Understanding immune signaling using advanced imaging techniques

Advanced imaging is key for visualizing the spatiotemporal regulation of immune signaling which is a complex process involving multiple players tightly regulated in space and time. Imaging techniques vary in their spatial resolution, spanning from nanometers to micrometers, and in their temporal resolution, ranging from microseconds to hours. In this review, we summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling. Finally, we discuss the limitations of current technologies and share our insights on how to overcome these limitations to visualize immune signaling with unprecedented fidelity

Temporal resolution of protein–protein interactions in the live-cell plasma membrane

We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053–1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section

TRM versus FRP in flexural strengthening of RC beams: behaviour at high temperatures

The flexural behaviour of RC beams strengthened with TRM and FRP composites was experimentally investigated and compared both at ambient and high temperatures. The investigated parameters were: (a) the strengthening material, namely TRM versus FRP, (b) the number of strengthening layers, (c) the textile surface condition (dry and coated), (d) the textile material (carbon, basalt or glass fibres) and (e) the end-anchorage of the flexural reinforcement. A total of 23 half-scale beams were constructed, strengthened in flexure and tested to assess these parameters and the effectiveness of the TRM versus FRP at high temperatures. TRM exhibited excellent performance as strengthening material in increasing the flexural capacity at high temperature; in fact, TRM maintained an average effectiveness of 55%, compared to its effectiveness at ambient temperature, contrary to FRP which totally lost its effectiveness when subjected to high temperature. In specific, from the high temperature test it was found that by increasing the number of layers, the TRM effectiveness was considerably enhanced and the failure mode was altered; coating enhanced the TRM effectiveness; and the end-anchorage at high temperature improved significantly the FRP and marginally the TRM effectiveness. Finally, the formula proposed by the Fib Model Code 2010 was used to predict the mean debonding stress in the TRM reinforcement, and using the experimental results obtained in this study, a reduction factor to account for the effect of high temperature on the flexural strengthening with TRM was proposed

Bond between TRM versus FRP composites and concrete at high temperatures

The use of fibre reinforced polymers (FRP) as a means of external reinforcement for strengthening the existing reinforced concrete (RC) structures nowadays is the most common technique. However, the use of epoxy resins limits the effectiveness of FRP technique, and therefore, unless protective (thermal insulation) systems are provided, the bond capacity at the FRP-concrete interface will be extremely low above the glass transition temperature (Tg). To address problems associated with epoxies and to provide cost-effectiveness and durability of the strengthening intervention, a new composite cement- based material, namely textile-reinforced mortar (TRM) has been developed the last decade. This paper for the first time examines the bond performance between the TRM and concrete interfaces at high temperatures and, also compares for the first time the bond of both FRP and TRM systems to concrete at ambient and high temperatures. The key parameters investigated include: (a) the matrix used to impregnate the fibres, namely resin or mortar, resulting in two strengthening systems (TRM or FRP), (b) the level of high temperature to which the specimens are exposed (20, 50, 75, 100, and 150 °C) for FRP-reinforced specimens, and (20, 50, 75, 100, 150, 200, 300, 400, and 500 °C) for TRM-strengthened specimens, (c) the number of FRP/TRM layers (3 and 4), and (d) the loading conditions (steady state and transient conditions). A total of 68 specimens (56 specimens tested in steady state condition, and 12 specimens tested in transient condition) were constructed, strengthened and tested under double- lap direct shear. The result showed that overall TRM exhibited excellent performance at high temperature. In steady state tests, TRM specimens maintained an average of 85% of their ambient bond strength up to 400 °C, whereas the corresponding value for FRP specimens was only 17% at 150 °C. In transient test condition, TRM also outperformed over FRP in terms of both the time they maintained the applied load and the temperature reached before failure

Textile-reinforced mortar (TRM) versus fibre-reinforced polymers (FRP) in flexural strengthening of RC beams

