Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation

The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.     

Distribution and diversity of Paraglomus spp. in tilled agricultural soils

Understanding of the ecology of arbuscular mycorrhizal fungi comes primarily from the order Glomerales, and relatively little is known of the ecology of other orders including the Paraglomerales. We investigated the distribution of the Paraglomerales across the English agricultural landscape under different management systems. Soils were collected from 11 tilled agricultural sites. Presence of Paraglomerales was assessed using PCR amplification of 18S/ITS region ribosomal DNA isolated from trap plants, terminal restriction fragment length polymorphism and cloning. Paraglomus spp. were detected in all samples from one location and sporadically in six more, but not at the other locations. Distribution was not related to soil physiochemical characteristics, but the Paraglomaceae were significantly more common in soils under organic management. Cloning of samples from three sites produced sequences closely related to Paraglomus laccatum but only distantly related to Paraglomus brasilianum and Paraglomus occultum. Individual sites had between 10 and 27 separate terminal restriction fragments (T-RFs). The large number of T-RFs reflected a significant sequence diversity in the ITS region. Paraglomerales were, therefore, widely distributed across the agricultural landscape, though with patchy distribution and low diversity. More intensive agricultural management appeared to impact negatively on Paraglomus spp.     

IgG responses to Anopheles gambiae salivary antigen gSG6 detect variation in exposure to malaria vectors and disease risk

Assessment of exposure to malaria vectors is important to our understanding of spatial and temporal variations in disease transmission and facilitates the targeting and evaluation of control efforts. Recently, an immunogenic Anopheles gambiae salivary protein (gSG6) was identified and proposed as the basis of an immuno-assay determining exposure to Afrotropical malaria vectors. In the present study, IgG responses to gSG6 and 6 malaria antigens (CSP, AMA-1, MSP-1, MSP-3, GLURP R1, and GLURP R2) were compared to Anopheles exposure and malaria incidence in a cohort of children from Korogwe district, Tanzania, an area of moderate and heterogeneous malaria transmission. Anti-gSG6 responses above the threshold for seropositivity were detected in 15% (96/636) of the children, and were positively associated with geographical variations in Anopheles exposure (OR 1.25, CI 1.01-1.54, p?=?0.04). Additionally, IgG responses to gSG6 in individual children showed a strong positive association with household level mosquito exposure. IgG levels for all antigens except AMA-1 were associated with the frequency of malaria episodes following sampling. gSG6 seropositivity was strongly positively associated with subsequent malaria incidence (test for trend p?=?0.004), comparable to malaria antigens MSP-1 and GLURP R2. Our results show that the gSG6 assay is sensitive to micro-epidemiological variations in exposure to Anopheles mosquitoes, and provides a correlate of malaria risk that is unrelated to immune protection. While the technique requires further evaluation in a range of malaria endemic settings, our findings suggest that the gSG6 assay may have a role in the evaluation and planning of targeted and preventative anti-malaria interventions.