Fluorescence Recovery After Photobleaching Research Articles

A General Autofluorescence Method to Characterize Polymerization Progress.

An autofluorescence technique to characterize polymerization progress in real time/in line was developed, which functioned in the absence of typical fluorogenic groups on the monomer or polymer. The monomer dicyclopentadiene and polymer polydicyclopentadiene are hydrocarbons that lack traditional functional groups for fluorescence spectroscopy. Here, the autofluorescence of formulations containing this monomer and polymer during ruthenium-catalyzed ring-opening metathesis polymerization (ROMP) was harnessed for reaction monitoring. The methods fluorescence recovery after photobleaching (FRAP) and here-developed fluorescence lifetime recovery after photobleaching (FLRAP) characterized polymerization progress in these native systems-without requiring exogenous fluorophore. (Auto)fluorescence lifetime recovery changes during polymerization correlated linearly to degree of cure, providing a quantitative link with reaction progress. These changing signals also provided relative rates of background polymerization, enabling comparison of 10 different catalyst-inhibitor-stabilized formulations. Multiple-well analysis demonstrated suitability for future high-throughput evaluation of formulations for thermosets. The central concept of the combined autofluorescence and FLRAP/FRAP method may be extendable to monitoring other polymerization reactions previously overlooked for lack of an obvious fluorescence handle.

Changes in local tissue microenvironment in response to subcutaneous long-acting delivery of tenofovir alafenamide in rats and non-human primates.

Several implantable long-acting (LA) delivery systems have been developed for sustained subcutaneous administration of tenofovir alafenamide (TAF), a potent and effective nucleotide reverse transcriptase inhibitor used for HIV pre-exposure prophylaxis (PrEP). LA platforms aim to address the lack of adherence to oral regimens, which has impaired PrEP efficacy. Despite extensive investigations in this field, tissue response to sustained subcutaneous TAF delivery remains to be elucidated as contrasting preclinical results have been reported in the literature. To this end, here we studied the local foreign body response (FBR) to sustained subdermal delivery of three forms of TAF, namely TAF free base (TAFfb), TAF fumarate salt (TAFfs), and TAFfb with urocanic acid (TAF-UA). Sustained constant drug release was achieved via titanium-silicon carbide nanofluidic implants previously shown to be bioinert. The analysis was conducted in both Sprague-Dawley (SD) rats and rhesus macaques over 1.5 and 3months, respectively. While visual observation did not reveal abnormal adverse tissue reaction at the implantation site, histopathology and Imaging Mass Cytometry (IMC) analyses exposed a local chronic inflammatory response to TAF. In rats, UA mitigated foreign body response to TAF in a concentration-dependent manner. This was not observed in macaques where TAFfb was better tolerated than TAFfs and TAF-UA. Notably, the level of FBR was tightly correlated with local TAF tissue concentration. Further, regardless of the degree of FBR, the fibrotic capsule (FC) surrounding the implants did not interfere with drug diffusion and systemic delivery, as evidenced by TAF PK results and fluorescence recovery after photobleaching (FRAP).

Effects of T217 Phosphorylation on Tau Localization and Microtubule Binding

AbstractBackgroundBiomarker‐based early detection of Alzheimer’s disease (AD) is necessary for developing therapies that counter AD symptom onset. Promising biomarkers include tau phosphorylated at T181, T217 or T231, levels of which can be measured in cerebrospinal fluid or plasma. Little is known about functional consequences of tau phosphorylation at these sites, though, especially for taupT217. We thus sought to identify where taupT217 is localized in the brains of human AD patients and AD model mice, and in cultured mouse neurons, and, in a more functional context, to determine if phosphorylation at T217 affects tau binding to microtubules.MethodBrain tissue from human AD patients and AD model mice, and cultured mouse neurons were analyzed by western blotting and immunofluorescence microscopy to compare the intracellular localization of taupT217, taupT181, taupT231 (AT180 epitope) and taupS396,pS404 (PHF1 epitope). EGFP‐tau (2N4R) was expressed in CV‐1 African green monkey kidney fibroblasts as wild type (WT), T217A non‐phosphorylatable, and T217E pseudo‐phosphorylated tau versions of the fluorescent fusion protein. Fluorescence recovery after photobleaching (FRAP) microscopy was used to assay turnover rates of the EGFP‐tau fusion proteins on microtubules, a surrogate measurement of affinity.ResultIn human AD brains and AD model mice, taupT217 was abundant in the somatodendritic compartment and co‐localized with neurofibrillary tangles. In cultured neurons, taupT217 was concentrated in somatodendritic puncta that were closely associated with dendritic spines. Both in vivo and in cultured neurons taupT217 co‐localized only partially with taupT181 and taupT231. Exposure of cultured neurons to extracellular oligomers of tau, but not amyloid‐β, increased intracellular taupT217. In CV‐1 cells, EGFP‐tauT217E recovered from photobleaching faster than EGFP‐tauT217A or EGFP‐tauT217.ConclusionIntracellular taupT217 is enriched at locations that normally lack much tau and is increased by extracellular tau oligomers. TaupT217 is enriched near dendritic spines, suggesting a possible role for T217 phosphorylation in altering synaptic activity. The faster fluorescence recovery of EGFP‐tauT217E compared to EGFP‐tauT217A or EGFP‐tauT217 implies that T217 phosphorylation reduces tau’s affinity for microtubules.

