Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers

The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer

Effects of axial ligands on the photophysical properties of silicon octaphenoxyphthalocyanine

The photochemistry and photophysics of six axially substituted silicon phthalocyanines are reported and show the importance of the axial groups in the photochemistry of these compounds. The fluorescence quantum yields are especially affected by the axial ligand. A very good correlation was found for the experimentally determined fluorescence lifetimes and the theoretically determined lifetimes using the Strickler-Berg equation for the unaggregated molecules

Time-resolved fluorescence anisotropy and molecular dynamics analysis of a novel GFP homo-FRET dimer

Förster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (〈E FRET exp〉 = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (〈E FRET MD〉 = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors 〈κ 2〉 = 0.17 ± 0.16 and 〈|κ|〉 = 0.35 ± 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time- and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET. </p

Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques

A time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo

Imaging the spatio-temporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using FRET measured by fluorescence lifetime imaging with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo

Scientists' warning to humanity on insect extinctions

Here we build on the manifesto ‘World Scientists’ Warning to Humanity, issued by the Alliance of World Scientists. As a group of conservation biologists deeply concerned about the decline of insect populations, we here review what we know about the drivers of insect extinctions, their consequences, and how extinctions can negatively impact humanity. We are causing insect extinctions by driving habitat loss, degradation, and fragmentation, use of polluting and harmful substances, the spread of invasive species, global climate change, direct overexploitation, and co-extinction of species dependent on other species. With insect extinctions, we lose much more than species. We lose abundance and biomass of insects, diversity across space and time with consequent homogenization, large parts of the tree of life, unique ecological functions and traits, and fundamental parts of extensive networks of biotic interactions. Such losses lead to the decline of key ecosystem services on which humanity depends. From pollination and decomposition, to being resources for new medicines, habitat quality indication and many others, insects provide essential and irreplaceable services. We appeal for urgent action to close key knowledge gaps and curb insect extinctions. An investment in research programs that generate local, regional and global strategies that counter this trend is essential. Solutions are available and implementable, but urgent action is needed now to match our intentions.Peer reviewe

Solutions for humanity on how to conserve insects

The fate of humans and insects intertwine, especially through the medium of plants. Global environmental change, including land transformation and contamination, is causing concerning insect diversity loss, articulated in the companion review Scientists' warning to humanity on insect extinctions. Yet, despite a sound philosophical foundation, recognized ethical values, and scientific evidence, globally we are performing poorly at instigating effective insect conservation. As insects are a major component of the tapestry of life, insect conservation would do well to integrate better with overall biodiversity conservation and climate change mitigation. This also involves popularizing insects, especially through use of iconic species, through more media coverage, and more inclusive education. Insect conservationists need to liaise better with decision makers, stakeholders, and land managers, especially at the conceptually familiar scale of the landscape. Enough evidence is now available, and synthesized here, which illustrates that multiple strategies work at local levels towards saving insects. We now need to expand these locally-crafted strategies globally. Tangible actions include ensuring maintenance of biotic complexity, especially through improving temporal and spatial heterogeneity, functional connectivity, and metapopulation dynamics, while maintaining unique habitats, across landscape mosaics, as well as instigating better communication. Key is to have more expansive sustainable agriculture and forestry, improved regulation and prevention of environmental risks, and greater recognition of protected areas alongside agro-ecology in novel landscapes. Future-proofing insect diversity is now critical, with the benefits far reaching, including continued provision of valuable ecosystem services and the conservation of a rich and impressive component of Earth's biodiversity.Peer reviewe

Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

Constitutive receptor tyrosine kinase phosphorylation requires regulation of kinase and phosphatase activity to prevent aberrant signal transduction. A dynamic mechanism is described here in which the adaptor protein, growth factor receptor–bound protein 2 (Grb2), controls fibroblast growth factor receptor 2 (FGFR2) signaling by regulating receptor kinase and SH2 domain–containing protein tyrosine phosphatase 2 (Shp2) phosphatase activity in the absence of extracellular stimulation. FGFR2 cycles between its kinase-active, partially phosphorylated, nonsignaling state and its Shp2-dephosphorylated state. Concurrently, Shp2 cycles between its FGFR2-phosphorylated and dephosphorylated forms. Both reciprocal activities of FGFR2 and Shp2 were inhibited by binding of Grb2 to the receptor. Phosphorylation of Grb2 by FGFR2 abrogated its binding to the receptor, resulting in up-regulation of both FGFR2’s kinase and Shp2’s phosphatase activity. Dephosphorylation of Grb2 by Shp2 rescued the FGFR2–Grb2 complex. This cycling of enzymatic activity results in a homeostatic, signaling-incompetent state. Growth factor binding perturbs this background cycling, promoting increased FGFR2 phosphorylation and kinase activity, Grb2 dissociation, and downstream signaling. Grb2 therefore exerts constitutive control over the mutually dependent activities of FGFR2 and Shp2