Communication codes in developmental signaling pathways

A handful of core intercellular signaling pathways play pivotal roles in a broad variety of developmental processes. It has remained puzzling how so few pathways can provide the precision and specificity of cell-cell communication required for multicellular development. Solving this requires us to quantitatively understand how developmentally relevant signaling information is actively sensed, transformed and spatially distributed by signaling pathways. Recently, single cell analysis and cell-based reconstitution, among other approaches, have begun to reveal the ‘communication codes’ through which information is represented in the identities, concentrations, combinations and dynamics of extracellular ligands. They have also revealed how signaling pathways decipher these features and control the spatial distribution of signaling in multicellular contexts. Here, we review recent work reporting the discovery and analysis of communication codes and discuss their implications for diverse developmental processes

Fluorescent-based nanosensors for selective detection of a wide range of biological macromolecules: A comprehensive review

Thanks to their unique attributes, such as good sensitivity, selectivity, high surface-to-volume ratio, and versatile optical and electronic properties, fluorescent-based bioprobes have been used to create highly sensitive nano -biosensors to detect various biological and chemical agents. These sensors are superior to other analytical instrumentation techniques like gas chromatography, high-performance liquid chromatography, and capillary electrophoresis for being biodegradable, eco-friendly, and more economical, operational, and cost-effective. Moreover, several reports have also highlighted their application in the early detection of biomarkers associ-ated with drug-induced organ damage such as liver, kidney, or lungs. In the present work, we comprehensively overviewed the electrochemical sensors that employ nanomaterials (nanoparticles/colloids or quantum dots, carbon dots, or nanoscaled metal-organic frameworks, etc.) to detect a variety of biological macromolecules based on fluorescent emission spectra. In addition, the most important mechanisms and methods to sense amino acids, protein, peptides, enzymes, carbohydrates, neurotransmitters, nucleic acids, vitamins, ions, metals, and electrolytes, blood gases, drugs (i.e., anti-inflammatory agents and antibiotics), toxins, alkaloids, antioxidants, cancer biomarkers, urinary metabolites (i.e., urea, uric acid, and creatinine), and pathogenic microorganisms were outlined and compared in terms of their selectivity and sensitivity. Altogether, the small dimensions and capability of these nanosensors for sensitive, label-free, real-time sensing of chemical, biological, and pharma-ceutical agents could be used in array-based screening and in-vitro or in-vivo diagnostics. Although fluorescent nanoprobes are widely applied in determining biological macromolecules, unfortunately, they present many challenges and limitations. Efforts must be made to minimize such limitations in utilizing such nanobiosensors with an emphasis on their commercial developments. We believe that the current review can foster the wider incorporation of nanomedicine and will be of particular interest to researchers working on fluorescence tech-nology, material chemistry, coordination polymers, and related research areas

Supramolecular Luminescent Sensors

There is great need for stand-alone luminescence-based chemosensors that exemplify selectivity, sensitivity, and applicability and that overcome the challenges that arise from complex, real-world media. Discussed herein are recent developments toward these goals in the field of supramolecular luminescent chemosensors, including macrocycles, polymers, and nanomaterials. Specific focus is placed on the development of new macrocycle hosts since 2010, coupled with considerations of the underlying principles of supramolecular chemistry as well as analytes of interest and common luminophores. State-of-the-art developments in the fields of polymer and nanomaterial sensors are also examined, and some remaining unsolved challenges in the area of chemosensors are discussed

Développement de biosenseurs fluorescents et d’inhibiteurs pour suivre et cibler CDK4/cycline D dans le mélanome

