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)

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)

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)

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 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 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)

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)

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)

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 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)

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)

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)

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.

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)

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.

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)

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)

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)

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)

Cryo‐electron microscopy allows the visualization of macromolecules in their native state. Combined with techniques of three‐dimensional reconstruction, cryo‐EM images of single molecules can be used to study macromolecular interactions. The ribosome, a large RNA–protein complex with multiple binding interactions, is an excellent test case illustrating the power of these new techniques. Conformational changes during the binding of tRNA and protein factors to the ribosome can now be studied without the interference of crystal packing. Now that the first X‐ray structures (...) of ribosomal subunits have become available, conformational changes observed by cryo‐EM in different functional states can be traced back to internal rearrangements of the underlying structural framework. Electron microscopy, X‐ray crystallography, and modeling should be used together in the endeavor to understand the functioning of the translational machinery. BioEssays 23:725–732, 2001. © 2001 John Wiley & Sons, Inc. (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)

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)

The sense of touch informs us of the physical properties of our surroundings and is a critical aspect of communication. Before touches are perceived, mechanical signals are transmitted quickly and reliably from the skin's surface to mechano‐electrical transduction channels embedded within specialized sensory neurons. We are just beginning to understand how soft tissues participate in force transmission and how they are deformed. Here, we review empirical and theoretical studies of single molecules and molecular ensembles thought to be involved in (...) mechanotransmission and apply the concepts emerging from this work to the sense of touch. We focus on the nematode Caenorhabditis elegans as a well‐studied model for touch sensation in which mechanics can be studied on the molecular, cellular, and systems level. Finally, we conclude that force transmission is an emergent property of macromolecular cellular structures that mutually stabilize one another. (shrink)

Biological psychiatry has been dominated by a psychopharmacologically-driven neurotransmitter dysfunction paradigm. The objective of this paper is to explore a reductionist assumption underlying this paradigm, and to suggest an improvement on it. The methods used are conceptual analysis with a comparative approach, particularly using illustrations from the history of both biological psychiatry and molecular biology. The results are that complete reduction to physicochemical explanations is not fruitful, at least in the initial stages of research in the medical and life sciences, (...) and that an appropriate (non-reducible) integrative principle - addressing a property of the whole system under study - is required for each domain of research. This is illustrated in Pauling's use of a topological integrative principle for the discovery of the functioning of proteins and in Watson and Crick's use of the notion of a genetic code as an integrative principle for the discovery of the structure of genes. The neurotransmitter dysfunction paradigm addresses single molecules and their neural pathways, yet their interactions within the CNS as a whole seem most pertinent to mental disorders such as schizophrenia. The lack within biological psychiatry of an integrative principle addressing a property of the CNS as a whole may be responsible for the empirical failure of orthomolecular psychiatry, as well as for the central role that serendipity has played in the study of mental disorders, which is dominated by the neurotransmitter paradigm. The conclusion is that research in biological psychiatry may benefit from using, at least initially, some integrative principle(s) addressing a property of the CNS as a whole, such as connectionism or a hierarchical notion. (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)

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)

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)

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)

Epithelia can be defined morphologically as tissues that line surfaces, and ultrastructurally with reference to their cells' apico‐basal polarity and possession of specific cell‐cell junctions. Defining the epithelial phenotype at a molecular level is more problematic ‐ while it is easy to name proteins (e.g. keratins) expressed by a “typical” epithelium, no known molecules are expressed by every epithelium but by no other tissues. Cells can differentiate to and from the epithelial state as part of normal development, as a response (...) to disease or when manipulated in culture. Many factors (matrix components, adhesion molecules, growth factors, transcription factors) have been identified that can trigger these transitions of phenotype in specific cases, but to date no general master regulators of the epithelial state have been found. The epithelial state may therefore be controlled by multiple regulatory genes so that there is no single molecule responsible for all of the diverse types of epithelium that exist in higher animals. (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)

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)

Structural biology has experienced several transformative technological advances in recent years. These include: development of extremely bright X-ray sources and the use of electrons to extend protein crystallography to ever decreasing crystal sizes; and an increase in the resolution attainable by cryo-electron microscopy. Here we discuss the use of these techniques in general terms and highlight their application for biological filament systems, an area that is severely underrepresented in atomic resolution structures. We assemble a model of a capped tropomyosin-actin minifilament (...) to demonstrate the utility of combining structures determined by different techniques. Finally, we survey the methods that attempt to transform high resolution structural biology into more physiological environments, such as the cell. Together these techniques promise a compelling decade for structural biology and, more importantly, they will provide exciting discoveries in understanding the designs and purposes of biological machines. Structural biology is undergoing technological advancements that now reveal the structures of previously inaccessible biological machines. Microfocus synchrotron beamlines, X-ray free electron lasers, and MicroED cope with vanishingly small crystals, while cryo-electron microscopy targets single molecules at near atomic resolution. These advances promise an exciting decade for structural biology. (shrink)

The nuclear export of mRNA through the nuclear pore complex (NPC) is a process required for the healthy functioning of human cells, making it a critical area of research. However, the geometries of mRNA and the NPC are well below the diffraction limit of light microscopy, thereby presenting significant challenges in evaluating the discrete interactions and dynamics involved in mRNA nuclear export through the native NPC. Recent advances in biotechnology and single‐molecule super‐resolution light microscopy have enabled researchers to (...) gain granular insight into the specific contributions made by discrete nucleoporins in the nuclear basket of the NPC to the export of mRNA. Specifically, by expanding upon the docking step facilitated by the protein TPR in the nuclear basket as well as identifying NUP153 as being the primary nuclear basket protein initiating export through the central channel of the NPC. (shrink)

