Outer membrane proteins (OMPs) maintain the viability of Gram‐negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi‐protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical (...) cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single‐molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis. (shrink)

New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter‐molecular (...) interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra‐molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio‐temporal protein‐protein interaction studies open new avenues for FRET in biological research. (shrink)

Conformational changes of proteins and other biomolecules play a fundamental role in their functional mechanism. Single pair Förster resonance energy transfer offers the possibility to detect these conformational changes and dynamics, and to characterize their underlying kinetics. Using spFRET on microscopes with different modes of detection, dynamic timescales ranging from nanoseconds to seconds can be quantified. Confocal microscopy can be used as a means to analyze dynamics in the range of nanoseconds to milliseconds, while total internal reflection fluorescence microscopy (...) offers information about conformational changes on timescales of milliseconds to seconds. While the existence of dynamics can be directly inferred from the FRET efficiency time trace or the correlation of FRET efficiency and fluorescence lifetime, additional computational approaches are required to extract the kinetic rates of these dynamics, a short overview of which is given in this review. The functional mechanism of proteins is often driven by underlying conformational changes. These intramolecular changes and dynamics can be investigated using single pair Förster resonance energy transfer. The kinetic timescales of the dynamics in the range of nanoseconds to seconds is then quantified using different experimental and analytical implementations. (shrink)

Macromolecular interactions play a central role in many biological processes. Protein‐protein interactions have mostly been studied by co‐immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real‐time at the single‐molecule level. This technique is called single‐molecule pull‐down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single‐molecule fluorescence microscopy. SiMPull (...) is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single‐molecule biochemical tool to perform functional studies on the pulled‐down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools. (shrink)

The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution‐based single molecule technique, fluorescence correlation spectroscopy (FCS). This time‐averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical (...) confidence. FCS has proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual‐color cross‐correlation and two‐photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research. BioEssays 24:758–764, 2002. © 2002 Wiley Periodicals, Inc. (shrink)

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact (...) of modern technologies is influenced not only by further hard‐ and software developments, but also by the availability and suitability of protein‐engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. (shrink)

A new field of gene expression regulation research is emerging that has previously been overlooked. This new area is concerned with distinguishing the expression of a single gene from the averaged expression of many gene copies within the cell population. This paper reviews research focused on individual genes in inducible gene expression systems. The main experimental strategy is to measure the gene expression level of a single cell containing a single reporter gene molecule. In contrast to (...) the commonly held belief, gene induction is found to be stochastic under certain conditions. The possible mechanisms and implications are discussed. (shrink)

Replication protein A (RPA), the major single‐stranded DNA‐binding protein in eukaryotic cells, is required for processing of single‐stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high‐affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single (...) class='Hi'>molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error‐prone repair of double‐stranded breaks and promote error‐free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. (shrink)

We have been using feather development as a model for understanding the molecular basis of pattern formation and to explore the roles of homeoproteins, retinoids and adhesion molecules in this process. Two kinds of homeobox (Hox) protein gradients in the skin have been identified: a ‘microgradient’ within a single feather bud and a ‘macrogradient’ across the feather tract. The asynchronous alignment of different Hox macrogradients establishes a unique repertoire of Hox expression patterns in skin appendages within the integument, designated (...) here as the ‘Hox codes of skin appendages’. It is hypothesized that these Hox codes contribute to the phenotypic determination of skin appendages. High doses of retinoic acid cause a morphological transformation between feather and scale, while low doses of retinoic acid cause an alteration of the axial orientation of skin appendages. We have tested the ability of molecules directly involved in the feather formation process to mediate the action of the Hox codes, and surmise that adhesion molecules are potential candidates. Using specific Fabs to suppress the activity of adhesion molecules, we have found that L‐CAM is involved in the formation of the hexagonal pattern, N‐CAM is involved in mediating dermal condensations, tenascin is involved in feather bud growth and elongation, and integrin β‐1 is essential for epithelial‐mesenchymal interactions. More work is in progress to fully understand the molecular pathways regulating the feather formation process. (shrink)

