Molecular Engineering of Near-Infrared Fluorescent Probes for Cell Membrane Imaging.

Cell membrane (CM) is a phospholipid bilayer that maintains integrity of a whole cell and relates to many physiological and pathological processes. Developing CM imaging tools is a feasible method for visualizing membrane-related events. In recent decades, small-molecular fluorescent probes in the near-infrared (NIR) region have been pursued extensively for CM staining to investigate its functions and related events. In this review, we summarize development of such probes from the aspect of design principles, CM-targeting mechanisms and biological applications. Moreover, at the end of this review, the challenges and future research directions in designing NIR CM-targeting probes are discussed. This review indicates that more efforts are required to design activatable NIR CM-targeting probes, easily prepared and biocompatible probes with long retention time regarding CM, super-resolution imaging probes for monitoring CM nanoscale organization and multifunctional probes with imaging and phototherapy effects.

Antimicrobial peptide magainin 2-induced rupture of single giant unilamellar vesicles comprising E. coli polar lipids.

Most antimicrobial peptides (AMPs) damage the cell membrane of bacterial cells and induce rapid leakage of the internal cell contents, which is a main cause of their bactericidal activity. One of the AMPs, magainin 2 (Mag), forms nanopores in giant unilamellar vesicles (GUVs) comprising phosphatidylcholine (PC) and phosphatidylglycerol (PG), inducing leakage of fluorescent probes. In this study, to elucidate the Mag-induced pore formation in lipid bilayer region in E. coli cell membrane, we examined the interaction of Mag with single GUVs comprising E. coli polar lipids (E. coli-lipid-GUVs). First, we investigated the Mag-induced leakage of a fluorescent probe AF488 from single E. coli-lipid-GUVs, and found that Mag caused rupture of GUVs, inducing rapid AF488 leakage. The rate constant of Mag-induced GUV rupture increased with the Mag concentration. Using fluorescence microscopy with a time resolution of 5 ms, we revealed the GUV rupture process: first, a small micropore was observed in the GUV membrane, then the pore radius increased within 50 ms without changing the GUV diameter, the thickness of the membrane at the pore rim concomitantly increased, and eventually membrane aggregates were formed. Mag bound to only the outer monolayer of the GUV before GUV rupture, which increased the area of the GUV bilayer. We also examined the physical properties of E. coli-lipid-GUVs themselves. We found that the rate constant of the constant tension-induced rupture of E. coli-lipid-GUVs was higher than that of PG/PC-GUVs. Based on these results, we discussed the Mag-induced rupture of E. coli-lipid-GUVs and its mechanism.

Cysteine Cathepsins in Breast Cancer: Promising Targets for Fluorescence-Guided Surgery.

The majority of breast cancer patients is treated with breast-conserving surgery (BCS) combined with adjuvant radiation therapy. Up to 40% of patients has a tumor-positive resection margin after BCS, which necessitates re-resection or additional boost radiation. Cathepsin-targeted near-infrared fluorescence imaging during BCS could be used to detect residual cancer in the surgical cavity and guide additional resection, thereby preventing tumor-positive resection margins and associated mutilating treatments. The cysteine cathepsins are a family of proteases that play a major role in normal cellular physiology and neoplastic transformation. In breast cancer, the increased enzymatic activity and aberrant localization of many of the cysteine cathepsins drive tumor progression, proliferation, invasion, and metastasis. The upregulation of cysteine cathepsins in breast cancer cells indicates their potential as a target for intraoperative fluorescence imaging. This review provides a summary of the current knowledge on the role and expression of the most important cysteine cathepsins in breast cancer to better understand their potential as a target for fluorescence-guided surgery (FGS). In addition, it gives an overview of the cathepsin-targeted fluorescent probes that have been investigated preclinically and in breast cancer patients. The current review underscores that cysteine cathepsins are highly suitable molecular targets for FGS because of favorable expression and activity patterns in virtually all breast cancer subtypes. This is confirmed by cathepsin-targeted fluorescent probes that have been shown to facilitate in vivo breast cancer visualization and tumor resection in mouse models and breast cancer patients. These findings indicate that cathepsin-targeted FGS has potential to improve treatment outcomes in breast cancer patients.