Phase separation is required for PML nuclear body biogenesis and function.

PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid-liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild-type (WT) NBs, PML A216V derived from arsenic-resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled-coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS-crippled and uncrippled NBs showed aggregation- and ring-like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS-driven NB formation was the prerequisite for partner recruitment, post-translational modifications (PTMs), and PML-driven cellular regulations, such as ROS stress control, mitochondria production, and PML-p53-mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis.

Green Nanoformulations of Polyvinylpyrrolidone-Capped Metal Nanoparticles: A Study at the Hybrid Interface with Biomimetic Cell Membranes and In Vitro Cell Models.

Noble metal nanoparticles (NP) with intrinsic antiangiogenic, antibacterial, and anti-inflammatory properties have great potential as potent chemotherapeutics, due to their unique features, including plasmonic properties for application in photothermal therapy, and their capability to slow down the migration/invasion speed of cancer cells and then suppress metastasis. In this work, gold (Au), silver (Ag), and palladium (Pd) NP were synthesized by a green redox chemistry method with the reduction of the metal salt precursor with glucose in the presence of polyvinylpyrrolidone (PVP) as stabilizing and capping agent. The physicochemical properties of the PVP-capped NP were investigated by UV-visible (UV-vis) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies, dynamic light scattering (DLS), and atomic force microscopy (AFM), to scrutinize the optical features and the interface between the metal surface and the capping polymer, the hydrodynamic size, and the morphology, respectively. Biophysical studies with model cell membranes were carried out by using laser scanning confocal microscopy (LSM) with fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) techniques. To this purpose, artificial cell membranes of supported lipid bilayers (SLBs) made with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) dye-labeled with 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD, FRET donor) and/or lissamine rhodamine B sulfonyl (Rh, FRET acceptor) were prepared. Proof-of-work in vitro cellular experiments were carried out with prostate cancer cells (PC-3 line) in terms of cytotoxicity, cell migration (wound scratch assay), NP cellular uptake, and cytoskeleton actin perturbation.

The Utility of Fluorescence Recovery after Photobleaching (FRAP) to Study the Plasma Membrane.

The plasma membrane of mammalian cells is involved in a wide variety of cellular processes, including, but not limited to, endocytosis and exocytosis, adhesion and migration, and signaling. The regulation of these processes requires the plasma membrane to be highly organized and dynamic. Much of the plasma membrane organization exists at temporal and spatial scales that cannot be directly observed with fluorescence microscopy. Therefore, approaches that report on the membrane's physical parameters must often be utilized to infer membrane organization. As discussed here, diffusion measurements are one such approach that has allowed researchers to understand the subresolution organization of the plasma membrane. Fluorescence recovery after photobleaching (or FRAP) is the most widely accessible method for measuring diffusion in a living cell and has proven to be a powerful tool in cell biology research. Here, we discuss the theoretical underpinnings that allow diffusion measurements to be used in elucidating the organization of the plasma membrane. We also discuss the basic FRAP methodology and the mathematical approaches for deriving quantitative measurements from FRAP recovery curves. FRAP is one of many methods used to measure diffusion in live cell membranes; thus, we compare FRAP with two other popular methods: fluorescence correlation microscopy and single-particle tracking. Lastly, we discuss various plasma membrane organization models developed and tested using diffusion measurements.