The Nanoessence project aims to examine life at a sub-cellular level, re-examining space and scale within the human context. A single HaCat skin cell is analysed with an Atomic Force Microscope (AFM) to explore comparisons between, life and death at a nano level. The humanistic discourse concerning life is now being challenged by nanotechnological research that brings into question the concepts of what constitutes living. The Nanoessence project research is based on data gathered as part of a residency at (...) SymbioticA, Centre of Excellence in Biological Arts, University of Western Australia and the Nanochemistry Research Institute, (NRI) Curtin University of Technology. The space of the body can be seen at an atomic level as having no defining boundaries. More generally, molecular self-assembly seeks to use concepts of supramolecular chemistry, and molecular recognition in particular, to cause single-molecule components to automatically arrange themselves into some useful conformation (reference Wikipedia). Nano assembly, or bottom-up approach, is the construction of a supramolecular chemistry by the assembly of nanoscopic particles, or even atoms and molecules. The proposal for nanotechnology to reshape nature atom by atom develops an interesting debate as to the constitution of life. The Nanoessence project aims to construct a physical experience to examine this scientific and metaphysical world. Nanoessence is an interactive audio-visual installation. In the Nanoessence installation the viewer will interface with the visual and sonic presentation through his or her own breath. In the context of the project breath has a strong conceptual and metaphorical link to a Biblical inception of life. The project attempts to maintain a high quality of authentic data to engage the viewer in a sensorial qualitative experience of quantitative data. (shrink)

The regulation of DNA replication is a fascinating biological problem both from a mechanistic angle—How is replication timing regulated?—and from an evolutionary one—Why is replication timing regulated? Recent work has provided significant insight into the first question. Detailed biochemical understanding of the mechanism and regulation of replication initiation has made possible robust hypotheses for how replication timing is regulated. Moreover, technical progress, including high‐throughput, single‐molecule mapping of replication initiation and single‐cell assays of replication timing, has allowed for (...) direct testing of these hypotheses in mammalian cells. This work has consolidated the conclusion that differential replication timing is a consequence of the varying probability of replication origin initiation. The second question is more difficult to directly address experimentally. Nonetheless, plausible hypotheses can be made and one—that replication timing contributes to the regulation of chromatin structure—has received new experimental support. (shrink)

The examination of the complex cell biology of the human malaria parasite Plasmodium falciparum usually relies on the time‐consuming generation of transgenic parasites. Here, metabolic labeling and click chemistry are employed as a fast transfection‐independent method for the microscopic examination of protein S‐palmitoylation, an important post‐translational modification during the asexual intraerythrocytic replication of P. falciparum. Applying various microscopy approaches such as confocal, single‐molecule switching, and electron microscopy, differences in the extent of labeling within the different asexual developmental stages (...) of P. falciparum and the host erythrocytes over time are observed. (shrink)

Molecular interactions are at the origin of life. How molecules get at different locations in the cell and how they locate their partners is a major and partially unresolved question in biology that is paramount to signaling. Spatio‐temporal correlations of fluctuating fluorescently tagged molecules reveal how they move, interact, and bind in the different cellular compartments. Methods based on fluctuations represent a remarkable technical advancement in biological imaging. Here we discuss image analysis methods based on spatial and temporal correlation of (...) fluctuations, raster image correlation spectroscopy, number and brightness, and spatial cross‐correlations that give us information about how individual molecules move in cells and interact with partners at the single molecule level. These methods can be implemented with a standard laser scanning microscope and produce a cellular level spatio‐temporal map of molecular interactions. (shrink)

We present a quantum-mechanical analysis of Szilard's famous single-molecule engine, showing that it is analogous to the double-slit experiment. We further show that the energy derived from the engine's operation is provided by the act of observing the molecule's location. The engine can be operated with no increase in physical entropy, and the second law of thermodynamics does not compel us to relate physical entropy to informational entropy. We conclude that information per seis a subjective, idealized, concept (...) separated from the physical realm. Physical entropy depends on physical objects and physical interactions, and any entropy change owing to observations is entirely a result of the entropy created in the physical apparatus by the process of observation. (shrink)

This treatise presents thoughts on the divide that exists in chemistry between those who seek their understanding within a universe wherein the laws of physics apply and those who prefer alternative universes wherein the laws are suspended or ‘bent’ to suit preconceived ideas. The former approach is embodied in the quantum theory of atoms in molecules (QTAIM), a theory based upon the properties of a system’s observable distribution of charge. Science is experimental observation followed by appeal to theory that, upon (...) occasion, leads to new experiments. This is the path that led to the development of the molecular structure hypothesis—that a molecule is a collection atoms with characteristic properties linked by a network of bonds that impart a structure—a concept forged in the crucible of nineteenth century experimental chemistry. One hundred and fifty years of experimental chemistry underlie the realization that the properties of some total system are the sum of its atomic contributions. The concept of a functional group, consisting of a single atom or a linked set of atoms, with characteristic additive properties forms the cornerstone of chemical thinking of both molecules and crystals and Dalton’s atomic hypothesis has emerged as the operational theory of chemistry. We recognize the presence of a functional group in a given system and predict its effect upon the static, reactive and spectroscopic properties of the system in terms of the characteristic properties assigned to that group. QTAM gives physical substance to the concept of a functional group. (shrink)