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Congratulations are in order to KC Park, the winner of this year's OXION Poster Prize.
Neurodegenerative diseases (NDs) including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease are incurable and affect millions of people worldwide. The development of treatments for this unmet clinical need is a major global research challenge. Computer-aided drug design (CADD) methods minimize the huge number of ligands that could be screened in biological assays, reducing the cost, time, and effort required to develop new drugs. In this review, we provide an introduction to CADD and examine the progress in applying CADD and other molecular docking studies to NDs. We provide an updated overview of potential therapeutic targets for various NDs and discuss some of the advantages and disadvantages of these tools.
Cardiac valve annulus manual segmentation using computer assisted visual feedback in three-dimensional image data.
Annulus manual segmentation is an important tool for the study of valve anatomy and physiology, for the four main valves of the heart (mitral, tricuspid, aortic and pulmonary). In this paper we review two traditional manual segmentation approaches: slice-by-slice and interpolating a sparse set of landmarks with a spline curve. We propose a new Spline Tool for the open source software platform Seg3D, that is fast and improves spatial coherence by providing visual feedback of the segmentation in real time. The Spline Tool was tested successfully on 14 rat hearts, on all four valves.
Mechano-sensitivity of cardiac pacemaker function: pathophysiological relevance, experimental implications, and conceptual integration with other mechanisms of rhythmicity.
Cardiac pacemaker cells exhibit spontaneous, rhythmic electrical excitation, termed automaticity. This automatic initiation of action potentials requires spontaneous diastolic depolarisation, whose rate determines normal rhythm generation in the heart. Pacemaker mechanisms have been split recently into: (i) cyclic changes in trans-sarcolemmal ion flows (termed the 'membrane-clock'), and (ii) rhythmic intracellular calcium cycling (the 'calcium-clock'). These two 'clocks'undoubtedly interact, as trans-sarcolemmal currents involved in pacemaking include calcium-carrying mechanisms, while intracellular calcium cycling requires trans-sarcolemmal ion flux as the mechanism by which it affects membrane potential. The split into separate 'clocks' is, therefore, somewhat arbitrary. Nonetheless, the 'clock' metaphor has been conceptually stimulating, in particular since there is evidence to support the view that either 'clock'could be sufficient in principle to set the rate of pacemaker activation. Of course, the same has also been shown for sub-sets of 'membrane-clock' ion currents, illustrating the redundancy of mechanisms involved in maintaining such basic functionality as the heartbeat, a theme that is common for vital physiological systems. Following the conceptual path of identifying individual groups of sub-mechanisms, it is important to remember that the heart is able to adapt pacemaker rate to changes in haemodynamic load, even after isolation or transplantation, and on a beat-by-beat basis. Neither the 'membrane-' nor the 'calcium-clock' do, as such, inherently account for this rapid adaptation to circulatory demand (cellular Ca²⁺ balance changes over multiple beats, while variation of sarcolemmal ion channel presence takes even longer). This suggests that a third set of mechanisms must be involved in setting the pace. These mechanisms are characterised by their sensitivity to the cyclically changing mechanical environment, and--in analogy to the above terminology--this might be considered a 'mechanics-clock'. In this review, we discuss possible roles of mechano-sensitive mechanisms for the entrainment of membrane current dynamics and calcium-handling. This can occur directly via stretch-activation of mechano-sensitive ion channels in the sarcolemma and/or in intracellular membrane compartments, as well as by modulation of 'standard' components of the 'membrane-' or 'calcium-clock'. Together, these mechanisms allow rapid adaptation to changes in haemodynamic load, on a beat-by-beat basis. Additional relevance arises from the fact that mechano-sensitivity of pacemaking may help to explain pacemaker dysfunction in mechanically over- or under-loaded tissue. As the combined contributions of the various underlying oscillatory mechanisms are integrated at the pacemaker cell level into a single output--a train of pacemaker action potentials--we will not adhere to a metaphor that implies separate time-keeping units ('clocks'), and rather focus on cardiac pacemaking as the result of interactions of a set of coupled oscillators, whose individual contributions vary depending on the pathophysiological context. We conclude by considering the utility and limitations of viewing the pacemaker as a coupled system of voltage-, calcium-, and mechanics-modulated oscillators that, by integrating a multitude of inputs, offers the high level of functional redundancy that is vitally important for cardiac automaticity.
Interrogation of living myocardium in multiple static deformation states with diffusion tensor and diffusion spectrum imaging.
