FlyBase: a guided tour of highlighted features

FlyBase provides a centralized resource for the genetic and genomic data of Drosophila melanogaster. As FlyBase enters our fourth decade of service to the research community, we reflect on our unique aspects and look forward to our continued collaboration with the larger research and model organism communities. In this study, we emphasize the dedicated reports and tools we have constructed to meet the specialized needs of fly researchers but also to facilitate use by other research communities. We also highlight ways that we support the fly community, including an external resources page, help resources, and multiple avenues by which researchers can interact with FlyBase.     

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σ^B), whose role in protection against acid stress is well established. Here, we investigated the involvement of the stressosome, a sensory hub, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σ^B and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was solely present retained the ability to induce σ^B activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1-T175A, -T209A, -T241A) and RsbS (rsbS-S56A), or the stressosome kinase RsbT (rsbT-N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1-T209A and rsbR1-T241A mutants displayed constitutive σ^B activity. Mild acid shock upregulates invasion genes inlAB and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σ^B in L. monocytogenes. Furthermore, they show that RsbR1 can function independently of its paralogues and signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209 but not T175. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.     

DendroCT – Dendrochronology without damage

The paper describes an evaluation of the applicability of computer tomography in archaeological dendrochronology. Two different computer tomographs were tested, a Siemens Somatom Emotion single slice scanner developed for medical use, and a Nikon Metrology model XT H 225 LC, which is an industrial type scanner. Both scanners were tested against air-dried, archaeological oak wood, and more limited experiments were made with waterlogged archaeological oak wood and archaeological oak wood which had been treated with high-molecular polyethyleneglycol as a conservation treatment. After scanning the resulting imagery were measured and analysed for dendrochronology using off-the-shelf software for handling and measuring on the images and the specialist programme DENDRO for the dendrochronological analyses.The results showed that only the industrial scanner produced sufficiently clear imagery to allow for dendrochronological analyses. In the scans it was possible to separate tree-rings down to 0.2 mm width, and it was possible to identify the sapwood–heartwood border when sufficient sapwood rings were present. It was found, however, that a visual inspection of the object was required to distinguish between sapwood and decayed wood. Comparisons between direct measurements of tree-rings and measurements based on CT-imagery revealed no significant differences. The scanning and subsequent dating of more than 90 objects showed that dendrochronological dating based on CT-scanning has a success rate equal to conventional dating, albeit more time consuming.The attempts to scan waterlogged and PEG-impregnated archaeological oak wood were unsuccessful, due to a low degree of contrast between the water/PEG and the preserved wood. The experiments were too limited to exclude, however, that a successful protocol can be developed also for these types of materials.     

An update on passive transport in and out of plant cells

Transport across membranes is critical for plant survival. Membranes are the interfaces at which plants interact with their environment. The transmission of energy and molecules into cells provides plants with the source material and power to grow, develop, defend, and move. An appreciation of the physical forces that drive transport processes is thus important for understanding the plant growth and development. We focus on the passive transport of molecules, describing the fundamental concepts and demonstrating how different levels of abstraction can lead to different interpretations of the driving forces. We summarize recent developments on quantitative frameworks for describing diffusive and bulk flow transport processes in and out of cells, with a more detailed focus on plasmodesmata, and outline open questions and challenges.

New insights enhance our understanding of the physical forces that drive passive transport across plant membranes highlights.     

Prevalence and Correlates of Physical Inactivity in Community-Dwelling Older Adults in Ireland

The public health challenges associated with rapid population ageing are likely to be exacerbated by poor physical activity levels. The purpose of this study was to identify correlates of physical inactivity in a population-representative sample of older adults in Ireland. This paper reports a secondary analysis of data from 4892 adults aged 60+ from the Irish Longitudinal Study on Ageing (TILDA). TILDA includes an assessment of the mental and physical health, and social and financial circumstances of participants assessed in a home interview and self-completion questionnaire. Chi squared statistics and forced entry logistic regression were used to identify factors associated with physical inactivity. Females were over twice as likely to be inactive as their male counterparts (Odds Ratio 2.2). Increasing old age was associated with inactivity among males and females. Those who reported above secondary level education, no reported falls in the last year and no fear of falling were less likely to be physically inactive. While older adults who noted poor/fair self-reported health, that they did not look after grandchildren, did not own a car or did not attend a course were also more likely to be inactive than those who reported positively for these items. Gender displayed a strong but often contrasting influence on factors that affect physical activity among older adults. Among females, living alone or in a rural area, retirement, fair/poor emotional health and activity being limited by illness were all significantly associated with inactivity. While cohabiting, being employed and residing in an urban area were related to low levels of activity in males. Our findings identify specific groups of the older Irish population who may be at particular risk of physical inactivity and thereby the associated physiological and psychological hazards. These results can support the development of tailored interventions to promote healthy ageing.     

Patterns of indirect protein interactions suggest a spatial organization to metabolism

It has long been believed that cells organize their cytoplasm so as to efficiently channel metabolites between sequential enzymes. This metabolic channeling has the potential to yield higher metabolic fluxes as well as better regulatory control over metabolism. One mechanism for achieving such channeling is to ensure that sequential enzymes in a pathway are physically close to each other in the cell. We present evidence that indirect protein interactions between related enzymes represent a global mechanism for achieving metabolic channeling; the intuition being that protein interactions between enzymes and non-enzymatic mediator proteins are a powerful means of physically associating enzymes in a modular fashion. By analyzing the metabolic and protein-protein interactions networks of Escherichia coli, yeast and humans, we are able to show that all three species have many more indirect protein interactions linking enzymes that share metabolites than would be expected by chance. Moreover, these interactions are distributed non-randomly in the metabolic network. Our analyses in yeast and E. coli show that reactions possessing such interactions also show higher flux than do those lacking them. On the basis of these observations, we suggest that an important role of protein interactions with mediator proteins is to contribute to the spatial organization of the cell. This hypothesis is supported by the fact that these mediator proteins are also enriched with annotations related to signal transduction, a system where scaffolding proteins are known to limit cross-talk by controlling spatial localization.     

