Computational Reconstruction of Multidomain Proteins Using Atomic Force Microscopy Data

SummaryClassical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures

Indigenous biosecurity: Māori responses to kauri dieback and myrtle rust in Aotearoa New Zealand

It is widely acknowledged that Indigenous peoples have traditional knowledge relevant to modern environmental management. By asserting roles within associated science and policy networks, such Indigenous Knowledge (IK) can be seen as part of the resistance to colonisation that includes protest, treaty making, political and economic empowerment, legislation, cultural renaissance and regulatory influence. In New Zealand, these achievements inform attempts by Māori (the Indigenous people of New Zealand) to manage forest ecosystems and cultural keystone species. This chapter presents two case studies of how indigenous participation in modern biosecurity through the example of Māori asserting and contributing to forest management. While progress is often frustratingly slow for indigenous participants, significant gains in acceptance of Māori cultural frameworks have been achieved

Can insect body pollen counts be used to estimate pollen deposition on pak choi stigmas?

Measuring pollen deposition onto stigmas by insects is one technique used to assess pollinator effectiveness, but it can be unpredictable and time-consuming as insects must visit test flowers. This study examined whether a measurement of pollen grains from flower-visiting insects could be used to predict pollen deposited on stigmas. Individuals were collected from four bee and six fly species as they visited pak choi flowers in commercial and trial seed fields to assess their body pollen. Pollen was removed from insects by pressing their bodies (excluding actively-collected pollen on bees' hind legs) with a cube of gelatine-fuchsin. In this study, there appears to be a strong correlation between mean estimated pollen counts for each insect species and previously published data recording mean number of pollen grains deposited on stigmas for the same species. Therefore, the measurement of pollen grains directly from flower-visiting insects shows potential as a quicker and easier technique to assess pollinator effectiveness as insects can be directly collected from flowers

Alternative pollinator taxa are equally efficient but not as effective as the honeybee in a mass flowering crop

1. The honeybee 'Apis mellifera' is currently in decline worldwide because of the combined impacts of Colony Collapse Disorder and the 'Varroa destructor' mite. In order to gain a balanced perspective of the importance of both wild and managed pollination services, it is essential to compare these services directly, a priori, within a cropping landscape. This process will determine the capacity of other flower visitors to act as honeybee replacements. 2. In a highly modified New Zealand agricultural landscape, we compared the pollination services provided by managed honeybees to unmanaged pollinator taxa (including flies) within a 'Brassica rapa var. chinensis' mass flowering crop. 3. We evaluate overall pollinator effectiveness by separating the pollination service into two components: efficiency (i.e. per visit pollen deposition) and visit rate (i.e. pollinator abundance per available flower and the number of flower visits per minute). 4. We observed 31 species attending flowers of 'B. rapa'. In addition to 'A. mellifera', seven insect species visited flowers frequently. These were three other bees ('Lasioglossum sordidum', 'Bombus terrestris' and 'Leioproctus' sp.) and four flies ('Dilophus nigrostigma', 'Melanostoma fasciatum', 'Melangyna novae'-zelandiae and 'Eristalis tenax'). 5. Two bee species, 'Bombus terrestris' and 'Leioproctus' sp. and one fly, 'Eristalis tenax' were as efficient as the honeybee and as effective (in terms of rate of flower visitation). A higher honeybee abundance, however, resulted in it being the more effective pollinator overall. 6. Synthesis and applications. Alternative land management practices that increase the population sizes of unmanaged pollinator taxa to levels resulting in visitation frequencies as high as 'A. mellifera', have the potential to replace services provided by the honeybee. This will require a thorough investigation of each taxon's intrinsic biology and a change in land management practices to ensure year round refuge, feeding, nesting and other resource requirements of pollinator taxa are met

An ancient and a recent colonization of islands by an Australian sap‐feeding insect

Aim: To assess the genetic structure, biogeography, and the potential for speciation, of a highly host‐specific insect pest with mainland and island populations. Location: East coast of Australia, Lord Howe Island (LHI), and New Zealand. Methods: We focussed on Mycopsylla fici, a plant sap‐feeding insect host‐specific to the fig tree Ficus macrophylla. We genotyped 152 insects from across the natural and extended host plant range at 14 microsatellite loci and analysed the data using standard population genetics statistics, Discriminant Analysis of Principal Components, genetic autocorrelation, and two Bayesian clustering approaches. Results: Genetic analyses revealed that the northeastern Australian mainland population (Brisbane) is the centre of genetic diversity. Northeastern and southeastern (Sydney) mainland populations are genetically differentiated and interconnected in a stepping‐stone pattern. The LHI population is the most distinct genetically and Bayesian estimates indicated that the most recent colonization occurred c. 2,000‐17,500 years ago from a northeastern mainland origin. In contrast, the New Zealand population is little differentiated from the Sydney population and probably diverged by colonization within the past 200 years. Main conclusions: The strong differentiation in nuclear microsatellites mirrors previous evidence for divergence of the LHI population from both mtDNA and endosymbiont DNA. The LHI population may be undergoing speciation from the mainland populations, with an oceanic barrier to gene flow. In contrast, the geographically isolated population in Auckland represents a far more recent colonization reflecting the contemporary naturalization of the plant host in New Zealand

Hyperaldosteronemia and activation of the epithelial sodium channel are not required for sodium retention in puromycin-induced nephrosis

Edema and ascites in nephrotic syndrome mainly result from increased Na+ reabsorption along connecting tubules and cortical collecting ducts (CCD). In puromycin aminonucleoside (PAN)-induced nephrosis, increased Na+ reabsorption is associated with increased activity of the epithelial sodium channel (ENaC) and Na+,K+-ATPase, two targets of aldosterone. Because plasma aldosterone increases in PAN-nephrotic rats, the aldosterone dependence of ENaC activation in PAN nephrosis was investigated. For this purpose, (1) the mechanism of ENaC activation was compared in nephrotic and sodium-depleted rats, and (2) ENaC activity in PAN-nephrotic rats was evaluated in the absence of hyperaldosteronemia. The mechanism of ENaC activation was similar in CCD from nephrotic and sodium-depleted rats, as demonstrated by (1) increased number of active ENaC evaluated by patch clamp, (2) recruitment of ENaC to the apical membrane determined by immunohistochemistry, (3) shift in the electrophoretic profile of gamma-ENaC, and (4) increased abundance of beta-ENaC mRNA. Corticosteroid clamp fully prevented all PAN-induced changes in ENaC but did not alter the development of a full-blown nephrotic syndrome with massive albuminuria, amiloride-sensitive sodium retention, induction of CCD Na+,K+-ATPase, and ascites. It is concluded that in PAN-nephrosis, (1) ENaC activation in CCD is secondary to hyperaldosteronemia, (2) sodium retention and induction of Na+,K+-ATPase in CCD are independent of hyperaldosteronemia, and (3) ENaC is not necessarily limiting for sodium reabsorption in the distal nephron