RNAP Promoter Search and Transcription Kinetics in Live E. coli Cells.

Bacterial transcription has been studied extensively in vitro, which has provided detailed molecular mechanisms of transcription. The in vivo cellular environment, however, may impose different rules on transcription than the homogeneous and well-controlled in vitro environment. How an RNA polymerase (RNAP) molecule searches rapidly through vast nonspecific chromosomal DNA in the three-dimensional nucleoid space and identifies a specific promoter sequence remains elusive. Transcription kinetics in vivo could also be impacted by specific cellular environments including nucleoid organization and nutrient availability. In this work, we investigated the promoter search dynamics and transcription kinetics of RNAP in live E. coli cells. Using single-molecule tracking (SMT) and fluorescence recovery after photobleaching (FRAP) across different genetic, drug inhibition, and growth conditions, we observed that RNAP's promoter search is facilitated by nonspecific DNA interactions and is largely independent of nucleoid organization, growth condition, transcription activity, or promoter class. RNAP's transcription kinetics, however, are sensitive to these conditions and mainly modulated at the levels of actively engaged RNAP and the promoter escape rate. Our work establishes a foundation for further mechanistic studies of bacterial transcription in live cells.

Interaction of LARP4 to filamin A mechanosensing domain regulates cell migrations.

Filamin A (FLNA) is an actin cross-linking protein that mediates mechanotransduction. Force-dependent conformational changes of FLNA molecule expose cryptic binding site of FLNA, allowing interaction with partners such as integrin, smoothelin, and fimbacin. Here, we identified La-related protein 4 (LARP4) as a new FLNA mechanobinding partner. LARP4 specifically interacts with the cleft formed by C and D strands of immunoglobulin-like repeat 21 (R21) which is blocked by A strand of R20 without force. We validated the interaction between LARP4 and FLNA R21 both in vivo and in vitro. We also determined the critical amino acid that is responsible for the interaction and generated the non-FLNA-binding mutant LARP4 (F277A in human: F273A in mouse Larp4) that disrupts the interaction. Fluorescence recovery after photobleaching (FRAP) of GFP-labeled LARP4 in living cells demonstrated that mutant LARP4 diffuses faster than WT LARP4. Proximity ligation assay (PLA) also confirmed their interaction and disruption of actin polymerization diminishes the interaction. Data mining of RNAseq analysis of LARP4 knockdown (KD) HEK293T cells suggested that LARP4 is involved in morphogenesis and cell motility. Consistent with this prediction, we found that KD of LARP4 increases cell migration speed and expression of the F277A mutant LARP4 in LARP4-KD cells also leads to a higher cell migration speed compared to WT LARP4. These results demonstrated that the LARP4 interaction with FLNA regulates cell migration.

Diffusivity of Human Cartilage Endplates in Healthy and Degenerated Intervertebral Disks.

The cartilage endplates (CEPs) on the superior and inferior surfaces of the intervertebral disk (IVD), are the primary nutrient transport pathways between the disk and the vertebral body. Passive diffusion is responsible for transporting small nutrient and metabolite molecules through the avascular CEPs. The baseline solute diffusivities in healthy CEPs have been previously studied, however alterations in CEP diffusion associated with IVD degeneration remain unclear. This study aimed to quantitatively compare the solute diffusion in healthy and degenerated human CEPs using a fluorescence recovery after photobleaching (FRAP) approach. Seven healthy CEPs and 22 degenerated CEPs were collected from five fresh-frozen human cadaveric spines and 17 patients undergoing spine fusion surgery, respectively. The sodium fluorescein diffusivities in CEP radial and vertical directions were measured using the FRAP method. The CEP calcification level was evaluated by measuring the average X-ray attenuation. No difference was found in solute diffusivities between radial and axial directions in healthy and degenerated CEPs. Compared to healthy CEPs, the average solute diffusivity was 44% lower in degenerated CEPs (Healthy: 29.07 μm2/s (CI: 23.96-33.62 μm2/s); degenerated: 16.32 μm2/s (CI: 13.84-18.84 μm2/s), p < 0.001). The average solute diffusivity had an inverse relationship with the degree of CEP calcification as determined by the normalized X-ray attenuation values (ß = -22.19, R2 = 0.633; p < 0.001). This study suggests that solute diffusion through the disk and vertebral body interface is significantly hindered by CEP calcification, providing clues to help further understand the mechanism of IVD degeneration.