Forsythiaside Protected H9c2 Cardiomyocytes from H2O2-Induced Oxidative Stress and Apoptosis via Activating Nrf2/HO-1 Signaling Pathway.

Forsythiaside, one of the main bioactive components of Chinese medicine Lian Qiao, exerts antioxidant, anti-bacterial, and anti-inflammatory effects. To date, the mechanism of Forsythiaside in cardiomyocyte injury remains unclear. However, the antioxidant effects of Forsythiaside on cardiac cells are currently unknown. This study investigated the effect and mechanism of Forsythiaside on oxidative stress in H9c2 cardiomyocytes. H9c2 cells were treated with H2O2 and Forsythiaside and then transfected with small-interfering RNA against nuclear factor erythroid 2-related factor 2 (siNrf2). Cell viability, apoptosis, accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential were measured using methyl thiazolyl tetrazolium (MTT), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123, respectively. The levels of oxidative stress-related markers were determined using their respective detection kits. Furthermore, the levels of apoptosis- and Nrf2 pathway-related molecules were determined via Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Forsythiaside had no obvious toxicity on H9c2 cells. H2O2 suppressed the viability, and reduced the levels of mitochondrial membrane potential, B-cell lymphoma-2 (Bcl-2), glutathione peroxidase (GSH-Px) and catalase (CAT) and superoxide dismutase (SOD), while promoted apoptosis, ROS accumulation, and elevated the levels of cleaved caspase 3, BCL2-Associated X (Bax) and malondialdehyde (MDA) in H9c2 cells. Contrarily, Forsythiaside reversed the aforementioned effects. H2O2 advanced the levels of cytoplasm Nrf2, heme oxygenase-1 (HO-1), and nucleus Nrf2 in H9c2 cells, whereas Forsythiaside enhanced these effects. SiNrf2 reversed the functions of H2O2 or Forsythiaside in cell viability, MDA, SOD, GSH-Px, CAT, Nrf2, and HO-1 in H9c2 cells, whereas Forsythiaside reversed the aforementioned effects of siNrf2. In sum, Forsythiaside protected H9c2 cells from oxidative stress and apoptosis induced by H2O2 by activating the Nrf2/HO-1 pathway.

The yin and yang of intracellular delivery of amphipathic optical probes using n-butyl charge masking.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

Designer exosomes for targeted and efficient ferroptosis induction in cancer via chemo-photodynamic therapy.

Background: Efficient and specific induction of cell death in liver cancer is urgently needed. In this study, we aimed to design an exosome-based platform to deliver ferroptosis inducer (Erastin, Er) and photosensitizer (Rose Bengal, RB) into tumor tissues with high specificity. Methods: Exosome donor cells (HEK293T) were transfected with control or CD47-overexpressing plasmid. Exosomes were isolated and loaded with Er and RB via sonication method. Hepa1-6 cell xenograft C57BL/6 model was injected with control and engineered exosomes via tail vein. In vivo distribution of the injected exosomes was analyzed via tracking the fluorescence labeled exosomes. Photodynamic therapy was conducted by 532 nm laser irradiation. The therapeutic effects on hepatocellular carcinoma and toxic side-effects were systemically analyzed. Results: CD47 was efficiently loaded on the exosomes from the donor cells when CD47 was forced expressed by transfection. CD47 surface functionalization (ExosCD47) made the exosomes effectively escape the phagocytosis of mononuclear phagocyte system (MPS), and thus increased the distribution in tumor tissues. Erastin and RB could be effectively encapsulated into exosomes after sonication, and the drug-loaded exosomes (Er/RB@ExosCD47) strongly induced ferroptosis both in vitro and in vivo in tumor cells after irradiation of 532 nm laser. Moreover, compared with the control exosomes (Er/RB@ExosCtrl), Er/RB@ExosCD47 displayed much lower toxicity in liver. Conclusion: The engineered exosomes composed of CD47, Erastin, and Rose Bengal, induce obvious ferroptosis in hepatocellular carcinoma (HCC) with minimized toxicity in liver and kidney. The proposed exosomes would provide a promising strategy to treat types of malignant tumors.