Insulin and IGF-1 signalling: longevity, protein homoeostasis and Alzheimer's disease

The quality control of protein homoeostasis deteriorates with aging, causing the accumulation of misfolded proteins and neurodegeneration. Thus, in AD (Alzheimer's disease), soluble oligomers, protofibrils and fibrils of the Aβ (amyloid β-peptide) and tau protein accumulate in specific brain regions. This is associated with the progressive destruction of synaptic circuits controlling memory and higher mental function. The primary signalling mechanisms that (i) become defective in AD to alter the normal proteostasis of Aβ and tau, and (ii) initiate a pathophysiological response to cause cognitive decline, are unclear. The IIS [insulin/IGF-1 (insulin-like growth factor 1)-like signalling] pathway is mechanistically linked to longevity, protein homoeostasis, learning and memory, and is emerging to be central to both (i) and (ii). This pathway is aberrantly overactivated in AD brain at the level of increased activation of the serine/threonine kinase Akt and the phosphorylation of its downstream targets, including mTOR (mammalian target of rapamycin). Feedback inhibition of normal insulin/IGF activation of the pathway also occurs in AD due to inactivation of IRS-1 (insulin receptor substrate 1) and decreased IRS-1/2 levels. Pathogenic forms of Aβ may induce aberrant sustained activation of the PI3K (phosphoinositide 3-kinase)/Akt signal in AD, also causing non-responsive insulin and IGF-1 receptor, and altered tau phosphorylation, conformation and function. Reducing IIS activity in animal models by decreasing IGF-1R levels or inhibiting mTOR activity alters Aβ and tau protein homoeostasis towards less toxic protein conformations, improves cognitive function and extends healthy lifespan. Thus normalizing IIS dysfunction may be therapeutically relevant in abrogating Aβ and tau proteotoxicity, synaptic dysfunction and cognitive decline in AD.     

Uptake of Voluntary Counselling and Testing among Young People Participating in an HIV Prevention Trial: Comparison of Opt-Out and Opt-In Strategies

Background

HIV voluntary counselling and testing (VCT) is an integral component of HIV prevention and treatment programmes. However, testing coverage in sub-Saharan Africa is still low, particularly among young people. As treatment becomes more widely available, strategies to expand VCT coverage are critically important. We compare VCT uptake using two delivery strategies (opt-in and opt-out) within the MEMA kwa Vijana trial in 20 communities in northwest Tanzania.

Methods

We analysed data from 12,590 young persons (median (IQR) age 22 years (20–23)) to assess the effect of delivery strategy on VCT uptake. Ten communities used an opt-in approach and 10 used opt-out, balanced across intervention and control. Conditional logistic regression was used to examine factors associated with uptake within each strategy.

Results

VCT uptake was significantly higher with the opt-out approach (90.9% vs 60.5%, prevalence ratio = 1.51, CI = 1.41–1.62). Among females, uptake in the opt-out approach was associated with decreased knowledge of HIV acquisition, sex with a casual partner, and being HSV-2 seronegative; among males, uptake was associated with lower education and increasing lifetime partners. In contrast, uptake using the opt-in approach varied by ethnic group, religion and marital status, and increased with increasing knowledge of STI acquisition (males) or pregnancy prevention (females).

Conclusion

VCT uptake among young people was extremely high when offered an opt-out strategy. Sociodemographic and knowledge factors affected uptake in different ways depending on delivery strategy. Increased knowledge may increase young persons'' self-efficacy, which may have a different impact on testing uptake, depending on the approach used.     

Differential release of high mannose structural isoforms by fungal and bacterial endo-β-N-acetylglucosaminidases

Endo-β-N-acetylglucosaminidases (ENGases) are widely used to remove N-linked oligosaccharides from glycoproteins for glycomic and proteomic studies and biopharmaceutical processes. Although several ENGases are widely available and their main oligosaccharide structural preferences are generally known (i.e. high mannose, hybrid or complex), the preferences of ENGases from different kingdoms for individual structural isoforms within the major classes of N-linked oligosaccharides have previously not been compared. In this work, a fungal ENGase (Endo Tv) was purified for the first time from a commercial Trichoderma viride chitinase mixture by sequential anion exchange and size exclusion chromatography, a commonly used strategy for purification of chitinases and endo enzymes. Oligosaccharides released from substrate glycoproteins by Endo Tv were identified and quantified by high pH anion exchange chromatography with pulsed amperometric detection and verified by mass spectrometric analysis. Unlike the widely-used bacterial ENGases, Endo H and Endo F1, Endo Tv released exclusively high mannose N-linked oligosaccharides from RNase B, ovalbumin, and yeast invertase. Endo Tv did not hydrolyze fucosylated, hybrid, complex type or bisecting N-acetylglucosamine-containing structures from bovine fetuin, ovalbumin and IgG. When compared to the bacterial ENGase, Endo H, the relative ratio of high-mannose oligosaccharide structural isoforms released from RNase B by Endo Tv was found to differ, with Endo Tv releasing more Man?GlcNAc and Man?GlcNAc isoform I and less Man(9)GlcNAc from RNase B. Based on these data, it is suggested that use of ENGases from multiple sources may serve to balance an introduced bias in quantitative analysis of released structural isoforms and may further prove valuable in biochemical structure-function studies.