Optimized genetic code expansion technology for time-dependent induction of adhesion GPCR-ligand engagement.

The introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair enables site-specific incorporation of unnatural amino acids (uAAs) with functionalized side chains into proteins of interest. Genetic Code Expansion (GCE) via amber codon suppression confers functionalities to proteins but can also be used to temporally control the incorporation of genetically encoded elements into proteins. Here, we report an optimized GCE system (GCEXpress) for efficient and fast uAA incorporation. We demonstrate that GCEXpress can be used to efficiently alter the subcellular localization of proteins within living cells. We show that click labeling can resolve co-labeling problems of intercellular adhesive protein complexes. We apply this strategy to study the adhesion G protein-coupled receptor (aGPCR) ADGRE5/CD97 and its ligand CD55/DAF that play central roles in immune functions and oncological processes. Furthermore, we use GCEXpress to analyze the time course of ADGRE5-CD55 ligation and replenishment of mature receptor-ligand complexes. Supported by fluorescence recovery after photobleaching (FRAP) experiments our results show that ADGRE5 and CD55 form stable intercellular contacts that may support transmission of mechanical forces onto ADGRE5 in a ligand-dependent manner. We conclude that GCE in combination with biophysical measurements can be a useful approach to analyze the adhesive, mechanical and signaling properties of aGPCRs and their ligand interactions. This article is protected by copyright. All rights reserved.

Membrane Models and Experiments Suitable for Studies of the Cholesterol Bilayer Domains.

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs.

Nutritional evaluation of some potential wild edible plants of North Eastern region of India.

India's north-eastern hill region (NEH) is one of the biodiversity hotspots, inhabited by several tribal communities still maintaining their traditional food habits. Much of their food resources are drawn from wild sources. Fourteen species of wild edible plants of high ethnic importance were collected from remote localities of Nagaland and Meghalaya states of the NEH region of India for nutritional profiling. Nutritional profiling of leaves of six species comprising Gynura cusimbua, Garcinia cowa, Herpetospermum operculatum, Plukenetia corniculata, Trichodesma khasianum, and Elatostemma sessile is conducted first time under present study. Samples were analyzed as per the Official Method of Analysis (AOAC) and standard methods. The range of variation in proximate composition was observed for moisture (72-92%), protein (1.71-6.66%), fat (0.22-1.36%), dietary fibre (5.16-14.58%), sugar (0.30-3.41%), and starch (0.07-2.14%). The highest protein content (6.66%) was recorded in Herpetospermum operculatum, followed by Trichodesma khasianum (5.89%) and Plukenetia corniculata (5.27%). Incidentally, two of these also have high iron (>7.0 mg/100 g) and high zinc (>2.0 mg/100 g) contents, except Trichodesma khasianum, which has low zinc content. High antioxidant activities in terms of gallic acid equivalent (GAE) by the cupric ion reducing antioxidant capacity (CUPRAC) method ranged from 1.10 to 8.40 mg/100 g, and by the Fluorescence recovery after photobleaching (FRAP) method ranged from 0.10 to 1.9 mg/100 g, while phenol content ranged between 0.30 and 6.00 mg/100 g. These wild vegetables have high potential because of their nutritional properties and are fully capable of enhancing sustainability and improving ecosystem services. Efforts were also initiated to mainstream these resources, mainly for widening the food basket of native peoples.

Real-Time Visualization of Photobrightening in Lead Halide Perovskites Using Confocal Laser Scanning Microscopy

Light induced changes in the optoelectronic properties affect the performance and the stability of halide perovskites. In this work, we report the real-time visualization of the photobrightening (PLB) effect using confocal laser scanning microscopy wherein the photon induced enhancement in photoluminescence is observed and their role in conductivity and photovoltaic properties are studied. The methodology is inspired from the Fluorescence Recovery After Photobleaching (FRAP) technique that is traditionally used to study biological cells. The role of composition, and surface/grain boundaries of perovskites, wavelengths, and intensity of illuminating photons, and time of illumination on the photobrightening or photobleaching is thoroughly investigated. The CH3NH3PbI3 exhibits a dominant photobrightening effect, with green photons showing more PLB than blue or red photons. The study of PLB between films and single crystals clearly shows the effect is a surface phenomenon. The presence of mixed iodide/bromide or pure bromide in the halide site and formamidinium or cesium in the A site suppressed the PLB. The strain relaxation in the organic site is found to be responsible for the PLB effect, and it enhanced the overall conductivity in material leading to better photovoltaic performance.