Plant extracts and betulin from Ligaria cuneifolia inhibit P-glycoprotein function in leukemia cells.

Overexpression of P-glycoprotein (P-gp), which is linked to multidrug resistance (MDR), is one of the underlying obstacles to the success of chemotherapy as it reduces the efficacy of anticancer drugs and the side effects of these increase as a result of any increased dose to achieve the therapeutic effect. To identify agents with P-gp inhibitory properties, ethanol extracts from 80 plants were screened for their ability to increase intracellular doxorubicin-associated fluorescence, and the extract of Ligaria cuneifolia was found to be the most effective. Its bioassay-guided isolation yielded the pentacyclic triterpene betulin as active agent. This efficiently inhibited P-gp mediated efflux, as demonstrated by the enhancement of the intracellular accumulation of doxorubicin and rhodamine 123 from 1.56 µM in the P-gp overexpressing MDR leukemia cell, Lucena 1. Betulin was also able to render Lucena 1 sensitive to Dox from 0.39 µM. The docking studies revealed that betulin tightly binds to a key region of the TMDs, with a binding mode overlapping one main site of doxorubicin and, more interestingly, emulating the same contacts as tariquidar, as revealed by the per-residue energetic analysis from molecular dynamics simulations. MTT assay using peripheral blood mononuclear cells and hemolysis assay showed that betulin is devoid of toxicity. These findings provide important evidence that betulin may be a safe and promising entity to be further investigated to develop agents able to overcome P-gp-mediated MDR, resulting in a more effective and less toxic chemotherapy.

Idioblasts and peltate hairs as distribution networks for water absorbed by xerophilous leaves.

Capparis odoratissima is a tree species native to semi-arid environments of South America where low soil water availability coexists with frequent night-time fog. A previous study showed that water applied to leaf surfaces enhanced leaf hydration, photosynthesis and growth, but the mechanisms of foliar water uptake are unknown. Here, we combine detailed anatomical evaluations with water and dye uptake experiments in the laboratory, and use immunolocalization of pectin and arabinogalactan protein epitopes to characterize water uptake pathways in leaves. Abaxially, the leaves of C. odoratissima are covered with peltate hairs, while the adaxial surfaces are glabrous. Both surfaces are able to absorb condensed water, but the abaxial surface has higher rates of water uptake. Thousands of idioblasts per cm2 , a higher density than stomata, connect the adaxial leaf surface and the abaxial peltate hairs, both of which contain hygroscopic substances such as arabinogalactan proteins and pectins. The highly specialized anatomy of the leaves of C odoratissima fulfils the dual function of minimizing water loss when stomata are closed, while maintaining the ability to absorb liquid water. Cell-wall related hygroscopic compounds in the peltate hairs and idioblasts create a network of microchannels that maintain leaf hydration and promote water uptake.

Choline Supplementation Sensitizes Legionella dumoffii to Galleria mellonella Apolipophorin III.

