Towards in vitro molecular diagnostics using nanostructures

Nanostructures appear to be promising for a number of applications in molecular diagnostics, mainly due to the increased surface-to-volume ratio they can offer, the very low limit of detection achievable, and the possibility to fabricate point-of-care diagnostic devices. In this paper, we review examples of the use of nanostructures as diagnostic tools that bring in marked improvements over prevalent classical assays. The focus is laid on the various sensing paradigms that possess the potential or have demonstrated the capability to replace or augment current analytical strategies. We start with a brief introduction of the various types of nanostructures and their physical properties that determine the transduction principle. This is followed by a concise collection of various functionalization protocols used to immobilize biomolecules on the nanostructure surface. The sensing paradigms are discussed in two contexts: the nanostructure acting as a label for detection, or the nanostructure acting as a support upon which the molecular recognition events take place. In order to be successful in the field of molecular diagnostics, it is important that the nanoanalytical tools be evaluated in the appropriate biological environment. The final section of the review compiles such examples, where the nanostructure-based diagnostic tools have been tested on realistic samples such as serum, demonstrating their analytical power even in the presence of complex matrix effects. The ability of nanodiagnostic tools to detect ultralow concentrations of one or more analytes coupled with portability and the use of low sample volumes is expected to have a broad impact in the field of molecular diagnostics.     

Structure and function of eukaryotic peptide transporters

The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.     

In vivo models of brain tumors: roles of genetically engineered mouse models in understanding tumor biology and use in preclinical studies

Although our knowledge of the biology of brain tumors has increased tremendously over the past decade, progress in treatment of these deadly diseases remains modest. Developing in vivo models that faithfully mirror human diseases is essential for the validation of new therapeutic approaches. Genetically engineered mouse models (GEMMs) provide elaborate temporally and genetically controlled systems to investigate the cellular origins of brain tumors and gene function in tumorigenesis. Furthermore, they can prove to be valuable tools for testing targeted therapies. In this review, we discuss GEMMs of brain tumors, focusing on gliomas and medulloblastomas. We describe how they provide critical insights into the molecular and cellular events involved in the initiation and maintenance of brain tumors, and illustrate their use in preclinical drug testing.     

Single cell biology beyond the era of antibodies: relevance,challenges, and promises in biomedical research

Research of the past two decades has proved the relevance of single cell biology in basic research and translational medicine. Successful detection and isolation of specific subsets is the key to understand their functional heterogeneity. Antibodies are conventionally used for this purpose, but their relevance in certain contexts is limited. In this review, we discuss some of these contexts, posing bottle neck for different fields of biology including biomedical research. With the advancement of chemistry, several methods have been introduced to overcome these problems. Even though microfluidics and microraft array are newer techniques exploited for single cell biology, fluorescence-activated cell sorting (FACS) remains the gold standard technique for isolation of cells for many biomedical applications, like stem cell therapy. Here, we present a comprehensive and comparative account of some of the probes that are useful in FACS. Further, we illustrate how these techniques could be applied in biomedical research. It is postulated that intracellular molecular markers like nucleostemin (GNL3), alkaline phosphatase (ALPL) and HIRA can be used for improving the outcome of cardiac as well as bone regeneration. Another field that could utilize intracellular markers is diagnostics, and we propose the use of specific peptide nucleic acid probes (PNPs) against certain miRNAs for cancer surgical margin prediction. The newer techniques for single cell biology, based on intracellular molecules, will immensely enhance the repertoire of possible markers for the isolation of cell types useful in biomedical research.     

Using computational models to discover and understand mechanisms

Areas of biology such as cell and molecular biology have been dominated by research directed at constructing mechanistic explanations that identify parts and operations that when organized appropriately are responsible for the various phenomena they investigate. Increasingly the mechanisms hypothesized involve non-sequential organization of non-linear operations and so exceed the ability of researchers to mentally rehearse their behavior. Accordingly, scientists rely on tools of computational modeling and dynamical systems theory in advancing dynamic mechanistic explanations. Using circadian rhythm research as an exemplar, this paper explores the variety of roles computational modeling is playing. They serve not just to determine whether the mechanism will produce the desired behavior, but in the discovery process of hypothesizing mechanisms and in understanding why proposed mechanisms behave as they do.     