Lipid droplets as flexible substrates for protein phase separation.

Biomolecular condensates have been shown to nucleate the formation of amyloid fibrils for many proteins implicated in neurodegenerative disorders, such as alpha synuclein in Parkinson's disease and fused in sarcoma (FUS) in amyotrophic lateral sclerosis. Within these fibrils, a distinct lipid signal is often observable, and indeed, a membrane environment is favorable for the formation of amyloid fibrils for a variety of biophysical reasons, including an increase in local protein concentration and conformational shift upon membrane binding. How might intracellular lipid substrates contribute to nucleation of phase separation? Here we observe for the first time that intrinsically disordered proteins can undergo phase separation on the surface of lipid droplets. We find that the low complexity domain of FUS (FUSLC) separates into protein-rich and protein-depleted phases at physiological salt (150mM) and protein concentrations (<5μM) when recruited to the surfaces of lipid droplets through a histidine-Ni-NTA headgroup interaction. The number of LDs showing FUSLC phase separation is strongly affected by variations in the protein and salt concentration, and it follows the trend seen previously with FUSLC droplets in solution. Further, these phase-separated patches demonstrate molecular mobility when assessed using fluorescence recovery after photobleaching (FRAP), which is indicative of a liquid-like state. This phenomenon extends to other well-known phase separating domains, such as LAF1-RGG. Overall, these results demonstrate the potential of monolayer substrates to nucleate protein phase separation and could have important implications for the assembly of intrinsically disordered lipid droplet-binding proteins.

Thick filament dynamics: A mechanism for protein replacement within the sarcomere.

The heart contracts continually throughout one's lifetime through antiparallel sliding of actin-based thin filaments along myosin-based thick filaments, organized within muscle sarcomeres. A recent biochemical study from our lab demonstrated that the half-life of cardiac thick filament proteins is ∼10 days in adult mice, suggesting that continual protein replacement supports structural and functional fidelity. These data further suggest that newly synthesized molecules are randomly incorporated into preexisting thick filaments by a stochastic mechanism. Therefore, we hypothesize that myosin replacement involves the dynamic exchange of single molecules into thick filaments in vivo. To test this hypothesis, we generated an adeno-associated virus that replaced 27±7% of the endogenous regulatory light chain (RLC) on myosin molecules with a fluorescent GFP-RLC. We then visualized myosin within intact hearts using two-photon microscopy. At high magnification, individual sarcomeres were observed with GFP-RLC incorporated into thick filaments. Next, we measured the movement of fluorescently labeled myosin within the heart by irreversibly photobleaching limited areas of the cells and quantifying fluorescence recovery after photobleaching (FRAP). No FRAP occurred within 30 minutes, when photobleaching regions encompassed entire sarcomeres. However, rapid FRAP occurred after photobleaching small portions of single sarcomeres at rates orders of magnitude faster than protein synthesis. Recovery appeared to come from the exchange of molecules in the unbleached portion of the sarcomere and was fastest near the tips of the thick filaments, suggesting enhanced exchange where myosin is less densely packed in the filament. Collectively, these data demonstrate dynamic thick filament structure in vivo with myosin molecules freely exchanging between thick filaments within single sarcomeres. Therefore, these large macromolecular complexes appear to be designed to allow for the rapid incorporation and release of myosin molecules while maintaining the structural integrity that is essential to support contractility.

Mechanisms of cardiac myosin replacement.