Eukaryotic genome DNA is wrapped around core histones and forms a nucleosome structure. Together with associated proteins and RNAs, these nucleosomes are organized three‐dimensionally in the cell as chromatin. Emerging evidence demonstrates that chromatin consists of rather irregular and variable nucleosome arrangements without the regular fiber structure and that its dynamic behavior plays a critical role in regulating various genome functions. Single‐nucleosome imaging is a promising method to investigate chromatin behavior in living cells. It reveals local chromatin motion, which (...) reflects chromatin organization not observed in chemically fixed cells. The motion data is like a gold mine. Data analyses from many aspects bring us more and more information that contributes to better understanding of genome functions. In this review article, we describe imaging of single‐nucleosomes and their tracked behavior through oblique illumination microscopy. We also discuss applications of this technique, especially in elucidating nucleolar organization in living cells. (shrink)

The traditional formalism of quantum mechanics is mainly used to describe ensembles of identical systems (with a density-operator formalism) or single isolated systems, but is not capable of describing single open quantum objects with many degrees of freedom showing pure-state stochastic dynamical behaviour. In particular, stochastic 'line-migration' as in single-molecule spectroscopy of defect molecules in a molecular matrix is not adequately described. Starting with the Bohr scenario of stochastic quantum jumps (between strict energy eigenstates), we try (...) to incorporate more general pure-state stochastic dynamical behaviour into the quantum mechanical formalism.Probability distributions of (approximately) pure states, arising through the stochastic pure-state dynamics for long times, give rise to appropriate decompositions of thermal density operators. These decompositions of density operators into pure states mediate between quantum mechanics for ensembles of molecules and quantum theory for single molecules (or single dressed quantum objects). We suggest that such decompositions should be consistent with infinite limits (e.g. the Born-Oppenheimer limit for infinite nuclear masses) in the sense that quantum fluctuations (around classical behaviour in the infinite limit) die out asymptotically. (shrink)

Rod photoreceptors are among the most sensitive light detectors in nature. They achieve their remarkable sensitivity across a wide variety of species through a number of essential adaptations: a specialized cellular geometry, a G‐protein cascade with an unusually stable receptor molecule, a low‐noise transduction mechanism, a nearly perfect effector enzyme, and highly evolved mechanisms of feedback control and receptor deactivation. Practically any change in protein expression, enzyme activity, or feedback control can be shown to impair photon detection, either by (...) decreasing sensitivity or signal‐to‐noise ratio, or by reducing temporal resolution. Comparison of mammals to amphibians suggests that rod outer‐segment morphology and the molecules and mechanism of transduction may have evolved together to optimize light sensitivity in darkness, which culminates in the extraordinary ability of these cells to respond to single photons at the ultimate limit of visual perception. (shrink)

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted (...) multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein–protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. BioEssays 23:447–455, 2001. © 2001 John Wiley & Sons, Inc. (shrink)

The neural cell adhesion molecule (N‐CAM) is believed to be a key regulator of adhesive events in the nervous system and skeletal muscle. The recent isolation of N‐CAM cDNAs from different tissues has identified a high degree of diversity in primary amino acid sequence between different isoforms. In this article, we review these recent studies and discuss methods for unravelling the functional consequences of the generation of multiple N‐CAM polypeptides using gene transfection approaches.