CDK/cyclins play a central role in coordinating cell cycle progression, and in sustaining proliferation of cancer cells, thereby constituting established cancer biomarkers and attractive pharmacological targets. In particular, CDK4/cyclin D, which is responsible for coordinating cell cycle progression through G1 into S phase, is a relevant target in several cancers including melanoma, associated with mutation of CDK4, cyclin D, p16INK4a and pRb.As there are no sensitive and direct approaches to probe CDK4/cyclin D activity in physiological and pathological conditions, the first goal of my thesis has consisted in engineering a fluorescent biosensor to probe this kinase in vitro and in cellulo. Once characterized and validated in vitro, the biosensor was applied to detect CDK4/cyclin D alterations in biopsies from human skin and melanoma xenografts in fluorescence-based activity assays, and in living cancer cells by fluorescence microscopy and timelapse imaging.Moreover, only few inhibitors are currently available to target CDK4/cyclin D and most of them bind the ATP pocket. As such, the second major goal of my thesis project has consisted in identifying non-ATP competitive inhibitors, either through rational design of peptides or by screening small molecule libraries. To this aim, two fluorescent biosensors were engineered which discriminate compounds that target the interface between CDK4 and cyclin D, or that perturb the conformational dynamics of CDK4, respectively, from ATP-pocket binding compounds. Fluorescence-based screening assays performed with these biosensors lead to identification of hits, which were validated and characterized in vitro and in cell proliferation assays, and which constitute promising candidates for selective chemotherapy in melanoma.Les CDK/cyclines jouent un rôle majeur dans la progression du cycle cellulaire et dans le maintien de la prolifération des cellules cancéreuses, constituant ainsi des biomarqueurs clés et des cibles pharmacologiques attractives. Plus particulièrement, l’activité de CDK4/cycline D, kinase responsable de la progression de la phase G1 et de la transition G1/S, est dérégulée dans de nombreux cancers dont le mélanome. Cette hyperactivation est associée à des mutations, à l’amplification ou à la surexpression de CDK4, cycline D, p16INK4a ou encore pRb.Comme aucune approche sensible et directe n’existe pour évaluer l’activité de CDK4/cycline D dans des conditions physiologiques et pathologiques, le premier objectif de ma thèse a consisté à développer un biosenseur fluorescent permettant d’étudier cette kinase in vitro et in cellulo. Une fois caractérisé et validé in vitro, le biosenseur a été appliqué à la détection d’altérations de CDK4/cycline D dans des biopsies de peau humaine et de xénogreffes de mélanome dans des essais fluorescents d’activité kinase, ainsi que dans des cellules cancéreuses vivantes par microscopie de fluorescence et vidéo microscopie.Par ailleurs, peu d’inhibiteurs sont actuellement disponibles pour inhiber CDK4/cycline D et la plupart d’entre eux ciblent la poche de fixation de l’ATP. C’est pourquoi le second objectif de ma thèse a consisté à identifier des inhibiteurs non compétitifs de l’ATP, soit par élaboration rationnelle de peptides, soit par criblage de petites molécules. A cette fin, deux biosenseurs fluorescents ont été développés qui permettent d’identifier respectivement des composés ciblant l’interface entre CDK4 et cycline D ou des inhibiteurs allostériques capables de perturber la dynamique conformationnelle de CDK4. Des essais de criblage par fluorescence réalisés avec ces biosenseurs ont conduit à l’identification de touches qui ont été validées et caractérisées in vitro et dans des essais de prolifération cellulaire, et qui constituent des candidats prometteurs pour une chimiothérapie sélective du mélanome

Molecular Probes for the Detection of Zn2+ and Fe3+ Ions

A number of molecular probes have been designed and synthesized for the detection of Zn2+ and Fe3+ ions. Two types of functional groups have been incorporated into the molecular scaffolds to utilize different fluorescent mechanisms. The first class of receptors contains a pyrene moiety. These molecular probes use the excimer mechanism for the detection of Zn2+ ion. The probes work well in an organic solvent with a detection limit of 20 nM (one ppb). Alternatives are made to make them water soluble, but this proved to be difficult. An interesting ion-induced self-assembly system will also be discussed. The second class of molecules is based on the “Off-On” mechanism. The rhodamine dyes are used in these molecular probes. This system is found to be selective for Fe3+ ions in a mixed organic/aqueous system. A protocol has also been developed to distinguish between Fe3+ and Al3+ ions. One of the rhodamine dyes has also been shown to detect Fe3+ ion in bacteria

