MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Marine and estuarine gradients — An overview

The Proceedings of the ECSA 21 symposium on Marine and Estuarine Gradients are reviewed. It is emphasised that this is probably the first time that a full set of papers on the tidal freshwater sections of estuaries has appeared. There is however some ambiguity in the terminology applied to such waters, and a more consistent terminology is proposed. In particular the estuary is defined as reaching upstream as far as the tidal limit, irrespective of salinity. A wide variety of gradients within estuaries and coastal waters are considered, but it is apparent that the crucial spatial gradients are based on salinity, oxygen and turbidity, and that many other gradients are co-variables with these parameters. Temperature is also important for temporal gradients.     

Seasonal growth of Atlantic cod: effects of temperature, feeding and reproduction

Growth of 2659 Atlantic cod Gadus morhua aged 4 to 9 years examined in Placentia Bay, Newfoundland, peaked in most cases in June and was at a minimum in October or November. Water temperature, partial fullness index ( I _P) and gonado‐somatic index ( I _G) explained between 31 and 52% of the monthly variability in growth. Temperature and I _P of capelin Mallotus villosus had significant effects on growth of all age groups and explained most of the variance for ages 6–8 and 4–5 years, respectively. The I _P of large invertebrates (ages 4 to 7 years), sandlance ( Ammodytes sp. age 6 years) and demersal fishes (age 9 years) had age‐specific effects in the model. Overall, amphipods, decapods and echinoderms dominated the Atlantic cod diet in most seasons, but fish consumption by Atlantic cod was high in June and July, particularly on capelin. The rapid increase in somatic mass during June and July occurred despite cold water temperatures ( < 3° C at 50 m) and moderate to high gonado‐somatic index. The findings of this study suggest that when food was not a limiting factor, growth tended to increase even when Atlantic cod occupied colder waters, but when food was limiting, the opposite may have occured.     

The effect of caffeine,different fixation regimes and low temperature on microtubules in the cells of higher plants

Caffeine, (1:3:7-tri-methyl-xanthine), either as a prefixation treatment or included with glutaralde-hyde as the primary fixative, destroys or disorganises the microtubules associated with the formation of secondary walls in fibres from the flowering stem of the grass Lolium temulentum L. There is no observable effect of caffeine treatment on the microtubules associated with primary wall formation in collenchyma and young fibres from L. temulentum or in root cap cells of Zea mays L. and Phaseolus vulgaris L. The microtubules associated with primary wall formation are destroyed by cold treatment but not those associated with secondary wall formation. Tannic acid included in the fixative shows the microtubules associated with secondary wall formation in fibres of L. temulentum to be composed of 13 subunits. Treatment with lanthanum hydroxide does not stain the core or the halo of the microtubules.Abbreviation PIPES Piperazine N-N- bis 2 ethanol sulphonic acid The Grassland Research Institute is financed through the Agricultural Research Council     

Maternal induction of larval diapause and its sensitive stage in the blow fly Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Lucilia sericata has a facultative diapause in the third larval instar after cessation of feeding. Induction of the diapause is influenced by the photoperiod and temperature conditions experienced by insects in the parental generation as well as those experienced by the larvae themselves. The sensitive stage of the parental generation for induction of diapause was examined using diapause‐averting conditions of 16 h light : 8 h darkness (LD 16:8) at 25°C and diapause‐inducing conditions of LD 12:12 at 20°C. The incidence of diapause in the progeny was predominantly determined by the conditions experienced by the parents in the adult stage. Moreover, the results of reciprocal crosses showed that only the mother's experience is involved in the induction of diapause in the progeny.     

