Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster

The larval body wall muscles of Drosophila melanogaster arise by fusion of founder myoblasts (FMs) and fusion-competent myoblasts (FCMs). Sticks-and-Stones (SNS) is expressed on the surface of all FCMs and mediates adhesion with FMs and developing syncytia. Intracellular components essential for myoblast fusion are then recruited to these adhesive contacts. In the studies herein, a functional analysis of the SNS cytodomain using the GAL4/UAS system identified sequences that direct myoblast fusion, presumably through recruitment of these intracellular components. An extensive series of deletion and site-directed mutations were evaluated for their ability to rescue the myoblast fusion defects of sns mutant embryos. Deletion studies revealed redundant functional domains within SNS. Surprisingly, highly conserved consensus sites for binding post-synaptic density-95/discs large/zonula occludens-1-domain-containing (PDZ) proteins and serines with a high probability of phosphorylation play no significant role in myoblast fusion. Biochemical studies establish that the SNS cytodomain is phosphorylated at multiple tyrosines and their site-directed mutagenesis compromises the ability of the corresponding transgenes to rescue myoblast fusion. Similar mutagenesis revealed a requirement for conserved proline-rich regions. This complexity and redundancy of multiple critical sequences within the SNS cytodomain suggest that it functions through a complex array of interactions that likely includes both phosphotyrosine-binding and SH3-domain-containing proteins.     

Are you what you eat? A highly transient and prey‐influenced gut microbiome in the grey house spider Badumna longinqua

Stable core microbial communities have been described in numerous animal species and are commonly associated with fitness benefits for their hosts. Recent research, however, highlights examples of species whose microbiota are transient and environmentally derived. Here, we test the effect of diet on gut microbial community assembly in the spider Badumna longinqua. Using 16S rRNA gene amplicon sequencing combined with quantitative PCR, we analyzed diversity and abundance of the spider's gut microbes, and simultaneously characterized its prey communities using nuclear rRNA markers. We found a clear correlation between community similarity of the spider's insect prey and gut microbial DNA, suggesting that microbiome assembly is primarily diet‐driven. This assumption is supported by a feeding experiment, in which two types of prey—crickets and fruit flies—both substantially altered microbial diversity and community similarity between spiders, but did so in different ways. After cricket consumption, numerous cricket‐derived microbes appeared in the spider's gut, resulting in a rapid homogenization of microbial communities among spiders. In contrast, few prey‐associated bacteria were detected after consumption of fruit flies; instead, the microbial community was remodelled by environmentally sourced microbes, or abundance shifts of rare taxa in the spider's gut. The reshaping of the microbiota by both prey taxa mimicked a stable core microbiome in the spiders for several weeks post feeding. Our results suggest that the spider's gut microbiome undergoes pronounced temporal fluctuations, that its assembly is dictated by the consumed prey, and that different prey taxa may remodel the microbiota in drastically different ways.     

The development of social behaviour between siblings and non-siblings of the voles Microtus ochrogaster and Microtus pennsylvanicus

It has been suggested that the strength and duration of sibling relations during the juvenile period may reflect the social structure of mammal species. Two Microtus species with contrasting social systems, M. ochrogaster and M. pennsylvanicus, were chosen for a comparative study of juvenile social behaviour. Young were observed in sibling and non-sibling dyadic encounters from the second until the seventh week of age. Social interactions consisted mainly of body nosing of the partner, and included behaviours interpreted as investigation, socialization and settling. Only minor sex differences in body nosing scores occurred, but some ontogenetic trends were noted. When the species were compared, M. ochrogaster received higher scores for body nosing and sitting in contact, and displayed more reciprocity in their nosing, than did M. pennsylvanicus, whether siblings or non-siblings were considered. When the sibling types were compared, non-sibling dyads received higher nosing scores than did siblings in both species, but the disparity was greater in M. ochrogaster than in M. pennsylvanicus. These results are interpreted with reference to known behavioural and developmental correlates of the extended family system in naturally occurring M. ochrogaster and the less communal social system in M. pennsylvanicus.     

