Protein kinase affinity reagents based on a 5-aminoindazole scaffold

Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be derivatized with diverse binding elements and immobilized analogs allow selective enrichment of protein kinases from complex mixtures.     

LaPO4 nanoparticles doped with actinium-225 that partially sequester daughter radionuclides

Nanoscale materials have been envisioned as carriers for various therapeutic drugs, including radioisotopes. Inorganic nanoparticles (NPs) are particularly appealing vehicles for targeted radiotherapy because they can package several radioactive atoms into a single carrier and can potentially retain daughter radioisotopes produced by in vivo generators such as actinium-225 ((225)Ac, t(1/2) = 10 d). Decay of this radioisotope to stable bismuth-209 proceeds through a chain of short-lived daughters accompanied by the emission of four α-particles that release >27 MeV of energy. The challenge in realizing the enhanced cytotoxic potential of in vivo generators lies in retaining the daughter nuclei at the therapy site. When (225)Ac is attached to targeting agents via standard chelate conjugation methods, all of the daughter radionuclides are released after the initial α-decay occurs. In this work, (225)Ac was incorporated into lanthanum phosphate NPs to determine whether the radioisotope and its daughters would be retained within the dense mineral lattice. Further, the (225)Ac-doped NPs were conjugated to the monoclonal antibody mAb 201B, which targets mouse lung endothelium through the vasculature, to ascertain the targeting efficacy and in vivo retention of radioisotopes. Standard biodistribution techniques and microSPECT/CT imaging of (225)Ac as well as the daughter radioisotopes showed that the NPs accumulated rapidly in mouse lung after intravenous injection. By showing that excess, competing, uncoupled antibodies or NPs coupled to control mAbs are deposited primarily in the liver and spleen, specific targeting of NP-mAb 201B conjugates was demonstrated. Biodistribution analysis showed that ～30% of the total injected dose of La((225)Ac)PO(4) NPs accumulated in mouse lungs 1 h postinjection, yielding a value of % ID/g >200. Furthermore, after 24 h, 80% of the (213)Bi daughter produced from (225)Ac decay was retained within the target organ and (213)Bi retention increased to ～87% at 120 h. In vitro analyses, conducted over a 1 month interval, demonstrated that ～50% of the daughters were retained within the La((225)Ac)PO(4) NPs at any point over that time frame. Although most of the γ-rays from radionuclides in the (225)Ac decay chain are too energetic to be captured efficiently by SPECT detectors, appropriate energy windows were found that provided dramatic microSPECT images of the NP distribution in vivo. We conclude that La((225)Ac)PO(4)-mAb 201B conjugates can be targeted efficiently to mouse lung while partially retaining daughter products and that targeting can be monitored by biodistribution techniques and microSPECT imaging.     

Boltzmann energy-based image analysis demonstrates that extracellular domain size differences explain protein segregation at immune synapses

Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell:cell communication and cell signalling, governing protein interactions and protein aggregation.     

Measured and estimated ground reaction forces for multi-segment foot models

Accurate measurement of ground reaction forces under discrete areas of the foot is important in the development of more advanced foot models, which can improve our understanding of foot and ankle function. To overcome current equipment limitations, a few investigators have proposed combining a pressure mat with a single force platform and using a proportionality assumption to estimate subarea shear forces and free moments. In this study, two adjacent force platforms were used to evaluate the accuracy of the proportionality assumption on a three segment foot model during normal gait. Seventeen right feet were tested using a targeted walking approach, isolating two separate joints: transverse tarsal and metatarsophalangeal. Root mean square (RMS) errors in shear forces up to 6% body weight (BW) were found using the proportionality assumption, with the highest errors (peak absolute errors up to 12% BW) occurring between the forefoot and toes in terminal stance. The hallux exerted a small braking force in opposition to the propulsive force of the forefoot, which was unaccounted for by the proportionality assumption. While the assumption may be suitable for specific applications (e.g. gait analysis models), it is important to understand that some information on foot function can be lost. The results help highlight possible limitations of the assumption. Measured ensemble average subarea shear forces during normal gait are also presented for the first time.     