The growth of Legionella dumoffii can be inhibited by Galleria mellonella apolipophorin III (apoLp-III) which is an insect homologue of human apolipoprotein E., and choline-cultured L. dumoffii cells are considerably more susceptible to apoLp-III than bacteria grown without choline supplementation. In the present study, the interactions of apoLp-III with intact L. dumoffii cells cultured without and with exogenous choline were analyzed to explain the basis of this difference. Fluorescently labeled apoLp-III (FITC-apoLp-III) bound more efficiently to choline-grown L. dumoffii, as revealed by laser scanning confocal microscopy. The cell envelope of these bacteria was penetrated more deeply by FITC-apoLp-III, as demonstrated by fluorescence lifetime imaging microscopy analyses. The increased susceptibility of the choline-cultured L. dumoffii to apoLp-III was also accompanied by alterations in the cell surface topography and nanomechanical properties. A detailed analysis of the interaction of apoLp-III with components of the L. dumoffii cells was carried out using both purified lipopolysaccharide (LPS) and liposomes composed of L. dumoffii phospholipids and LPS. A single micelle of L. dumoffii LPS was formed from 12 to 29 monomeric LPS molecules and one L. dumoffii LPS micelle bound two molecules of apoLp-III. ApoLp-III exhibited the strongest interactions with liposomes with incorporated LPS formed of phospholipids isolated from bacteria cultured on exogenous choline. These results indicated that the differences in the phospholipid content in the cell membrane, especially PC, and LPS affected the interactions of apoLp-III with bacterial cells and suggested that these differences contributed to the increased susceptibility of the choline-cultured L. dumoffii to G. mellonella apoLp-III.

Enzyme-responsive turn-on nanoprobes for in situ fluorescence imaging and localized photothermal treatment of multidrug-resistant bacterial infections.

Sensitive diagnosis and elimination of multidrug-resistant bacterial infections at an early stage remain paramount challenges. Herein, we present a gelatinase-responsive turn-on nanoprobe for in situ near-infrared (NIR) fluorescence imaging and localized photothermal treatment (PTT) of in vivo methicillin-resistant Staphylococcus aureus (MRSA) infections. The designed nanoprobe (named AuNS-Apt-Cy) is based on gold nanostars functionalized with MRSA-identifiable aptamer and gelatinase-responsive heptapeptide linker (CPLGVRG)-cypate complexes. The AuNS-Apt-Cy nanoprobe is non-fluorescent in aqueous environments due to the fluorescence resonance energy transfer between the gold nanostar core and cypate dye. We demonstrate that the AuNS-Apt-Cy nanoprobe can achieve MRSA targeting and accumulation as well as gelatinase (overexpressed in MRSA environments)-responsive turn-on NIR fluorescence due to the cleavage of the CPLGVRG linker and localized in vitro PTT via a mechanism involving bacterial cell wall and membrane disruption. In vivo experiments show that the AuNS-Apt-Cy nanoprobe can enable rapid (1 h post-administration) and in situ turn-on NIR fluorescence imaging with high sensitivity (105 colony-forming units) in diabetic wound and implanted bone plate mouse models. Remarkably, the AuNS-Apt-Cy nanoprobe can afford efficient localized PTT of diabetic wound and implanted bone plate-associated MRSA infections under the guidance of turn-on NIR fluorescence imaging, showing robust capability for early diagnosis and treatment of in vivo MRSA infections. In addition, the nanoprobe exhibits negligible damage to surrounding healthy tissues during PTT due to its targeted accumulation in the MRSA-infected site, guaranteeing its excellent in vivo biocompatibility and solving the main bottlenecks that hinder the clinical application of PTT-based antibacterial strategies.

H2 O2 -induced oxidative stress disrupts mitochondrial functions and impairs migratory potential of human epidermal melanocytes.

Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.

Dual-Reporting Transcriptionally Linked Genetically Encoded Fluorescent Indicators Resolve the Spatiotemporal Coordination of Cytosolic Abscisic Acid and Second Messenger Dynamics in Arabidopsis.