A recent mass spectrometry-based proteomic study from our lab demonstrated that newly synthesized myosin molecules are randomly inserted into preexisting thick filaments in adult mouse hearts in vivo. Therefore, we hypothesized that myosin molecules exist in a dynamic equilibrium between a filamentous pool required for contractility, and a monomeric pool required for myosin replacement. To test this hypothesis, we replaced 27 ± 7% of the cardiac myosin regulatory light chain (RLC) in adult mice with fluorescent RLC (GFP-RLC), via adeno-associated viral transfection. We visualized the random distribution of GFP-RLC within thick filaments in intact hearts with 2-photon microscopy, and the exchange of GFP-RLC between these filaments by quantifying fluorescence recovery after photobleaching (FRAP). Rapid FRAP occurred within minutes, when photobleaching small, ∼1-µm3 areas, of partial sarcomeres. These data demonstrate that the GFP-RLC exchanges between the thick filaments and a monomeric pool on a timescale that is orders of magnitude faster than that of protein replacement (t1/2 = 10.8 days). To determine whether the mechanism of this exchange may involve the folding of monomeric myosin, GFP-labeled myosin molecules were extracted from the transfected hearts and their Stokes radii were measured over a range of ionic strengths, using microfluidic diffusional sizing. At low ionic strengths (150 mM KCl), filament formation was favored, but the monomeric fraction of GFP-labeled myosin demonstrated a radius of 12.6±1.1 nm. At higher ionic strengths (400-600 mM KCl), the radius was increased to 18.0 nm. Similar radii have been reported for smooth muscle myosin monomers in folded and extended conformations. Together, these data suggest that myosin molecules exist in rapid equilibrium between a filamentous and monomeric pool within the sarcomere, and individual monomers may adopt a folded, compact conformation to regulate their exchange into, and out of filaments.

P009 IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

Abstract Background The pathogenesis of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC) is still incompletely understood. However, despite having been described back in the 1980s and reported to be involved in chronic inflammatory arthritis, the role of interleukin 3 (IL-3) signaling via the IL-3 receptor (IL-3R) in the development and perpetuation of chronic intestinal inflammation remains largely unexplored. In this study, we therefore aimed to explore the role of IL-3 signaling via IL-3R in experimental colitis as well as human IBD. Methods We analysed IL-3 expression in lamina propria mononuclear cells (LPMCs) from patients with IBD and from mice with experimental colitis. We compared the development of T cell transfer colitis in Rag1-/- mice after transfer of Il3r-/- and Il3r+/+ naïve T cells and investigated the underlying mechanisms by immunofluorescence and functional cell trafficking assays. The mechanical properties of Il3r-/- and Il3r+/+ T cells were determined by real-time deformability cytometry (RT-DC) and atomic force microscopy (AFM) and cytoskeleton structure was analysed by scanning electron microscopy (SEM) and fluorescence recovery after photobleaching (FRAP). Results The expression of IL-3 and IL3+ T cells were increased in the gut of patients with IBD and IL-3 expression was associated to shorter flare-free survival. In vivo, experimental chronic colitis upon T cell transfer was exacerbated in the absence of IL-3 or IL-3R signalling. This was attributable to IL-3-induced changes in kinase signalling and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of regulatory T (Treg) cells from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Treg cells. (A) IL3 mRNA expression in colon tissue from patients.(B) Flare-free survival of IBD patients.(C) Colitis in Rag1–/– mice after transfer of naïve CD4+ T cells from Il3r+/+ and Il3r-/- mice. Mini-endoscopy (left) and IVIS (right).(D) RT-DC plot (left) and quantification (right) of thymic lymphocytes from Il3r-/- and Il3r+/+ mice.(E) Lightsheet microscopy (left) and quantification of TReg index (right, TReg fraction in Ozanimod/DATK32 per fraction in placebo) in mLNs from Rag1-/- mice with transfer colitis induced by Il3r-/- or Il3r+/+ T cells. Conclusion We uncover a crucial role of IL-3R signalling in regulating TReg mechanobiology and tissue egress. Together, our data demonstrate a central – probably counter-regulatory – role for IL-3 receptor signalling in Treg cells in the pathogenesis of murine and human chronic intestinal inflammation and therefore suggest that IL-3 might be a novel target for future therapeutic approaches in IBD.

Odor Discrimination by Lipid Membranes.

