Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy

Fluorescence fluctuation methods such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis (FIDA) have proven to be versatile tools for studying molecular interactions with single molecule sensitivity. Another well-known fluorescence technique is the measurement of the fluorescence lifetime. Here, we introduce a method that combines the benefits of both FIDA and fluorescence lifetime analysis. It is based on fitting the two-dimensional histogram of the number of photons detected in counting time intervals of given width and the sum of excitation to detection delay times of these photons. Referred to as fluorescence intensity and lifetime distribution analysis (FILDA), the technique distinguishes fluorescence species on the basis of both their specific molecular brightness and the lifetime of the excited state and is also able to determine absolute fluorophore concentrations. The combined information yielded by FILDA results in significantly increased accuracy compared to that of FIDA or fluorescence lifetime analysis alone. In this paper, the theory of FILDA is elaborated and applied to both simulated and experimental data. The outstanding power of this technique in resolving different species is shown by quantifying the binding of calmodulin to a peptide ligand, thus indicating the potential for application of FILDA to similar problems in the life sciences.     

Crystallization and preliminary crystallographic analysis of manganese superoxide dismutase from Bacillus halodenitrificans

Manganese superoxide dismutase (GP-MnSOD), a component of the so-called 'green protein' (green protein complex) from the facultative anaerobic halodenitrifier Bacillus halodenitrificans, has been crystallized using the hanging-drop vapor diffusion method. Crystals have unit-cell parameters a=b=93.4 A, c=65.0 A, and belong to the space group P4(3)2(1)2. Preliminary analysis indicates there is one monomer in each asymmetric unit. The structural information from this enzyme will enrich our knowledge on its high catalytic activity and its possible role in green protein complex.     

Cdc25C expression in meiotically competent and incompetent goat oocytes

Change in Cdc25C expression and localization during maturation and meiotic competence acquisition was investigated in goat oocytes. Western blot analysis revealed that Cdc25C is constitutively expressed throughout meiosis in competent goat oocytes, with changes in its phosphorylation level. Cdc25C was detected at 55 and 70 kDa, representing the nonphosphorylated form and the hyperphosphorylated active form, respectively. During the G2-M transition at meiosis resumption, Cdc25C was hyperphosphorylated as evidenced by a clear shift from 55 to 70 kDa. Okadaic acid which induced premature meiosis resumption associated with MPF activation also involved a premature shift from 55 to 70 kDa in goat competent oocytes. After artificial activation of goat oocytes, Cdc25C returned to its 55 kDa form. By indirect immunofluorescence, Cdc25C was found essentially localized in the nucleus at the germinal vesicle stage, suggesting that Cdc25C functions within the nucleus to regulate MPF activation. Concomitantly with germinal vesicle breakdown, Cdc25C was redistributed throughout the cytoplasm. The amount of Cdc25C, very low in incompetent oocytes, increased with meiosis competence acquisition. On the other hand, during oocyte growth while the expression of Cdc25C increased, its phosphorylation level increased concomitantly as well as its nuclear translocation. These results suggest that meiosis resumption needs a sufficient amount of Cdc25C which must be completely phosphorylated and nuclear and that the amount of Cdc25C may be a limiting factor for meiotic competence acquisition. We could consider that Cdc25C nuclear translocation and phosphorylation, during oocyte growth, prepare the oocytes in advance for the G2-M phase transition occurring during meiosis resumption.     

The indestructible insect: Velvet ants from across the United States avoid predation by representatives from all major tetrapod clades

Velvet ants are a group of parasitic wasps that are well known for a suite of defensive adaptations including bright coloration and a formidable sting. While these adaptations are presumed to function in antipredator defense, observations between potential predators and this group are lacking. We conducted a series of experiments to determine the risk of velvet ants to a host of potential predators including amphibians, reptiles, birds, and small mammals. Velvet ants from across the United States were tested with predator's representative of the velvet ants native range. All interactions between lizards, free‐ranging birds, and a mole resulted in the velvet ants survival, and ultimate avoidance by the predator. Two shrews did injure a velvet ant, but this occurred only after multiple failed attacks. The only predator to successfully consume a velvet ant was a single American toad (Anaxyrus americanus). These results indicate that the suite of defenses possessed by velvet ants, including aposematic coloration, stridulations, a chemical alarm signal, a hard exoskeleton, and powerful sting are effective defenses against potential predators. Female velvet ants appear to be nearly impervious to predation by many species whose diet is heavily derived of invertebrate prey.     