We here review primary methods used in quantifying and mapping 5‐hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme‐based detection, and methods involving DNA‐enrichment strategies and the genome‐wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5‐methylcytosine (5mC) by ten‐eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single‐molecule imaging technique that (...) can detect and quantify 5hmC from trace samples and thus offer information regarding the distance‐based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer. (shrink)

The astonishing efficiency and accuracy of DNA replication has long suggested that refined rules enforce a single highly reproducible sequence of molecular events during the process. This view was solidified by early demonstrations that DNA unwinding and synthesis are coupled within a stable molecular factory, known as the replisome, which consists of conserved components that each play unique and complementary roles. However, recent single-molecule observations of replisome dynamics have begun to challenge this view, revealing that replication may (...) not be defined by a uniform sequence of events. Instead, multiple exchange pathways, pauses, and DNA loop types appear to dominate replisome function. These observations suggest we must rethink our fundamental assumptions and acknowledge that each replication cycle may involve sampling of alternative, sometimes parallel, pathways. Here, we review our current mechanistic understanding of DNA replication while highlighting findings that exemplify multi-pathway aspects of replisome function and considering the broader implications. Recent single-molecule studies have revealed that replisome function is dominated by stochastic sampling of multiple molecular pathways. In the context of these new findings, we re-examine the foundational assumptions and seminal experiments that have guided our understanding of replication mechanism and reconcile several long-debated coordination models. (shrink)

Dans le titre un peu provocateur de ce livre, les lecteurs reconnaîtront sans peine un clin d’œil fait à la parabole du « fils prodigue » de l’évangile de Luc. D’emblée, l’auteure explique l’intérêt de ce récit biblique : alors que le fils dont il est question, revient à la maison, domaine privilégié des femmes, la parabole ne parle jamais d’elles. L’histoire de Luc ne concerne que des hommes. Pour Élisabeth Parmentier, ce silence est significatif de l’expérience des femmes dans (...) la « maison-É... (shrink)

J’ai rencontré Marie-Jeanne Bérère à Lyon en 1994. C’était une petite femme vive, à la parole claire et précise, avec un bel accent bourguignon. Elle avait accepté une interview, à son domicile lyonnais, le 23 mars 1995. Je souhaitais la revoir pour ce numéro de CLIO, Histoire, Femmes et Sociétés sur les Chrétiennes mais elle est décédée dans l’été 2000, à 73 ans, sans avoir eu le temps de rédiger ses mémoires. Son itinéraire de femme engagée dans l’Église catholique est (...) celui d’une chrétienn... (shrink)

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two‐dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High‐throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser‐induced fluorescence detection of labeled DNA strands have been of key importance for (...) the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH‐driven mobilization, provides efficient separations and on‐line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single‐cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single‐molecule detection. (shrink)

Since 1990, the multinational public accounting firms have all adopted flexible work arrangement policies. In part, the firms are doing this to fulfill an ethical obligation in creating an appropriate professional environment for their employees. This study examines the effect of participation in a flexible work arrangement program on an individual''s professional success and anticipated turnover as perceived by the participant''s peers and superiors. Subjects from one Big Five accounting firm read a description of a manager and answered a series (...) of questions about the likelihood of the manager''s promotion to partner, voluntary and involuntary turnover, and desirability on a job. Gender and participation in a flexible work arrangement were manipulated in a 2×2 design. The results indicate that participation in a flexible work arrangement evoked significantly more pessimistic predictions on all of the dimensions. Gender did not have an effect on the likelihood ratings. Follow-up questions about the factors that enhance and hinder individuals career success in each work scenario indicated that the perceived ability to "juggle" and the ability to "pull one''s weight" potentially affects evaluations of what it takes to be a successful professional in the financial services environment. Implications for professional and ethics practice and research are also presented. (shrink)