Deciphering signal transduction processes is crucial for understanding how plants sense and respond to environmental changes. Various chemical compounds function as central messengers within deeply intertwined signaling networks. How such compounds act in concert remains to be elucidated. We have developed dual-reporting transcriptionally linked genetically encoded fluorescent indicators (2-in-1-GEFIs) for multiparametric in vivo analyses of the phytohormone abscisic acid (ABA), Ca2+, protons (H+), chloride (anions), the glutathione redox potential, and H2O2 Simultaneous analyses of two signaling compounds in Arabidopsis (Arabidopsis thaliana) roots revealed that ABA treatment and uptake did not trigger rapid cytosolic Ca2+ or H+ dynamics. Glutamate, ATP, Arabidopsis PLANT ELICITOR PEPTIDE, and glutathione disulfide (GSSG) treatments induced rapid spatiotemporally overlapping cytosolic Ca2+, H+, and anion dynamics, but except for GSSG, only weakly affected the cytosolic redox state. Overall, 2-in-1-GEFIs enable complementary, high-resolution in vivo analyses of signaling compound dynamics and facilitate an advanced understanding of the spatiotemporal coordination of signal transduction processes in Arabidopsis.

Visualising Silicon in Plants: Histochemistry, Silica Sculptures and Elemental Imaging.

Silicon is a non-essential element for plants and is available in biota as silicic acid. Its presence has been associated with a general improvement of plant vigour and response to exogenous stresses. Plants accumulate silicon in their tissues as amorphous silica and cell walls are preferential sites. While several papers have been published on the mitigatory effects that silicon has on plants under stress, there has been less research on imaging silicon in plant tissues. Imaging offers important complementary results to molecular data, since it provides spatial information. Herein, the focus is on histochemistry coupled to optical microscopy, fluorescence and scanning electron microscopy of microwave acid extracted plant silica, techniques based on particle-induced X-ray emission, X-ray fluorescence spectrometry and mass spectrometry imaging (NanoSIMS). Sample preparation procedures will not be discussed in detail, as several reviews have already treated this subject extensively. We focus instead on the information that each technique provides by offering, for each imaging approach, examples from both silicifiers (giant horsetail and rice) and non-accumulators (Cannabis sativa L.).

A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids.

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.

Selenium-doped two-photon fluorescent carbon nanodots for in-situ free radical scavenging in mitochondria.

Mitochondrial oxidative stress is associated with the occurrence and development of a wide range of human diseases. The development of methodologies to alleviate oxidative stress-mediated injury may have therapeutic potential. Herein, we report the design and preparation of triphenylphosphonium-functionalized selenium-doped carbon nanodots (TPP-Se-CDs) that can efficiently scavenging hydroxyl radicals (OH) and superoxide anions (O2-) in mitochondria region. Se-CDs with two-photon blue fluorescence were initially prepared by facile hydrothermal treatment of selenomethionine, followed by the covalent conjugation with TPP. The as-obtained TPP-Se-CDs showed high colloidal stability, strong scavenging abilities towards OH and O2-. Moreover, TPP-Se-CDs exhibited low cytotoxicity and mitochondria targeting ability. Taking advantages of these prominent features, TPP-Se-CDs have been successfully applied to combat H2O2 and phorbol 12-myristate 13-acetate (PMA) induced oxidative stress in mitochondria.

Perturbing Circadian Oscillations in an In Vitro Suprachiasmatic Nucleus With Magnetic Stimulation.

Many neurological disorders are associated with abnormal oscillatory dynamics. The suprachiasmatic nucleus (SCN) is responsible for the timing and synchronization of physiological processes. We performed experiments on PERIOD2::LUCIFERASE transgenic "knock-in" mice. In these mice, a gene that is expressed in a circadian pattern is fused to an inserted gene that codes for luciferase, which is a bioluminescent enzyme. A one-time 3 min magnetic stimulation (MS) was applied to excised slices of the SCN. The MS consisted of a 50-mT field that was turned on and off 4,500 times. The rise time and fall time of the field were 75 µs. A photon count that extended over the full 5 days that the slice remained viable, subsequently revealed how the MS affected the circadian cycle. The MS was applied at points in the circadian cycle that correspond to either maximal or minimal bioluminescence. It was found that both the amplitude and period of the endogenous circadian oscillation are affected by MS and that the effects strongly depend on where in the circadian cycle the stimulation was applied. Our MS dose is in the same range as clinically applied doses, and our findings imply that transcranial MS may be instrumental in remedying disorders that originate in circadian rhythm abnormalities. Bioelectromagnetics. 2020;41:63-72 © 2019 Wiley Periodicals, Inc.