Hammerhead ribozyme design and application

The emerging knowledge about RNA-based enzymes has already had great impact on our concept of evolutionary history, making the ‘RNA world’ more likely. It may well have an equally important impact on the diagnostic and therapeutic practices of human and veterinary medicine in the next decade. We are not quite there yet. This review addresses the design and application of hammerhead ribozymes, two aspects of a conserved and most commonly studied and used enzymatically active entity among the RNA enzymes. The emerging picture is one of great diversity. There is at this stage no general cell model nor a clearly preferable ribozyme structure. Each and every cell line (and tissue) may be unique in that they vary with respect to structural requirements for optimal uptake, activity and stability of ribozymes. We may have seen only the tip of the iceberg when it comes to RNA-based enzymes and their roles in biology and medicine. Received 3 June 1998; received after revision 28 July 1998; accepted 28 July 1998     

Plant mitochondrial carriers: an overview

In the two last decades, biochemical studies using mitochondrial swelling experiments or direct solute uptake in isolated mitochondria have lead to the identification of different transport systems at the level of the plant mitochondrial inner membrane. Although most of them have been found to have similar features to those identified in animal mitochondria, some differences have been observed between plant and animal transporters. More recently, molecular biology studies have revealed that most of the mitochondrial exchanges are performed by nuclear encoded proteins, which form a superfamily. Members of this family have been reported in animals, yeast as well as plants. This review attempts to give an overview of the present knowledge concerning the biochemical and molecular characterisation of plant members of the mitochondrial carrier family and, when possible, a comparison with carriers from other organisms.     

Prediction of individual response to anticancer therapy: historical and future perspectives

Since the introduction of chemotherapy for cancer treatment in the early 20th century considerable efforts have been made to maximize drug efficiency and at the same time minimize side effects. As there is a great interpatient variability in response to chemotherapy, the development of predictive biomarkers is an ambitious aim for the rapidly growing research area of personalized molecular medicine. The individual prediction of response will improve treatment and thus increase survival and life quality of patients. In the past, cell cultures were used as in vitro models to predict in vivo response to chemotherapy. Several in vitro chemosensitivity assays served as tools to measure miscellaneous endpoints such as DNA damage, apoptosis and cytotoxicity or growth inhibition. Twenty years ago, the development of high-throughput technologies, e.g. cDNA microarrays enabled a more detailed analysis of drug responses. Thousands of genes were screened and expression levels were correlated to drug responses. In addition, mutation analysis became more and more important for the prediction of therapeutic success. Today, as research enters the area of -omics technologies, identification of signaling pathways is a tool to understand molecular mechanism underlying drug resistance. Combining new tissue models, e.g. 3D organoid cultures with modern technologies for biomarker discovery will offer new opportunities to identify new drug targets and in parallel predict individual responses to anticancer therapy. In this review, we present different currently used chemosensitivity assays including 2D and 3D cell culture models and several –omics approaches for the discovery of predictive biomarkers. Furthermore, we discuss the potential of these assays and biomarkers to predict the clinical outcome of individual patients and future perspectives.     

Biomimetic actuators: where technology and cell biology merge

The structural and functional analysis of biological macromolecules has reached a level of resolution that allows mechanistic interpretations of molecular action, giving rise to the view of enzymes as molecular machines. This machine analogy is not merely metaphorical, as bio-analogous molecular machines actually are being used as motors in the fields of nanotechnology and robotics. As the borderline between molecular cell biology and technology blurs, developments in the engineering and material sciences become increasingly instructive sources of models and concepts for biologists. In this review, we provide a--necessarily selective--summary of recent progress in the usage of biological and biomimetic materials as actuators in artificial environments, focussing on motors built from DNA, classical cellular motor systems (tubulin/kinesin, actin/myosin), the rotary motor F1F0-ATPase and protein-based 'smart' materials.     

The functional role of sodium taurocholate cotransporting polypeptide NTCP in the life cycle of hepatitis B,C and D viruses

Chronic hepatitis B, C and D virus (HBV, HCV and HDV) infections are a major cause of liver disease and cancer worldwide. Despite employing distinct replication strategies, the three viruses are exclusively hepatotropic, and therefore depend on hepatocyte-specific host factors. The sodium taurocholate co-transporting polypeptide (NTCP), a transmembrane protein highly expressed in human hepatocytes that mediates the transport of bile acids, plays a key role in HBV and HDV entry into hepatocytes. Recently, NTCP has been shown to modulate HCV infection of hepatocytes by regulating innate antiviral immune responses in the liver. Here, we review the current knowledge of the functional role and the molecular and cellular biology of NTCP in the life cycle of the three major hepatotropic viruses, highlight the impact of NTCP as an antiviral target and discuss future avenues of research.     