Flexible work arrangements (FWAs) are widely offered in public accounting as a tool to retain valued professional staff. Previous research has shown that participants in FWAs are perceived to be less likely to succeed in their careers in public accounting than individuals in public accounting who do not participate in FWAs (Cohen and Single, 2001). Research has also documented an increasing backlash against family–friendly policies in the workplace as placing unfair burdens on individuals without children. Building directly on a (...) previous study in this journal (Cohen and Single, 2001), this study addresses the issue of whether the documented perceptions toward FWA participants are the result of electing to take part in the FWA or the result of bias against employees with children. The research questions are addressed in a 3 × 2 experimental setting in which we manipulate FWA participation, along with family status and gender of a hypothetical manager in a public accounting firm. Our findings indicate that FWA participants are viewed as less likely to advance and as less committed than individuals without children or individuals who had children but who were not taking part in a FWA. Male FWA participants are viewed as less likely to succeed than female FWA participants. This effect appears to arise from a perception that FWA participants are willing to make sacrifices in their careers to accommodate family needs and thus may not be as committed to making the sacrifices perceived as necessary to meet the rigorous demands of the public accounting environment. This raises the ethical question of what could be done to change the culture in public accounting to foster a substantive support system for individuals who want to balance a family and a career. (shrink)

Flexible work arrangements (FWAs) are widely offered in public accounting as a tool to retain valued professional staff. Previous research has shown that participants in FWAs are perceived to be less likely to succeed in their careers in public accounting than individuals in public accounting who do not participate in FWAs (Cohen and Single, 2001). Research has also documented an increasing backlash against family–friendly policies in the workplace as placing unfair burdens on individuals without children. Building directly on a (...) previous study in this journal (Cohen and Single, 2001), this study addresses the issue of whether the documented perceptions toward FWA participants are the result of electing to take part in the FWA or the result of bias against employees with children. The research questions are addressed in a 3 × 2 experimental setting in which we manipulate FWA participation, along with family status and gender of a hypothetical manager in a public accounting firm. Our findings indicate that FWA participants are viewed as less likely to advance and as less committed than individuals without children or individuals who had children but who were not taking part in a FWA. Male FWA participants are viewed as less likely to succeed than female FWA participants. This effect appears to arise from a perception that FWA participants are willing to make sacrifices in their careers to accommodate family needs and thus may not be as committed to making the sacrifices perceived as necessary to meet the rigorous demands of the public accounting environment. This raises the ethical question of what could be done to change the culture in public accounting to foster a substantive support system for individuals who want to balance a family and a career. (shrink)

In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single‐vesicle experiments involving SNARE proteins have been performed only with lipid‐mixing, but not content‐mixing indicators. Through simultaneous detection of lipid and small content‐mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50‐seconds observation period, for Ca2+‐triggered fusion with SNAREs, full‐length synaptotagmin‐1, and complexin. Our results illustrate the caveats (...) of commonly used bulk lipid‐mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content‐mixing indicators in addition to, or in place of, lipid‐mixing indicators. (shrink)

