. To better inform current policy, we reviewed epidemiologic studies published from 2003 to 2008 that investigated the human health consequences of DDT and/or DDE (dichlorodiphenyldichloroethylene) exposure. Data sources and extraction: We conducted a PubMed search in October 2008 and retrieved 494 studies. Data synthesis: Use restrictions have been successful in lowering human exposure to DDT, but blood concentrations of DDT and DDE are high in countries where DDT is currently being used or was more recently restricted. The recent literature shows a growing body of evidence that exposure to DDT and its breakdown product DDE may be associated with adverse health outcomes such as breast cancer, diabetes, decreased semen quality, spontaneous abortion, and impaired neurodevelopment in children. Conclusions: Although we provide evidence to suggest that DDT and DDE may pose a risk to human health, we also highlight the lack of knowledge about human exposure and health effects in communities where DDT is currently being sprayed for malaria control. We recommend research to address this gap and to develop safe and effective alternatives to DDT.Dichlorodiphenyltrichloroethane (DDT) was used worldwide until the 1970s, when concerns about its toxic effects, its environmental persistence, and its concentration in the food supply led to use restrictions and prohibitions. In 2001, more than 100 countries signed the Stockholm Convention on Persistent Organic Pollutants (POPs), committing to eliminate the use of 12 POPs of greatest concern. However, DDT use was allowed for disease vector control. In 2006, the World Health Organization and the U.S. Agency for International Development endorsed indoor DDT spraying to control malaria. To better inform current policy, we reviewed epidemiologic studies published from 2003 to 2008 that investigated the human health consequences of DDT and/or DDE (dichlorodiphenyldichloroethylene) exposure. Data sources and extraction We conducted a PubMed search in October 2008 and retrieved 494 studies. Data synthesis Use restrictions have been successful in lowering human exposure to DDT, but blood concentrations of DDT and DDE are high in countries where DDT is currently being used or was more recently restricted. The recent literature shows a growing body of evidence that exposure to DDT and its breakdown product DDE may be associated with adverse health outcomes such as breast cancer, diabetes, decreased semen quality, spontaneous abortion, and impaired neurodevelopment in children. Conclusions Although we provide evidence to suggest that DDT and DDE may pose a risk to human health, we also highlight the lack of knowledge about human exposure and health effects in communities where DDT is currently being sprayed for malaria control. We recommend research to address this gap and to develop safe and effective alternatives to DDThttp://dx.doi.org/10.1289/ehp.1174

Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

BACKGROUND: [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. CONCLUSIONS/SIGNIFICANCE: [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy

Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

The cellular and molecular mechanisms underlying the pathogenesis of cataracts leading to visual impairment remain poorly understood. In recent studies, several mutations in the cytoplasmic sterile-α-motif (SAM) domain of human EPHA2 on chromosome 1p36 have been associated with hereditary cataracts in several families. Here, we have investigated how these SAM domain mutations affect EPHA2 activity. We showed that the SAM domain mutations dramatically destabilized the EPHA2 protein in a proteasome-dependent pathway, as evidenced by the increase of EPHA2 receptor levels in the presence of the proteasome inhibitor MG132. In addition, the expression of wild-type EPHA2 promoted the migration of the mouse lens epithelial αTN4-1 cells in the absence of ligand stimulation, whereas the mutants exhibited significantly reduced activity. In contrast, stimulation of EPHA2 with its ligand ephrin-A5 eradicates the enhancement of cell migration accompanied by Akt activation. Taken together, our studies suggest that the SAM domain of the EPHA2 protein plays critical roles in enhancing the stability of EPHA2 by modulating the proteasome-dependent process. Furthermore, activation of Akt switches EPHA2 from promoting to inhibiting cell migration upon ephrin-A5 binding. Our results provide the first report of multiple EPHA2 cataract mutations contributing to the destabilization of the receptor and causing the loss of cell migration activity

History, epidemiology and regional diversities of urolithiasis

Archeological findings give profound evidence that humans have suffered from kidney and bladder stones for centuries. Bladder stones were more prevalent during older ages, but kidney stones became more prevalent during the past 100 years, at least in the more developed countries. Also, treatment options and conservative measures, as well as ‘surgical’ interventions have also been known for a long time. Our current preventive measures are definitively comparable to those of our predecessors. Stone removal, first lithotomy for bladder stones, followed by transurethral methods, was definitively painful and had severe side effects. Then, as now, the incidence of urolithiasis in a given population was dependent on the geographic area, racial distribution, socio-economic status and dietary habits. Changes in the latter factors during the past decades have affected the incidence and also the site and chemical composition of calculi, with calcium oxalate stones being now the most prevalent. Major differences in frequency of other constituents, particularly uric acid and struvite, reflect eating habits and infection risk factors specific to certain populations. Extensive epidemiological observations have emphasized the importance of nutritional factors in the pathogenesis of urolithiasis, and specific dietary advice is, nowadays, often the most appropriate for prevention and treatment of urolithiasis