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Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

Background Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer’s disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function. Methods We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays. Results PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function. Conclusions The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. K D values broadly agree with published biochemical measurements.

Analyses of families affected by cold urticaria, immunodeficiency, and autoimmunity implicate mutations that activate phospholipase Cγ2 (PLCγ2), an enzyme pivotal to the translation of binding events at the cell surface to the intracellular milieu, as a cause of the disease. The genetic dissection of unique inflammatory phenotypes can identify and elucidate immunologic pathways and mechanisms. Such investigations have ultimately led to findings whose significance extends beyond the monogenic diseases harboring the mutations. Examples include the recognition that FOXP3 is essential for the differentiation of regulatory T cells in Scurfy mice and in patients with profound immune dysregulation, 1 – 4 the demonstration of a critical role for AIRE in thymic negative selection of T cells in patients with a specific autoimmune polyendocrinopathy, 5 and the identification of NLRP3 as a critical regulator of interleukin-1 in families with cold-induced inflammation. 6 Cold-induced urticaria is a . . .

Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been hindered by challenges involved in their expression and extraction in a homogeneous, functionally-active form. The inherent enzymatic activity of receptor tyrosine kinases (RTKs) presents additional challenges. Oncogenic fusions of RTKs with heterologous partners represent a particularly difficult-to-express protein subtype due to their high flexibility, aggregation propensity and the lack of a known method for extraction within the native lipid environment. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesis in vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 h. The resultant protein was reconstituted using SMA copolymers with a specific yield of 300 µg/mL of lysate. Functionally, the protein demonstrated significant kinase domain phosphorylation ( t  <  0.0001 ). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins.

The auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome is a rare primary immunodeficiency caused by a gain-of-function mutation S707Y in the gene previously described in two patients from one family. The APLAID patients presented with early-onset blistering skin lesions, posterior uveitis, inflammatory bowel disease (IBD) and recurrent sinopulmonary infections caused by a humoral defect, but lacked circulating autoantibodies and had no cold-induced urticaria, contrary to the patients with the related PLAID syndrome. We describe a new APLAID patient who presented with vesiculopustular rash in the 1st weeks of life, followed by IBD, posterior uveitis, recurrent chest infections, interstitial pneumonitis, and also had sensorineural deafness and cutis laxa. Her disease has been refractory to most treatments, including IL1 blockers and a trial with ruxolitinib has been attempted. In this patient, we found a unique heterozygous missense L848P mutation in the gene, predicted to affect the PLCγ2 structure. Similarly to S707Y, the L848P mutation led to the increased basal and EGF-stimulated PLCγ2 activity . Whole blood assays showed reduced production of IFN-γ and IL-17 in response to polyclonal T-cell stimulation and reduced production of IL-10 and IL-1β after LPS stimulation. Reduced IL-1β levels and the lack of clinical response to treatment with IL-1 blockers argue against NLRP3 inflammasome hyperactivation being the main mechanism mediating the APLAID pathogenesis. Our findings indicate that L848P is novel a gain-of-function mutation that leads to PLCγ2 activation and suggest cutis laxa as a possible clinical manifestations of the APLAID syndrome.

Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demonstrated by both ex vivo calcium responses of the patient’s B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation.

Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections-achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.

Phospholipase Cε (PLCε) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLCε in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLCε in Ras-driven cancers, we generated two new mouse strains: one carrying a targeted deletion of Plce (Plce-/-) and the other carrying mutant alleles of Plce unable to bind to Ras (PlceRAm/RAm). The Plce-/- and, to a lesser degree, PlceRAm/RAm transgenic mice exhibited increased susceptibility to tumor formation in the two-stage skin carcinogenesis protocol, revealing a tumor suppressor function for this PLC. This result also suggests that in this context Ras binding in part regulates functions of PLCε. Although significant differences were not seen in the LSL-KrasG12D nonsmall cell lung carcinoma model, down-regulation of PLCε was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLCε on cell growth requires intact lipase activity and is likely mediated by protein kinase C enzymes. Further cellular studies suggest involvement of histone deacetylase in the mechanism of PLCε down-regulation. Taken together, our results show a previously unidentified tumor suppressor role for this PLC in animal models and, together with observations of marked down-regulation in colorectal, lung, and skin tumors, suggest its use as a biological marker in cancer.

Key Points The phosphoinositide signalling system can be viewed as a network of interconverting enzymes, phospholipid messengers and their binding proteins. Interactions between phosphoinositides and their binding proteins are key to their regulatory actions. Control of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) signals in cell survival, proliferation and growth is frequently dysfunctional in cancer, resulting in enhanced PtdIns(3,4,5)P 3 signalling. The phosphoinositide enzymes PI3KIα and 3-phosphatase PTEN stringently control this signalling. Some important downstream targets of PtdIns(3,4,5)P 3 , such as the protein kinase Akt, are also regulated by PtdIns(3,4)P 2 . The phosphoinositide enzymes that generate PtdIns(3,4)P 2 from PtdIns(3,4,5)P 3 , phosphoinositide 5-phosphatases, and enzymes that degrade PtdIns(3,4)P 2 to PtdIns3P, phosphoinositide 4-phosphatases, are also implicated in cancer. The most abundant phosphoinositide, PtdIns(4,5)P 2 , binds to proteins important for actin polymerization, formation and turnover of focal contacts and cell–cell adhesion. These proteins could be regulated by local changes in the PtdIns(4,5)P 2 levels through the action of enzymes such as PtdIns4P-5 kinases (PIPKIγ) and PLC (PLCγ) that are implicated in the regulation of cancer cell motility. In addition to the local regulation of PtdIns(4,5)P 2 levels, diverse functions of PLC enzymes in cancer could be mediated by the generation of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate or in some instances by their function as signalling scaffolds. The most important outstanding task is to further evaluate the role and extent of involvement of different phosphoinositide enzymes in the generation and progression of human tumours. Key roles in cancer development have been established for PI3K and PTEN — enzymes that regulate the levels of phosphatidylinositol-3,4,5-trisphosphate, but several other phosphoinositide-modifying enzymes have been implicated in the generation and progression of tumours. New insights into the mechanisms and the extent of their involvement in cancer are summarized in this Review. There are numerous studies that suggest multiple links between the cellular phosphoinositide system and cancer. As key roles in cancer have been established for PI3K and PTEN — enzymes that regulate the levels of phosphatidylinositol-3,4,5-trisphosphate — compounds targeting this pathway are entering the clinic at a rapid pace. Several other phosphoinositide-modifying enzymes, including phosphoinositide kinases, phosphatases and phospholipase C enzymes, have been implicated in the generation and progression of tumours. Studies of these enzymes are providing new insights into the mechanisms and the extent of their involvement in cancer, highlighting new potential targets for therapeutic intervention.