Evolutionary epistemology has experienced a continuous rise over the last decades. Important new theoretical considerations and novel empirical findings have been integrated into the existing framework. In this paper, I would like to suggest three lines of research that I believe will significantly contribute to further advance EE: ontogenetic considerations, key ideas from cognitive biology, and the framework of the Extended Evolutionary Synthesis. EE, in particular the program of the evolution of epistemological mechanisms, seeks to provide a phylogenetic account of (...) the generation of cognitive processes underlying knowledge creation. Traditionally, EE and EEM have been oriented towards an account of evolutionary theory that mainly drew from the tenets of the Modern Synthesis. The Modern Synthesis largely dismisses ontogenetic processes and considers them irrelevant for evolutionary explanations. If anything, the role of development in evolution is believed to be that of a constraint. There is, however, ample evidence for a tight intertwinement of developmental and evolutionary processes. Organisms employ their cognitive apparatus to interact with the environment in order to achieve a fully functioning perceptual and cognitive nervous system. Also, ontogeny provides generative potentials to enable variations that natural selection can act upon. EEM’s agenda may, therefore, strongly benefit from bringing together ontogenetic and phylogenetic approaches. To grapple with this challenge, an alternative vision of the evolutionary theory termed Extended Evolutionary Synthesis could be used. This extended evolutionary theory explores relationships between the processes of individual development and phenotypic change during evolution and can provide a more suitable framework for EEM to draw from. In recent years, cognitive biology has gained momentum as an independent research field. Cognitive biology builds on the concepts of EEM and understands knowledge as a biogenic phenomenon. Its main objective is also the formulation of substantiated interrelations between cognition and evolution but it focuses on cognitive functionality at all levels of biological organization. It thus employs a “vertical” approach that encompasses nested hierarchies which span from single molecules, cells, and tissues to the organismal level, communities, and societies. In contrast to cognitive biology, EEM is here understood to adopt a “horizontal” approach that focuses on phylogenetic explanations of cognition and knowledge acquisition. Linking EEM with the key ideas of cognitive biology could make EEM’s research program stronger as it can more easily accommodate phylogenetic and ontogenetic questions within a hierarchical, multilevel perspective. This is of particular importance for a more comprehensive account of cognition since living systems are subject to context-dependent causal influences from different organizational levels. In addition to EEM, there is a second program of EE. This program has been labeled evolutionary epistemology of theories and understands the increase in human knowledge, such as scientific theories, as naturalistic accounts of evolution. Both, EEM and EET initially drew from the core concepts of the Modern Synthesis. Several scholars have severely criticized the analogies made between EET and the Neo-Darwinian key processes of evolution. In particular processes of random mutation, the rate of variation, natural selection as the unique driving force, and the adaptationist agenda are believed to reveal disanalogies. In contrast to the Modern Synthesis, the Extended Evolutionary Synthesis not only recognizes developmental processes but also ecological interactions and systems dynamics as well as social and cultural evolutionary reciprocity as important evolutionary processes. Concepts of the Extended Evolutionary Synthesis are therefore expected to be more fruitful for re-conceptualizing parallels between scientific theorizing and biological evolution. (shrink)

DNA topoisomerases, capable of manipulating DNA topology, are ubiquitous and indispensable for cellular survival due to the numerous roles they play during DNA metabolism. As we review here, current structural approaches have revealed unprecedented insights into the complex DNA‐topoisomerase interaction and strand passage mechanism, helping to advance our understanding of their activities in vivo. This has been complemented by single‐molecule techniques, which have facilitated the detailed dissection of the various topoisomerase reactions. Recent work has also revealed the importance (...) of topoisomerase interactions with accessory proteins and other DNA‐associated proteins, supporting the idea that they often function as part of multi‐enzyme assemblies in vivo. In addition, novel topoisomerases have been identified and explored, such as topo VIII and Mini‐A. These new findings are advancing our understanding of DNA‐related processes and the vital functions topos fulfil, demonstrating their indispensability in virtually every aspect of DNA metabolism. (shrink)

New Mechanist philosophical models of "phenomenon reconstitution" understand the process to be driven by explanatory considerations. Here I discuss an episode of phenomenon reconstitution that occurred entirely within an experimental program dedicated to characterizing the phenomenon of kinesin motility. Rather than being driven by explanatory considerations, as standard mechanist views maintain, I argue that the phenomenon of kinesin motility was reconstituted to enhance researchers’ primary experimental tool—the single molecule motility assay.

Landauer's Principle asserts that there is an unavoidable cost in thermodynamic entropy creation when data is erased. It is usually derived from incorrect assumptions, most notably, that erasure must compress the phase space of a memory device or that thermodynamic entropy arises from the probabilistic uncertainty of random data. Recent work seeks to prove Landauer’s Principle without using these assumptions. I show that the processes assumed in the proof, and in the thermodynamics of computation more generally, can be combined to (...) produce devices that both violate the second law and erase data without entropy cost, indicating an inconsistency in the theoretical system. Worse, the standard repertoire of processes selectively neglects thermal fluctuations. Concrete proposals for how we might measure dissipationlessly and expand single molecule gases reversibly are shown to be fatally disrupted by fluctuations. Reversible, isothermal processes on molecular scales are shown to be disrupted by fluctuations that can only be overcome by introducing entropy creating, dissipative processes. (shrink)