The effects of articulated figure skates on jump landing forces

Lower extremity injuries in figure skating have long been linked to skating boot stiffness, and recent increases in jump practice time may be influencing the frequency and seriousness of these injuries. It is hypothesized that stiff boots compromise skaters' abilities to attenuate jump landing forces. Decreasing boot stiffness by adding an articulation at the ankle may reduce the rate and magnitude of landing forces. Prototype articulated figure skating boots were tested in this study to determine their effectiveness in enabling skaters to land with lower peak impact forces. Nine competitive figure skaters, who trained in standard boots and subsequently in articulated boots, performed off-ice jump simulations and on-ice axels, double toe loops, and double axels. Analysis of the off-ice simulations showed decreases in peak heel force and loading rate with use of the articulated boot, although the exact kinematic mechanisms responsible for these decreases are still unclear. Analysis of the on-ice jumps revealed few kinematic differences between boot types, implying that the skaters did not use the articulation. Greater adaptation and training time is likely needed for the results seen off-ice to transfer to difficult on-ice jumps.     

Mechanosensing in T lymphocyte activation

Mechanical forces play an increasingly recognized role in modulating cell function. This report demonstrates mechanosensing by T cells, using polyacrylamide gels presenting ligands to CD3 and CD28. Naive CD4 T cells exhibited stronger activation, as measured by attachment and secretion of IL-2, with increasing substrate elastic modulus over the range of 10–200 kPa. By presenting these ligands on different surfaces, this report further demonstrates that mechanosensing is more strongly associated with CD3 rather than CD28 signaling. Finally, phospho-specific staining for Zap70 and Src family kinase proteins suggests that sensing of substrate rigidity occurs at least in part by processes downstream of T-cell receptor activation. The ability of T cells to quantitatively respond to substrate rigidly provides an intriguing new model for mechanobiology.     

Explosive radiation of a bacterial species group

The current diversity of life on earth is the product of macroevolutionary processes that have shaped the dynamics of diversification. Although the tempo of diversification has been studied extensively in macroorganisms, much less is known about the rates of diversification in the exceedingly diverse and species-rich microbiota. Decreases in diversification rates over time, a signature of explosive radiations, are commonly observed in plant and animal lineages. However, the few existing analyses of microbial lineages suggest that the tempo of diversification in prokaryotes may be fundamentally different. Here, we use multilocus and genomic sequence data to test hypotheses about the rate of diversification in a well-studied pathogenic bacterial lineage, Borrelia burgdorferi sensu lato (sl). Our analyses support the hypothesis that an explosive radiation of lineages occurred near the origin of the clade, followed by a sharp decay in diversification rates. These results suggest that explosive radiations may be a general feature of evolutionary history across the tree of life.     

Probabilistic modeling of selective stimulation of the human sciatic nerve with a flat interface nerve electrode

Ankle control is critical to both standing balance and efficient walking. The hypothesis presented in this paper is that a Flat Interface Nerve Electrode (FINE) placed around the sciatic nerve with a fixed number of contacts at predetermined locations and without a priori knowledge of the nerve's underlying neuroanatomy can selectively control each ankle motion. Models of the human sciatic nerve surrounded by a FINE of varying size were created and used to calculate the probability of selective activation of axons within any arbitrarily designated, contiguous group of fascicles. Simulations support the hypothesis and suggest that currently available implantable technology cannot selectively recruit each target plantar flexor individually but can restore plantar flexion or dorsiflexion from a site on the sciatic nerve without spillover to antagonists. Successful activation of individual ankle muscles in 90% of the population can be achieved by utilizing bipolar stimulation and/or by using a cuff with at least 20 contacts.     

Fluctuating environments, sexual selection and the evolution of flexible mate choice in birds

Environmentally-induced fluctuation in the form and strength of natural selection can drive the evolution of morphology, physiology, and behavior. Here we test the idea that fluctuating climatic conditions may also influence the process of sexual selection by inducing unexpected reversals in the relative quality or sexual attractiveness of potential breeding partners. Although this phenomenon, known as 'ecological cross-over', has been documented in a variety of species, it remains unclear the extent to which it has driven the evolution of major interspecific differences in reproductive behavior. We show that after controlling for potentially influential life history and demographic variables, there are significant positive associations between the variability and predictability of annual climatic cycles and the prevalence of infidelity and divorce within populations of a taxonomically diverse array of socially monogamous birds. Our results are consistent with the hypothesis that environmental factors have shaped the evolution of reproductive flexibility and suggest that in the absence of severe time constraints, secondary mate choice behaviors can help prevent, correct, or minimize the negative consequences of ecological cross-overs. Our findings also illustrate how a basic evolutionary process like sexual selection is susceptible to the increasing variability and unpredictability of climatic conditions that is resulting from climate change.