Studies in Physical Biology: Exploring Allosteric Regulation, Enzymatic Error Correction, and Cytoskeletal Self-Organization Using Theory and Modeling

Physical biology offers powerful tools for quantitatively dissecting the various aspects of cellular life that one cannot attribute to inanimate matter. Signature examples of living matter include adaptation, self-organization, and division. In this thesis, we explore different interconnected facets of these processes using statistical mechanics, nonequilibrium thermodynamics, and biophysical modeling. One of the key mechanisms underlying physiological and evolutionary adaptation is allosteric regulation. It allows cells to dynamically respond to changes in the state of the environment often expressed through altered levels of different environmental cues. The first thread of our work is dedicated to exploring the combinatorial diversity of responses available to allosteric proteins that are subject to multi-ligand regulation. We demonstrate that proteins characterized through the Monod-Wyman-Changeux model of allostery and operating at thermodynamic equilibrium are capable of eliciting a wide range of response behaviors which include the kinds known from the field of digital circuits (e.g., NAND logic response), as well as more sophisticated computations such as ratiometric sensing. Despite the fact that biomolecules at thermodynamic equilibrium are able to orchestrate a variety of fascinating behaviors, the cell is ultimately 'alive' because it constantly metabolizes nutrients and generates energy to drive functions that cannot be sustained in the absence of energy consumption. One prominent example of such a function is nonequilibrium error correction present in high-fidelity processes such as protein synthesis, DNA replication, or pathogen recognition. We begin the second thread of our work by providing a conceptual understanding of the prevailing mechanism used in explaining this high-fidelity behavior, namely that of kinetic proofreading. Specifically, we develop an allostery-based mechanochemical model of a kinetic proofreader where chemical driving is replaced with a mechanical engine with tunable knobs which allow modulating the amount of dissipation in a transparent way. We demonstrate how varying levels of error correction can be attained at different regimes of dissipation and offer intuitive interpretations for the conditions required for efficient biological proofreading. We then extend the notion of error correction to equilibrium enzymes not endowed with structural features typically required for proofreading. We show that, under physiological conditions, purely diffusing enzymes can take advantage of the existing nonequilibrium organization of their substrates in space and enhance the fidelity of catalysis. Our proposed mechanism called spatial proofreading offers a novel perspective on spatial structures and compartmentalization in cells as a route to specificity. In the last thread of the thesis, we make a transition from molecular-scale studies to the mesoscopic scale, and explore the principles of self-organization in nonequilibrium structures formed in reconstituted microtubule-motor mixtures. In particular, we develop a theoretical framework that predicts the spatial distribution of kinesin motors in radially symmetric microtubule asters formed under various conditions using optogenetic control. The model manages to accurately recapitulate the experimentally measured motor profiles through effective parameters that are specific for each kind of kinesin motor used. Our theoretical work of rigorously assessing the motor distribution therefore offers an avenue for understanding the link between the microscopic motor properties (e.g., processivity or binding affinity) and the large-scale structures they create. In all, the thesis encompasses a series of case studies with shared themes of allostery and nonequilibrium, highlighting the capacity of living matter to perform remarkable tasks inaccessible to nonliving materials.</p

Understanding the Logistics for the Distribution of Heme in Cells

[Image: see text] Heme is essential for the survival of virtually all living systems—from bacteria, fungi, and yeast, through plants to animals. No eukaryote has been identified that can survive without heme. There are thousands of different proteins that require heme in order to function properly, and these are responsible for processes such as oxygen transport, electron transfer, oxidative stress response, respiration, and catalysis. Further to this, in the past few years, heme has been shown to have an important regulatory role in cells, in processes such as transcription, regulation of the circadian clock, and the gating of ion channels. To act in a regulatory capacity, heme needs to move from its place of synthesis (in mitochondria) to other locations in cells. But while there is detailed information on how the heme lifecycle begins (heme synthesis), and how it ends (heme degradation), what happens in between is largely a mystery. Here we summarize recent information on the quantification of heme in cells, and we present a discussion of a mechanistic framework that could meet the logistical challenge of heme distribution