Small molecule modifiers of circadian clocks

Circadian clocks orchestrate 24-h oscillations of essential physiological and behavioral processes in response to daily environmental changes. These clocks are remarkably precise under constant conditions yet highly responsive to resetting signals. With the molecular composition of the core oscillator largely established, recent research has increasingly focused on clock-modifying mechanisms/molecules. In particular, small molecule modifiers, intrinsic or extrinsic, are emerging as powerful tools for understanding basic clock biology as well as developing putative therapeutic agents for clock-associated diseases. In this review, we will focus on synthetic compounds capable of modifying the period, phase, or amplitude of circadian clocks, with particular emphasis on the mammalian clock. We will discuss the potential of exploiting these small molecule modifiers in both basic and translational research.     

Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries

Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.     

Computational approaches to structural and functional analysis of plastocyanin and other blue copper proteins

Computational techniques are becoming increasingly important in structural and functional biology, in particular as tools to aid the interpretation of experimental results and the design of new systems. This review reports on recent studies employing a variety of computational approaches to unravel the microscopic details of the structure-function relationships in plastocyanin and other proteins belonging to the blue copper superfamily. Aspects covered include protein recognition, electron transfer and protein-solvent interaction properties of the blue copper protein family. The relevance of integrating diverse computational approaches to address the analysis of a complex protein system, such as a cupredoxin metalloprotein, is emphasized.Received 9 May 2003; received after revision 24 November 2003; accepted 28 November 2003     

Structure and function of RGD peptides involved in bone biology

This review focuses on recent papers that describe the involvement of the RGD sequence in bone biology and incorporate the use of synthetic RGD peptides to develop new drugs or control the bioactivity of materials used for bone regeneration. Because in vivo bone function is completely dependent on angiogenesis and vessels, the present publication is focused on physiology, pathophysiology and therapeutics of RGD peptides dedicated to bone cells and endothelial systems. It appears that alphavbeta3, alphavbeta5 and alphaIIbbeta3 are the integrins most reported to be involved in bone function and RGD sequence binding. The specificity of RGD peptides depends on backbone conformation, orientations of the charged side chains of Arg and Asp residues, and hydrophobic moieties flanking the Asp residue. Despite of recent progress in integrins and RGD peptide structures and function, future work should focus on integrin selectivity of RGD-based agents, model structure and activity-selectivity relationships.     

Laminin: loss-of-function studies

Laminin, one of the most widely expressed extracellular matrix proteins, exerts many important functions in multiple organs/systems and at various developmental stages. Although its critical roles in embryonic development have been demonstrated, laminin’s functions at later stages remain largely unknown, mainly due to its intrinsic complexity and lack of research tools (most laminin mutants are embryonic lethal). With the advance of genetic and molecular techniques, many new laminin mutants have been generated recently. These new mutants usually have a longer lifespan and show previously unidentified phenotypes. Not only do these studies suggest novel functions of laminin, but also they provide invaluable animal models that allow investigation of laminin’s functions at late stages. Here, I first briefly introduce the nomenclature, structure, and biochemistry of laminin in general. Next, all the loss-of-function mutants/models for each laminin chain are discussed and their phenotypes compared. I hope to provide a comprehensive review on laminin functions and its loss-of-function models, which could serve as a reference for future research in this understudied field.     

Fluctuation and response in biology

In 1905, Albert Einstein proposed that the forces that cause the random Brownian motion of a particle also underlie the resistance to macroscopic motion when a force is applied. This insight, of a coupling between fluctuation (stochastic behavior) and responsiveness (non-stochastic behavior), founded an important branch of physics. Here we argue that his insight may also be relevant for understanding evolved biological systems, and we present a ‘fluctuation–response relationship’ for biology. The relationship is consistent with the idea that biological systems are similarly canalized to stochastic, environmental, and genetic perturbations. It is also supported by in silico evolution experiments, and by the observation that ‘noisy’ gene expression is often both more responsive and more ‘evolvable’. More generally, we argue that in biology there is (and always has been) an important role for macroscopic theory that considers the general behavior of systems without concern for their intimate molecular details.     

Cellular copper distribution: a mechanistic systems biology approach

Copper is an essential but potentially harmful trace element required in many enzymatic processes involving redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by regulating copper transport through the copper import CTR proteins and the copper exporters ATP7A and ATP7B. Once copper is imported into the cell, several pathways involving a number of copper proteins are responsible for trafficking it specifically where it is required for cellular life, thus avoiding the release of harmful free copper ions. In this study we review recent progress made in understanding the molecular mechanisms of copper transport in cells by analyzing structural features of copper proteins, their mode of interaction, and their thermodynamic and kinetic parameters, thus contributing to systems biology of copper within the cell.