The aim of this paper is to compare the flexural performance of reinforced concrete (RC) beams strengthened with textile-reinforced mortar (TRM) and fibre-reinforced polymers (FRP). The investigated parameters included the strengthening material, namely TRM or FRP; the number of TRM/FRP layers; the textile surface condition (coated and uncoated); the textile fibre material (carbon, coated basalt or glass fibres); and the end-anchorage system of the external reinforcement. Thirteen RC beams were fabricated, strengthened and tested in four-point bending. One beam served as control specimen, seven beams strengthened with TRM, and five with FRP. It was mainly found that: (a) TRM was generally inferior to FRP in enhancing the flexural capacity of RC beams, with the effectiveness ratio between the two systems varying from 0.46 to 0.80, depending on the parameters examined, (b) by tripling the number of TRM layers (from one to three), the TRM versus FRP effectiveness ratio was almost doubled, (c) providing coating to the dry textile enhanced the TRM effectiveness and altered the failure mode; (d) different textile materials, having approximately same axial stiffness, resulted in different flexural capacity increases; and (e) providing end-anchorage had a limited effect on the performance of TRM-retrofitted beams. Finally, a simple formula proposed by fib Model Code 2010 for FRP reinforcement was used to predict the mean debonding stress developed in the TRM reinforcement. It was found that this formula is in a good agreement with the average stress calculated based on the experimental results when failure was similar to FRP-strengthened beams

Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in ‘membrane-lipid therapy’ to combat many various protein-misfolding diseases associated with aging

Membrane fluidity matters: Hyperthermia from the aspects of lipids and membranes

Hyperthermia is a promising treatment modality for cancer in combination both with radio- and chemotherapy. In spite of its great therapeutic potential, the underlying molecular mechanisms still remain to be clarified. Due to lipid imbalances and 'membrane defects' most of the tumour cells possess elevated membrane fluidity. However, further increasing membrane fluidity to sensitise to chemo-or radiotherapy could have some other effects. In fact, hyperfluidisation of cell membrane induced by membrane fluidiser initiates a stress response as the heat shock protein response, which may modulate positively or negatively apoptotic cell death. Overviewing some recent findings based on a technology allowing direct imaging of lipid rafts in live cells and lipidomics, novel aspects of the intimate relationship between the 'membrane stress' of tumour cells and the cellular heat shock response will be highlighted. Our findings lend support to both the importance of membrane remodelling and the release of lipid signals initiating stress protein response, which can operate in tandem to control the extent of the ultimate cellular thermosensitivity. Overall, we suggest that the fluidity variable of membranes should be used as an independent factor for predicting the efficacy of combinational cancer therapies

The importance of the cellular stress response in the pathogenesis and treatment of type 2 diabetes

Organisms have evolved to survive rigorous environments and are not prepared to thrive in a world of caloric excess and sedentary behavior. A realization that physical exercise (or lack of it) plays a pivotal role in both the pathogenesis and therapy of type 2 diabetes mellitus (t2DM) has led to the provocative concept of therapeutic exercise mimetics. A decade ago, we attempted to simulate the beneficial effects of exercise by treating t2DM patients with 3 weeks of daily hyperthermia, induced by hot tub immersion. The short-term intervention had remarkable success, with a 1 % drop in HbA1, a trend toward weight loss, and improvement in diabetic neuropathic symptoms. An explanation for the beneficial effects of exercise and hyperthermia centers upon their ability to induce the cellular stress response (the heat shock response) and restore cellular homeostasis. Impaired stress response precedes major metabolic defects associated with t2DM and may be a near seminal event in the pathogenesis of the disease, tipping the balance from health into disease. Heat shock protein inducers share metabolic pathways associated with exercise with activation of AMPK, PGC1-a, and sirtuins. Diabetic therapies that induce the stress response, whether via heat, bioactive compounds, or genetic manipulation, improve or prevent all of the morbidities and comorbidities associated with the disease. The agents reduce insulin resistance, inflammatory cytokines, visceral adiposity, and body weight while increasing mitochondrial activity, normalizing membrane structure and lipid composition, and preserving organ function. Therapies restoring the stress response can re-tip the balance from disease into health and address the multifaceted defects associated with the disease

Examination at a Material and Structural Level of the Fatigue Life of Beams Strengthened with Mineral or Epoxy Bonded FRPs: The State of the Art

This paper presents a state of the art review of different material combinations and applications of mineral-based and epoxy-based bonded Fiber Reinforced Polymers (FRP), used for the strengthening of concrete structures subjected to fatigue loading. In this review, models of the fatigue life at the material and structural level are presented. This study examines the mechanical behavior of the FRP-material, surface bonding behavior and concrete beams strengthened under fatigue loading with different types of FRP-systems. The parameters that are investigated are applied load value, time dependent effects, type of strengthened structures (shear, flexural or combined) and the configuration of sheets or plates. The building codes and researchers' recommendations are also discussed. As a result of this review, the reader will obtains an overview of suitable materials and methods for strengthening structures subjected to fatigue loading by referring to the estimated fatigue life of material and strengthening structures at various applied stress levels.Validerad; 2013; 20130823 (thojoh