Molecular Probes, Chemosensors, and Nanosensors for Optical Detection of Biorelevant Molecules and Ions in Aqueous Media and Biofluids

Synthetic molecular probes, chemosensors, and nanosensors used in combination with innovative assay protocols hold great potential for the development of robust, low-cost, and fast-responding sensors that are applicable in biofluids (urine, blood, and saliva). Particularly, the development of sensors for metabolites, neurotransmitters, drugs, and inorganic ions is highly desirable due to a lack of suitable biosensors. In addition, the monitoring and analysis of metabolic and signaling networks in cells and organisms by optical probes and chemosensors is becoming increasingly important in molecular biology and medicine. Thus, new perspectives for personalized diagnostics, theranostics, and biochemical/medical research will be unlocked when standing limitations of artificial binders and receptors are overcome. In this review, we survey synthetic sensing systems that have promising (future) application potential for the detection of small molecules, cations, and anions in aqueous media and biofluids. Special attention was given to sensing systems that provide a readily measurable optical signal through dynamic covalent chemistry, supramolecular host–guest interactions, or nanoparticles featuring plasmonic effects. This review shall also enable the reader to evaluate the current performance of molecular probes, chemosensors, and nanosensors in terms of sensitivity and selectivity with respect to practical requirement, and thereby inspiring new ideas for the development of further advanced systems

IST Austria Thesis

Plant hormone auxin and its transport between cells belong to the most important mechanisms controlling plant development. Auxin itself could change localization of PINs and thereby control direction of its own flow. We performed an expression profiling experiment in Arabidopsis roots to identify potential regulators of PIN polarity which are transcriptionally regulated by auxin signalling. We identified several novel regulators and performed a detailed characterization of the transcription factor WRKY23 (At2g47260) and its role in auxin feedback on PIN polarity. Gain-of-function and dominant-negative mutants revealed that WRKY23 plays a crucial role in mediating the auxin effect on PIN polarity. In concordance, typical polar auxin transport processes such as gravitropism and leaf vascular pattern formation were disturbed by interfering with WRKY23 function. In order to identify direct targets of WRKY23, we performed consequential expression profiling experiments using a WRKY23 inducible gain-of-function line and dominant-negative WRKY23 line that is defunct in PIN re-arrangement. Among several genes mostly related to the groups of cell wall and defense process regulators, we identified LYSINE-HISTIDINE TRANSPORTER 1 (LHT1; At5g40780), a small amino acid permease gene from the amino acid/auxin permease family (AAAP), we present its detailed characterisation in auxin feedback on PIN repolarization, identified its transcriptional regulation, we propose a potential mechanism of its action. Moreover, we identified also a member of receptor-like protein kinase LRR-RLK (LEUCINE-RICH REPEAT TRANSMEMBRANE PROTEIN KINASE PROTEIN 1; LRRK1; At1g05700), which also affects auxin-dependent PIN re-arrangement. We described its transcriptional behaviour, subcellular localization. Based on global expression data, we tried to identify ligand responsible for mechanism of signalling and suggest signalling partner and interactors. Additionally, we described role of novel phytohormone group, strigolactone, in auxin-dependent PIN re-arrangement, that could be a fundament for future studies in this field. Our results provide first insights into an auxin transcriptional network targeting PIN localization and thus regulating plant development. We highlighted WRKY23 transcriptional network and characterised its mediatory role in plant development. We identified direct effectors of this network, LHT1 and LRRK1, and describe their roles in PIN re-arrangement and PIN-dependent auxin transport processes