Quantification of residual ophthalmic viscosurgical device after irrigation/aspiration in experimental cataract surgery in vitro.

PURPOSE: To determine the distribution and quantity of ophthalmic viscosurgical device (OVD) retained in the lens capsular bag after irrigation/aspiration (I/A) in experimental cataract surgery. SETTING: Department of Ophthalmology, Kashiwa Hospital, Jikei University School of Medicine, Japan. DESIGN: Experimental study. METHODS: Fifteen freshly enucleated porcine eyes were used. Sodium hyaluronate 1.0% (Healon) was mixed with a fixed concentration of silica nanoparticles encapsulating fluorescein isothiocyanate (labeled OVD), and it was injected separately into the capsular bag and injector cartridge for intraocular lens (IOL) insertion. After a 3-piece IOL (YA-60BBR) or a 1-piece IOL (iSert 255) was implanted within the capsular bag, OVD was removed by thorough I/A. Eyes that were injected with the labeled OVD into the capsular bag without IOL insertion were used as controls. The distribution of residual OVD in the capsular bag was observed under ultraviolet irradiation using Miyake-Apple view. Then, the excised capsular bag was dissolved in hydrogen fluoride, and silica concentration was measured quantitatively by elemental analysis. RESULTS: The quantity of residual OVD in the capsular bag was 243.1 µg ± 1.3 (SD) in the 3-piece IOL-implanted group, 383.8 ± 11.1 µg in the 1-piece IOL-implanted group, and 99.0 ± 1.3 µg in the control group. In the 1-piece IOL-implanted eyes, OVD in the form of clumps tended to remain near the center of the optic on the posterior side, and the quantity of residual OVD was significantly greater than in 3-piece IOL-implanted eyes (P < .05). CONCLUSION: The quantity of residual OVD after I/A could be determined indirectly using labeled OVD, and the quantity was significantly greater in 1-piece IOL-implanted eyes than in 3-piece IOL-implanted eyes.

Labelled chemical probes for demonstrating direct target engagement in living systems.

Demonstrating target engagement in living systems can help drive successful drug discovery. Target engagement and occupancy studies in cells confirm direct binding of a ligand to its intended target protein and provide the binding affinity. Combined with biomarkers to measure the functional consequences of target engagement, these experiments can increase confidence in the relationship between in vitro pharmacology and observed biological effects. In this review, we focus on chemically and radioactively labelled probes as key reagents for performing such experiments. Using recent examples, we examine how the labelled probes have been employed in combination with unlabelled ligands to quantify target engagement in cells and in animals. Finally, we consider future developments of this emerging methodology.

Nucleic Acid Platform Technologies.

Every microarray experiment is based on a common format. First, a large number of nucleotide "spots" are arrayed onto a substrate, typically a glass slide, a silicon chip, or microbeads. Second, a complex population of nucleic acids (isolated from cells, selected from in vitro-synthesized libraries, or obtained from another source) is labeled, typically with fluorescent dyes. Third, the labeled nucleic acids are allowed to hybridize to their complementary spot(s) on the microarray. Fourth, the hybridized microarray is washed, allowing the amount of hybridized label to then be quantified. Analysis of the raw data generates a readout of the levels of each species of RNA in the original complex population. This introduction includes several examples of microarray applications and provides a discussion of the basic steps of most microarray experiments.

Transient Gene Expression as a Tool to Monitor and Manipulate the Levels of Acidic Phospholipids in Plant Cells.

Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.