Socio-ecological correlates of neophobia in corvids

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The effect of dielectric polarization of the electrode on anomalous temperature effects in the electrical double layer

Monte Carlo simulations and a modified Poisson–Boltzmann (MPB) theory are used to investigate the temperature dependence of the capacitance (around the potential of zero charge) of an electric double layer in the presence of surface polarization due to a dielectric boundary. Within the context of the restricted primitive model planar double layer, whose solvent dielectric constant is ε₂, the cases when the electrode is an insulator (ε₁ = 1), when the electrode and the electrolyte have the same permittivity (ε₁ = ε₂, no polarization), and when the electrode is a conductor (ε₁ → ∞) are studied for the case where the electrolyte concentration is 0.1 M. The simulations reveal a capacitance anomaly, that is, a positive temperature dependence of the capacitance at low temperatures for the former two situations. The MPB theory also shows this effect for these two situations and is in qualitative or better agreement with the simulation data. In these two cases, both the simulations and theory show a dramatic increase of the diffuse layer potential in the temperature regime where capacitance anomaly occurs. However, in the latter situation, where the electrode is metallic, the capacitance always has a negative temperature derivative for the MPB theory and probably also for the simulation data.     

A simple,band‐mounted device to measure behaviorally modified thermal microclimates experienced by birds

Birds encounter climate at the scale of microclimates that can vary rapidly in time and space and so understanding potential vulnerability and adaptations to those microclimates requires fine‐scale measurements that accurately track thermal exposures. However, few options exist for recording the microclimates actually experienced by birds (realized microclimates). We constructed and tested a simple, low‐cost, temperature logger for recording the realized microclimates of ground‐nesting birds. We developed attachment protocols for band‐mounting Thermochron iButtons on Ring‐billed Gull (Larus delawarensis) chicks. We tested these mounted, temperature‐logging devices on 20 chicks weighing > 200 g (device weight was 4 g), attaching devices for 48 h and observing behavior before and after attachment and removal. Devices recorded temperature immediately surrounding the leg at 2‐min intervals. Recorded temperatures were strong predictors of observed thermoregulatory behaviors (panting and sitting), outperforming predictions based on air temperatures measured by basic, static approaches. Through comparison with matched controls (chicks with just a band), we detected no adverse physiological effects of devices, no effects on social or feeding behavior, and only a short‐term decrease in inactivity immediately after device attachment (likely due to increased preening). By attaching iButtons to the legs of birds, we quantified realized thermal exposure, integrating air temperature, modes of environmental heat transfer, and bird behavior at microclimatic scales. Although not yet validated for broader use, our approach (including possible miniaturization) should be suitable to measure thermal exposure of adults, not just chicks, allowing collection of data concerning thermal exposures during flight under field conditions. At ~ $25 USD per device, our approach facilitates experimental protocols with robust sample sizes, even for relatively modest budgets.     

The natural history, thermal physiology, and ecological impacts of intertidal mesopredators, Oedoparena spp. (Diptera: Dryomyzidae)

Abstract. The ecological roles of small (1–1000 mg) predators in benthic marine systems are poorly understood. We investigated the natural history and predatory impact of one group of such mesopredators—larvae of dipteran flies in the genus Oedoparena —which prey on intertidal barnacles. We 1) quantified patterns of larval Oedoparena distribution and abundance in the Northwest Straits of Washington State, USA, 2) determined larval physiological tolerance limits in the laboratory, and 3) conducted a manipulative field experiment to assess the role of microhabitat temperature on predation rates in Oedoparena . Members of Oedoparena in Washington are univoltine, with peak larval abundance in late spring and early summer. Infestation frequencies in the barnacles Balanus glandula and Chthamalus dalli were as high as 22% and 35%, respectively. In laboratory studies, larvae of O . glauca were able to tolerate temperatures up to 37°C; however, this temperature is often exceeded in high intertidal habitats. In a field manipulation using experimental shades, we demonstrate that the alleviation of physiological stress greatly increased the abundance of larvae of Oedoparena spp. As a result of increased larval densities under shades, adult B. glandula mortality increased from 5% to nearly 30%, and C. dalli mortality increased from less than 20% to over 60%. Because high intertidal barnacles serve as food and habitat for a diverse array of species, Oedoparena spp. have the potential to play a major role in structuring high intertidal communities, particularly in cooler microhabitats.