Odor detection and discrimination in mammals is known to be initiated by membrane-bound G-protein-coupled receptors (GPCRs). The role that the lipid membrane may play in odor discrimination, however, is less well understood. Here, we used model membrane systems to test the hypothesis that phospholipid bilayer membranes may be capable of odor discrimination. The effect of S-carvone, R-carvone, and racemic lilial on the model membrane systems was investigated. The odorants were found to affect the fluidity of supported lipid bilayers as measured by fluorescence recovery after photobleaching (FRAP). The effect of odorants on surface-supported lipid multilayer microarrays of different dimensions was also investigated. The lipid multilayer micro- and nanostructure was highly sensitive to exposure to these odorants. Fluorescently-labeled lipid multilayer droplets of 5-micron diameter were more responsive to these odorants than ethanol controls. Arrays of lipid multilayer diffraction gratings distinguished S-carvone from R-carvone in an artificial nose assay. Our results suggest that lipid bilayer membranes may play a role in odorant discrimination and molecular recognition in general.

Cell-mimicking polyethylene glycol-diacrylate based nanolipogel for encapsulation and delivery of hydrophilic biomolecule.

Lipid based nanoparticulate formulations have been widely used for the encapsulation and sustain release of hydrophilic drugs, but they still face challenges such as high initial burst release. Nanolipogel (NLG) emerges as a potential system to encapsulate and deliver hydrophilic drug while suppressing its initial burst release. However, there is a lack of characterization of the drug release mechanism from NLGs. In this work, we present a study on the release mechanism of hydrophilic Dextran-Fluorescein Isothiocyanate (DFITC) from Poly (ethylene glycol) Diacrylate (PEGDA) NLGs by using different molecular weights of PEGDA to vary the mesh size of the nanogel core, drawing inspiration from the macromolecular crowding effect in cells, which can be viewed as a mesh network of undefined sizes. The effect is then further characterized and validated by studying the diffusion of DFITC within the nanogel core using Fluorescence Recovery after Photobleaching (FRAP), on our newly developed cell derived microlipogels (MLG). This is in contrast to conventional FRAP works on cells or bulk hydrogels, which is limited in our application. Our work showed that the mesh size of the NLGs can be controlled by using different Mw of PEGDA, such as using a smaller MW to achieve higher crosslinking density, which will lead to having smaller mesh size for the crosslinked nanogel, and the release of hydrophilic DFITC can be sustained while suppressing the initial burst release, up to 10-fold more for crosslinked PEGDA 575 NLGs. This is further validated by FRAP which showed that the diffusion of DFITC is hindered by the decreasing mesh sizes in the NLGs, as a result of lower mobile fractions. These findings will be useful for guiding the design of PEGDA NLGs to have different degree of suppression of the initial burst release as well as the cumulative release, for a wide array of applications. This can also be extended to other different types of nanogel cores and other nanogel core-based nanoparticles for encapsulation and release of hydrophilic biomolecules.

Localization, induction, and cellular effects of tau phosphorylated at threonine 217.

Tau phosphorylation at T217 is a promising Alzheimer's disease (AD) biomarker, but its functional consequences were unknown. Human brain and cultured mouse neurons were analyzed by immunoblotting and immunofluorescence for total tau, taupT217 , taupT181 , taupT231 , and taupS396/pS404 . Direct stochastic optical reconstruction microscopy (dSTORM) super resolution microscopy was used to localize taupT217 in cultured neurons. Enhanced green fluorescent protein (EGFP)-tau was expressed in fibroblasts as wild type and T217E pseudo-phosphorylated tau, and fluorescence recovery after photobleaching (FRAP) reported tau turnover rates on microtubules. In the brain, taupT217 appears in neurons at Braak stages I and II, becomes more prevalent later, and co-localizes partially with other phospho-tau epitopes. In cultured neurons, taupT217 is increased by extracellular tau oligomers (xcTauOs) and is associated with developing post-synaptic sites. FRAP recovery was fastest for EGFP-tauT217E . TaupT217 increases in the brain as AD progresses and is induced by xcTauOs. Post-synaptic taupT217 suggests a role for T217 phosphorylation in synapse impairment. T217 phosphorylation reduces tau's affinity for microtubules. Validation of anti-tau phosphorylated at threonine-217 (taupT217 ) specificity is essential due to epitope redundancy. taupT217 increases as Alzheimer's disease progresses and is found throughout diseased neurons. taupT217 is associated with developing post-synaptic sites in cultured neurons. Extracellular oligomers of tau, but not amyloid beta, increase intracellular taupT217 . T217E pseudo-phosphorylation reduces tau's affinity for microtubules.