Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K_ds) of biomolecules. The determination of a K_d depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ～0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the K_d.     

Structural and Functional Characterization of a Novel Homodimeric Three-finger Neurotoxin from the Venom of Ophiophagus hannah (King Cobra)

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (αβγδ) and neuronal (α₇, α₃β₂, and α₄β₂) nicotinic acetylcholine receptors (nAChRs) with highest affinity for α₇-nAChRs. The high resolution (1.5 Å) crystal structure revealed haditoxin to be a homodimer, like κ-neurotoxins, which target neuronal α₃β₂- and α₄β₂-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain α-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain α-neurotoxins and κ-neurotoxins notwithstanding, haditoxin exhibited unique blockade of α₇-nAChRs (IC₅₀ 180 nm), which is recognized by neither short-chain α-neurotoxins nor κ-neurotoxins. This is the first report of a dimeric short-chain α-neurotoxin interacting with neuronal α₇-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.     

Evolution of resistance to anti-cancer therapy during general dosing schedules

Anti-cancer drugs targeted to specific oncogenic pathways have shown promising therapeutic results in the past few years; however, drug resistance remains an important obstacle for these therapies. Resistance to these drugs can emerge due to a variety of reasons including genetic or epigenetic changes which alter the binding site of the drug target, cellular metabolism or export mechanisms. Obtaining a better understanding of the evolution of resistant populations during therapy may enable the design of more effective therapeutic regimens which prevent or delay progression of disease due to resistance. In this paper, we use stochastic mathematical models to study the evolutionary dynamics of resistance under time-varying dosing schedules and pharmacokinetic effects. The populations of sensitive and resistant cells are modeled as multi-type non-homogeneous birth-death processes in which the drug concentration affects the birth and death rates of both the sensitive and resistant cell populations in continuous time. This flexible model allows us to consider the effects of generalized treatment strategies as well as detailed pharmacokinetic phenomena such as drug elimination and accumulation over multiple doses. We develop estimates for the probability of developing resistance and moments of the size of the resistant cell population. With these estimates, we optimize treatment schedules over a subspace of tolerated schedules to minimize the risk of disease progression due to resistance as well as locate ideal schedules for controlling the population size of resistant clones in situations where resistance is inevitable. Our methodology can be used to describe dynamics of resistance arising due to a single (epi)genetic alteration in any tumor type.     

Limitations of cross‐ and multigenerational plasticity for marine invertebrates faced with global climate change

Although cross generation (CGP) and multigenerational (MGP) plasticity have been identified as mechanisms of acclimation to global change, the weight of evidence indicates that parental conditioning over generations is not a panacea to rescue stress sensitivity in offspring. For many species, there were no benefits of parental conditioning. Even when improved performance was observed, this waned over time within a generation or across generations and fitness declined. CGP and MGP studies identified resilient species with stress tolerant genotypes in wild populations and selected family lines. Several bivalves possess favourable stress tolerance and phenotypically plastic traits potentially associated with genetic adaptation to life in habitats where they routinely experience temperature and/or acidification stress. These traits will be important to help ‘climate proof’ shellfish ventures. Species that are naturally stress tolerant and those that naturally experience a broad range of environmental conditions are good candidates to provide insights into the physiological and molecular mechanisms involved in CGP and MGP. It is challenging to conduct ecologically relevant global change experiments over the long times commensurate with the pace of changing climate. As a result, many studies present stressors in a shock‐type exposure at rates much faster than projected scenarios. With more gradual stressor introduction over longer experimental durations and in context with conditions species are currently acclimatized and/or adapted to, the outcomes for sensitive species might differ. We highlight the importance to understand primordial germ cell development and the timing of gametogenesis with respect to stressor exposure. Although multigenerational exposure to global change stressors currently appears limited as a universal tool to rescue species in the face of changing climate, natural proxies of future conditions (upwelling zones, CO₂ vents, naturally warm habitats) show that phenotypic adjustment and/or beneficial genetic selection is possible for some species, indicating complex plasticity–adaptation interactions.     

Auxin influences strigolactones in pea mycorrhizal symbiosis

Hormone interactions are essential for the control of many developmental processes, including intracellular symbioses. The interaction between auxin and the new plant hormone strigolactone in the regulation of arbuscular mycorrhizal symbiosis was examined in one of the few auxin deficient mutants available in a mycorrhizal species, the auxin-deficient bsh mutant of pea (Pisum sativum). Mycorrhizal colonisation with the fungus Glomus intraradices was significantly reduced in the low auxin bsh mutant. The bsh mutant also exhibited a reduction in strigolactone exudation and the expression of a key strigolactone biosynthesis gene (PsCCD8). Strigolactone exudation was also reduced in wild type plants when the auxin content was reduced by stem girdling. Low strigolactone levels appear to be at least partially responsible for the reduced colonisation of the bsh mutant, as application of the synthetic strigolactone GR24 could partially rescue the mycorrhizal phenotype of bsh mutants. Data presented here indicates root auxin content was correlated with strigolactone exudation in both mutant and wild type plants. Mutant studies suggest that auxin may regulate early events in the formation of arbuscular mycorrhizal symbiosis by controlling strigolactone levels, both in the rhizosphere and possibly during early root colonisation.     

Strigolactones: New Physiological Roles for an Ancient Signal

Strigolactones are an ancient group of plant signalling molecules. They play a critical role in the rhizosphere where they facilitate the formation of symbioses with fungi, crucial for the acquisition of plant nutrients in over 80 % of land plant species. Strigolactones have also been exploited by parasitic weeds as a rhizosphere signal indicating the presence of a host species, resulting in devastating losses in some agricultural systems. Recently, they have also been shown to act endogenously as plant hormones controlling shoot branching and have been implicated in a wide range of other physiological processes, including root growth, root-hair elongation, adventitious rooting, secondary growth, photomorphogenesis, seed germination, nodulation, and protonemal development in mosses. Here, we discuss the evidence for the involvement of strigolactones as endogenous regulators of these processes and highlight some examples where the evidence is inconclusive. One major gap in our understanding is the identity of the endogenous strigolactone(s) that are biologically active. A discussion of the interactions between the different plant hormones and the possible role of strigolactones as integrators of the root-to-shoot balance, nutrient acquisition, and thus resource allocation illustrates some important future directions for this area of research.     

Strigolactones promote nodulation in pea

Strigolactones are recently defined plant hormones with roles in mycorrhizal symbiosis and shoot and root architecture. Their potential role in controlling nodulation, the related symbiosis between legumes and Rhizobium bacteria, was explored using the strigolactone-deficient rms1 mutant in pea (Pisum sativum L.). This work indicates that endogenous strigolactones are positive regulators of nodulation in pea, required for optimal nodule number but not for nodule formation per se. rms1 mutant root exudates and root tissue are almost completely deficient in strigolactones, and rms1 mutant plants have approximately 40% fewer nodules than wild-type plants. Treatment with the synthetic strigolactone GR24 elevated nodule number in wild-type pea plants and also elevated nodule number in rms1 mutant plants to a level similar to that seen in untreated wild-type plants. Grafting studies revealed that nodule number and strigolactone levels in root tissue of rms1 roots were unaffected by grafting to wild-type scions indicating that strigolactones in the root, but not shoot-derived factors, regulate nodule number and provide the first direct evidence that the shoot does not make a major contribution to root strigolactone levels.     

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.     

IL-33 activates B1 cells and exacerbates contact sensitivity

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33-activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33-activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2(-/-) mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.