Above‐ and belowground biotic interactions facilitate relocation of plants into cooler environments

One important but largely unanswered question about floristic responses to climate change is how interactions such as competition, facilitation and plant–soil feedbacks will influence the ability of species to track shifting climates. In a rugged and moisture‐limited region that has recently warmed by 2° (Siskiyou Mountains, OR, USA), we planted three species into cooler aspects and elevations than those they currently inhabit, with and without removal of neighbouring plants, and tracked them over 2 years. Two species had higher success in cooler topographic locations, and this success was enhanced by neighbouring plants, which appeared to modulate minimum growing season temperatures. One species' success was also facilitated by the higher soil organic matter found in cooler sites. These results are a novel experimental demonstration of two important factors that may buffer climate change impacts on plants: rugged topography and plant–plant facilitation.     

A disease state mutation unfolds the parkin ubiquitin-like domain

E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease.     

Next-generation phenotyping: requirements and strategies for enhancing our understanding of genotype–phenotype relationships and its relevance to crop improvement

More accurate and precise phenotyping strategies are necessary to empower high-resolution linkage mapping and genome-wide association studies and for training genomic selection models in plant improvement. Within this framework, the objective of modern phenotyping is to increase the accuracy, precision and throughput of phenotypic estimation at all levels of biological organization while reducing costs and minimizing labor through automation, remote sensing, improved data integration and experimental design. Much like the efforts to optimize genotyping during the 1980s and 1990s, designing effective phenotyping initiatives today requires multi-faceted collaborations between biologists, computer scientists, statisticians and engineers. Robust phenotyping systems are needed to characterize the full suite of genetic factors that contribute to quantitative phenotypic variation across cells, organs and tissues, developmental stages, years, environments, species and research programs. Next-generation phenotyping generates significantly more data than previously and requires novel data management, access and storage systems, increased use of ontologies to facilitate data integration, and new statistical tools for enhancing experimental design and extracting biologically meaningful signal from environmental and experimental noise. To ensure relevance, the implementation of efficient and informative phenotyping experiments also requires familiarity with diverse germplasm resources, population structures, and target populations of environments. Today, phenotyping is quickly emerging as the major operational bottleneck limiting the power of genetic analysis and genomic prediction. The challenge for the next generation of quantitative geneticists and plant breeders is not only to understand the genetic basis of complex trait variation, but also to use that knowledge to efficiently synthesize twenty-first century crop varieties.     

Measuring sub-lethal effects on freshwater crayfish (<Emphasis Type="Italic">Paranephrops planifrons</Emphasis>) behaviour and physiology: laboratory and in situ exposure to modified zeolite

Various behavioural and physiological tests were applied to determine whether an aluminised modified zeolite clay product (Z2G1), used to cap phosphorus release from lake sediments, had sub-lethal effects on adult freshwater crayfish (Paranephrops planifrons). In situ exposures of caged crayfish before and after application of modified zeolite to Lake Okaro, Central North Island, New Zealand, at a nominal application concentration of 350 g m⁻², did not impact the survival of freshwater crayfish or produce any significant sub-lethal effects on crayfish mobility or physiology that we could detect. In laboratory sediment tests, crayfish did not show any consistent effect from short-term (10 days) exposure to modified zeolite (at concentrations of 350, 700 or 2,100 g m⁻²) or to alum. When combined, the field and laboratory data provide strong evidence that applications of 350 g m⁻² modified zeolite will have no short-term effect on adult crayfish, with a ‘margin of safety’ for higher application rates. Chronic exposure effects of capping agents and the indirect impacts of lake treatment (e.g. food web responses) are yet to be determined. Quantitative measures of shade- and shelter-seeking behaviour, righting behaviour, and length of escape response, could be improved by the addition of standardised behavioural observations. Physiological measures of ammoniacal-N production and oxygen consumption are less variable than behavioural tests, but comparisons between treatments are best conducted simultaneously. A suite of behavioural and physiological sub-lethal measures using field and laboratory exposures is recommended to provide an integrated assessment of crayfish health.     

Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen

Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redox-active cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (lambda max = 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required.     

The effect of sample handling on cross sectional HIV incidence testing results

Objective(s)

To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays.

Methods

Serum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4°C, 25°C and 37°C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay).

Results

Compared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4°C or 25°C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37°C prior to testing.

Conclusions

Samples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4°C or 25°C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays.     

Structure and Dynamics of a Compact State of a Multidomain Protein,the Mercuric Ion Reductase

The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.