Inferring the origin and genetic diversity of the introduced wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) populations in Argentina: an approach from mitochondrial markers

The Eurasian wild boar (Sus scrofa Linnaeus, 1758) was introduced into Argentina at the beginning of the twentieth century when individuals from Europe were taken to La Pampa province for hunting purposes. Starting from there, a dispersal process began due to the invasive characteristics of the species and to human-mediated translocations. The main objective of this study was to characterize for the first time, the phylogenetic relationships among wild boars from Argentina with those from Uruguay, Europe, Asia, and the Near East, along with diverse domestic pig breeds in order to corroborate the historical information about the origin of the local populations. To this end, we used mitochondrial Control Region and Cytochrome b sequences from sampled Argentinian wild boars and retrieved from GenBank. The results showed that the majority of the Argentinian wild boar populations descend from European lineages, in particular of the E1 clade, according to the historical records. Remarkably, the population of El Palmar National Park had Asian origin that could be attributed to hybridization with local domestic pigs or to unrecorded translocations. Finally, genetic diversity in Argentinian populations was lower than in Europe and Uruguay meaning that wild boar in Argentina is still under the influence of founder effect and has experienced minor genetic introgression from domestic pigs, representing in this sense a reservoir of the original wild boar genetic variability.     

The IKK‐related kinase TBK1 activates mTORC1 directly in response to growth factors and innate immune agonists

The innate immune kinase TBK1 initiates inflammatory responses to combat infectious pathogens by driving production of type I interferons. TBK1 also controls metabolic processes and promotes oncogene‐induced cell proliferation and survival. Here, we demonstrate that TBK1 activates mTOR complex 1 (mTORC1) directly. In cultured cells, TBK1 associates with and activates mTORC1 through site‐specific mTOR phosphorylation (on S2159) in response to certain growth factor receptors (i.e., EGF‐receptor but not insulin receptor) and pathogen recognition receptors (PRRs) (i.e., TLR3; TLR4), revealing a stimulus‐selective role for TBK1 in mTORC1 regulation. By studying cultured macrophages and those isolated from genome edited mTOR S2159A knock‐in mice, we show that mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFN‐β production. These data demonstrate a direct mechanistic link between TBK1 and mTORC1 function as well as physiologic significance of the TBK1‐mTORC1 axis in control of innate immune function. These data unveil TBK1 as a direct mTORC1 activator and suggest unanticipated roles for mTORC1 downstream of TBK1 in control of innate immunity, tumorigenesis, and disorders linked to chronic inflammation.     

Terpenes associated with resistance against the gall wasp,Leptocybe invasa,in Eucalyptus grandis

Leptocybe invasa is an insect pest causing gall formation on oviposited shoot tips and leaves of Eucalyptus trees leading to leaf deformation, stunting, and death in severe cases. We previously observed different constitutive and induced terpenes, plant specialized metabolites that may act as attractants or repellents to insects, in a resistant and susceptible clone of Eucalyptus challenged with L. invasa. We tested the hypothesis that specific terpenes are associated with pest resistance in a Eucalyptus grandis half‐sib population. Insect damage was scored over 2 infestation cycles, and leaves were harvested for near‐infrared reflectance (NIR) and terpene measurements. We used Bayesian model averaging for terpene selection and obtained partial least squares NIR models to predict terpene content and L. invasa infestation damage. In our optimal model, 29% of the phenotypic variation could be explained by 7 terpenes, and the monoterpene combination, limonene, α‐terpineol, and 1,8‐cineole, could be predicted with an NIR prediction ability of  .67. Bayesian model averaging supported α‐pinene, γ‐terpinene, and iso‐pinocarveol as important for predicting L. invasa infestation. Susceptibility was associated with increased γ‐terpinene and α‐pinene, which may act as a pest attractant, whereas reduced susceptibility was associated with iso‐pinocarveol, which may act to recruit parasitoids or have direct toxic effects.     

The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release

Background

Connexin43 hemichannels have been implicated in many inflammatory diseases including diabetic retinopathy (DR). Particularly, hemichannel-mediated ATP release has been associated with inflammasome pathway activation. Using an in vitro cell culture model, we evaluated hemichannel roles in response to inflammatory cytokines under high glucose (HG) conditions and propose a mechanism by which a connexin43 hemichannel-mediated autocrine ATP feedback loop augments chronic inflammatory disease.