The paper describes the change from molecular genetics to postgenomic biology. It focuses on phenomena in the regulation of gene expression that provide a break with the central dogma, according to which sequence specificity for a gene product must be template derived. In its place we find what is called here ‘constitutive molecular epigenesis’. Its three classes of phenomena, which I call sequence ‘activation’, ‘selection’ and ‘creation’, are exemplified by processes such as transcriptional activation, alternative cis- and trans-splicing, and RNA (...) editing. These phenomena support the following main theses of the paper: 1. Other molecular resources share the causal role of ‘genes’: the ‘causal specificity’ for the linear sequence of any gene product is distributed between the coding sequence, cis-acting sequences, trans-acting factors, environmental signals, and the contingent history of the cell (the cellular code) (thesis of distributed causal specificity). 2. These multiple and overlapping processing and targeting mechanisms amplify the repertoire of RNA and protein products specified through the eukaryotic genome, expanding the possibilities specified by the literal code of DNA (thesis of genetic underdeterminism). 3. These mechanisms of gene expression change the focus of postgenomic research from single molecules and their molecular, biochemical and intrinsic function to their cellular, constituent, component or contextual function due to their recruitment and organization in complex cellular networks. In other words, all agents involved in the regulation of gene expression, including DNA, must interact with other agents to achieve full specificity, which is imposed by regulated recruitment and combinatorial control (theses of regulated recruitment and of system analysis). I conclude from these three main theses that the complexity of higher organisms lies not in its number of genes but in the flexibility, versatility and reactivity of its whole genome. (shrink)

It is suggested that if Cultural-Historical Activity Theory (CHAT) is to fulfil its potential as an approach to cultural and historical science in general, then an interdisciplinary concept of activity is needed. Such a concept of activity would provide a common foundation for all the human sciences, underpinning concepts of, for example, state and social movement equally as, for example, learning and personality. For this is needed a clear conception of the ‘unit of analysis’ of activity, i.e., of what constitutes (...) ‘ an activity’, and a clear distinction between the unit of analysis and the substance , i.e., ultimate reality underlying all the human sciences: artifact-mediated joint activity. It is claimed that the concept of ‘project collaboration’ – the interaction between two or more persons in pursuit of a common objective – forms such a unit of activity, the single ‘molecule’ in terms of which both sociological and psychological phenomena can be theorised. It is suggested that such a clarification of the notion of activity allows us to see how individual actions and societal activities mutually constitute one another and are each construed in the light of the other. (shrink)

Biological processes span several scales in space, from the single molecules to organisms and ecosystems. Multiscale modelling approaches in biology are useful to take into account the complex interactions between different organisation levels in those systems. We review several single- and multiscale models, from the most simple to the complex ones, and discuss their properties from a multiscale point of view. Approaches based on master equations for stochastic processes, individual-based models, hybrid continuous-discrete models and structured PDE models are (...) presented. (shrink)

Eukaryotic DNA replication initiates at multiple origins. In early fly and frog embryos, chromosomal replication is very rapid and initiates without sequence specificity. Despite this apparent randomness, the spacing of these numerous initiation sites must be sufficiently regular for the genome to be completely replicated on time. Studies in various eukaryotes have revealed that there is a strict temporal separation of origin “licensing” prior to S phase and origin activation during S phase. This may suggest that replicon size must be (...) already established at the licensing stage. However, recent experiments suggest that a large excess of potential origins are assembled along chromatin during licensing. Thus, a regular replicon size may result from the selection of origins during S phase. We review single molecule analyses of origin activation and other experiments addressing this issue and their general significance for eukaryotic DNA replication. BioEssays 25:116–125, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

Clathrin‐mediated endocytosis is a major route for the retrieval of plasma‐membrane cargoes, and defects of this process can cause catastrophic human dysfunctions. However, the processes governing how a clathrin‐coated profile (ccp) is initiated are still murky. Despite an ever‐growing cast of molecules proposed as triggers of ccp nucleation and increasingly sophisticated bioimaging techniques examining clathrin‐mediated endocytosis, it is yet unknown if ccp formation is governed by a universal mechanism. A recent paper by Cocucci et al. has tracked single‐molecule (...) events to identify that stable accumulation of ccps requires the near‐simultaneous arrival of two AP2 adaptors bridged by one clathrin triskelion. This commentary examines the role of AP2 in cargo‐mediated endocytosis in the light of recent advances in biophotonics, chemical inhibitors and genetics, examines the claims of other molecules to be the initiators of ccp formation and proposes future directions in research into this topic.Editor's suggested further reading in BioEssays: The evolution of dynamin to regulate clathrin‐mediated endocytosis AbstractClathrin‐mediated endocytosis: What works for small, also works for big Abstract. (shrink)

We clarify different definitions of the density matrix by proposing the use of different names, the full density matrix for a single-closed quantum system, the compressed density matrix for the averaged single molecule state from an ensemble of molecules, and the reduced density matrix for a part of an entangled quantum system, respectively. We show that ensembles with the same compressed density matrix can be physically distinguished by observing fluctuations of various observables. This is in contrast to (...) a general belief that ensembles with the same compressed density matrix are identical. Explicit expression for the fluctuation of an observable in a specified ensemble is given. We have discussed the nature of nuclear magnetic resonance quantum computing. We show that the conclusion that there is no quantum entanglement in the current nuclear magnetic resonance quantum computing experiment is based on the unjustified belief that ensembles having the same compressed density matrix are identical physically. Related issues in quantum communication are also discussed. (shrink)

The two theories of pancreatic enzyme secretion, those of exocytosis and transmembrane flow, are described. Data thought to support the theory of transmembrane flow of single molecules from pancreatic acinar cells are first reviewed, and the conditions which could allow these data to be explained by the theory of exocytosis of enzyme quanta, i.e. secretory granules, are then discussed.The evidence suggesting short‐term modulation of the composition of pancreatic juice is also considered, and its possible explanations at the organ and (...) cellular level are discussed, in the light of suggested theories on enzyme secretion pathways. (shrink)

This thesis investigates the epistemological and metaphysical relations between chemistry and quantum mechanics. These relations are examined with respect to how chemistry and quantum mechanics each describe a single inert molecule. A review of how these relations are understood in the literature shows that there is a proliferation of positions which focus on how chemistry is separate from quantum mechanics. This proliferation is accompanied by a tendency within the philosophy of chemistry community to connect the legitimacy of the (...) field with the autonomy of chemistry. First, it is argued that this connection should not be made. Secondly, it is argued that chemistry and quantum mechanics are unified in accordance with Harold Kincaid’s model of non-reductive unity because the two theories exhibit particular epistemic and metaphysical interconnections. Thirdly, a metaphysical account is examined which is incompatible with Kincaid’s model of unity; namely strong emergence as understood by Robin Hendry. According to Hendry, the structure of a single inert molecule strongly emerges from its quantum mechanical entities in the sense that there is downward causation. However, Hendry’s defense of this account faces certain problems. Moreover, the putative empirical evidence for his understanding of strong emergence can be explained without invoking strong emergence. This is shown by considering how quantum mechanics assumes an idealized understanding of a molecule’s stability and structure. In the light of the philosophical literature on idealizations, this idealization can be interpreted in two different ways, both of which explain why quantum mechanics describes the structure of a single molecule the way it does, without assuming strong emergence in Hendry’s sense. Each interpretation has philosophical implications regarding the nature of chemical properties, and the relation of chemistry and quantum mechanics. These implications are consistent with Kincaid’s model of unity and thus further support chemistry’s unity with quantum mechanics (as per Kincaid). (shrink)

Pointillistic based super‐resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super‐resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations (...) in photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair‐correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described. (shrink)