Diffusion tensor magnetic resonance imaging (MRI) reveals valuable insights into tissue histo-anatomy and microstructure, and has steadily gained traction in the cardiac community. Its wider use in small animal cardiac imaging in vivo has been constrained by its extreme sensitivity to motion, exaggerated by the high heart rates usually seen in rodents. Imaging of the isolated heart eliminates respiratory motion and, if conducted on arrested hearts, cardiac pulsation. This serves as an important intermediate step for basic and translational studies. However, investigating the micro-structural basis of cardiac deformation in the same heart requires observations in different deformation states. Here, we illustrate the imaging of isolated rat hearts in three mechanical states mimicking diastole (cardioplegic arrest), left-ventricular (LV) volume overload (cardioplegic arrest plus LV balloon inflation), and peak systole (lithium-induced contracture). An optimised MRI-compatible Langendorff perfusion setup with the radio-frequency (RF) coil integrated into the wet chamber was developed for use in a 9.4T horizontal bore scanner. Signal-to-noise ratio improved significantly, by 75% compared to a previous design with external RF coil, and stability tests showed no significant changes in mean T1, T2 or LV wall thickness over a 170 min period. In contracture, we observed a significant reduction in mean fractional anisotropy from 0.32 ± 0.02 to 0.28 ± 0.02, as well as a significant rightward shift in helix angles with a decrease in the proportion of left-handed fibres, as referring to the locally prevailing cell orientation in the heart, from 24.9% to 23.3%, and an increase in the proportion of right-handed fibres from 25.5% to 28.4%. LV overload, in contrast, gave rise to a decrease in the proportion of left-handed fibres from 24.9% to 21.4% and an increase in the proportion of right-handed fibres from 25.5% to 26.0%. The modified perfusion and coil setup offers better performance and control over cardiac contraction states. We subsequently performed high-resolution diffusion spectrum imaging (DSI) and 3D whole heart fibre tracking in fixed ex vivo rat hearts in slack state and contracture. As a model-free method, DSI augmented the measurements of water diffusion by also informing on multiple intra-voxel diffusion orientations and non-Gaussian diffusion. This enabled us to identify the transition from right- to left-handed fibres from the subendocardium to the subepicardium, as well as voxels in apical regions that were traversed by multiple fibres. We observed that both the mean generalised fractional anisotropy and mean kurtosis were lower in hearts in contracture compared to the slack state, by 23% and 9.3%, respectively. While its heavy acquisition burden currently limits the application of DSI in vivo, ongoing work in acceleration techniques may enable its use in live animals and patients. This would provide access to the as yet unexplored dimension of non-Gaussian diffusion that could serve as a highly sensitive marker of cardiac micro-structural integrity.
Multi-parametric electrophysiological measurements using optical methods have become a highly valued standard in cardiac research. Most published optical mapping systems are expensive and complex. Although some applications demand high-cost components and complex designs, many can be tackled with simpler solutions. Here, we describe (1) a camera-based voltage and calcium imaging system using a single 'economy' electron-multiplying charge-coupled device camera and demonstrate the possibility of using a consumer camera for imaging calcium transients of the heart, and (2) a photodiode-based voltage and calcium high temporal resolution measurement system using single-element photodiodes and an optical fibre. High-throughput drug testing represents an application where system scalability is particularly attractive. Therefore, we tested our systems on tissue exposed to a well-characterized and clinically relevant calcium channel blocker, nifedipine, which has been used to treat angina and hypertension. As experimental models, we used the Langendorff-perfused whole-heart and thin ventricular tissue slices, a preparation gaining renewed interest by the cardiac research community. Using our simplified systems, we were able to monitor simultaneously the marked changes in the voltage and calcium transients that are responsible for the negative inotropic effect of the compound.
Integrated approach for the study of anatomical variability in the cardiac Purkinje system: from high resolution MRI to electrophysiology simulation.
The ordered electrical stimulation of the ventricles is achieved by a specialized network of fibres known as the Purkinje system. The gross anatomy and basic functional role of the Purkinje system is well understood. However, very little is known about the detailed anatomy of the Purkinje system, its inter-individual variability and the implications of the variability in ventricular function, in part due to limitations in experimental techniques. In this study, we aim to provide new insight into the inter-individual variability of the free running Purkinje system anatomy and its impact on ventricular electrophysiological function. As a first step towards achieving this aim, high resolution magnetic resonance imaging (MRI) datasets of rat and the rabbit ventricles are obtained and analysed using a novel semi-automatic image processing algorithm for segmentation of the free-running Purkinje system. Segmented geometry from the MRI datasets is used to construct a computational model of the Purkinje system, which is incorporated in to an anatomically-based ventricular geometry to simulate ventricular electrophysiological activity.
Simultaneous measurement and modulation of multiple physiological parameters in the isolated heart using optical techniques
Whole-heart multi-parametric optical mapping has provided valuable insight into the interplay of electrophysiological parameters, and this technology will continue to thrive as dyes are improved and technical solutions for imaging become simpler and cheaper. Here, we show the advantage of using improved 2nd-generation voltage dyes, provide a simple solution to panoramic multi-parametric mapping, and illustrate the application of flash photolysis of caged compounds for studies in the whole heart. For proof of principle, we used the isolated rat whole-heart model. After characterising the blue and green isosbestic points of di-4-ANBDQBS and di-4-ANBDQPQ, respectively, two voltage and calcium mapping systems are described. With two newly custom-made multi-band optical filters, (1) di-4-ANBDQBS and fluo-4 and (2) di-4-ANBDQPQ and rhod-2 mapping are demonstrated. Furthermore, we demonstrate three-parameter mapping using di-4-ANBDQPQ, rhod-2 and NADH. Using off-the-shelf optics and the di-4- ANBDQPQ and rhod-2 combination, we demonstrate panoramic multi-parametric mapping, affording a 360° spatiotemporal record of activity. Finally, local optical perturbation of calcium dynamics in the whole heart is demonstrated using the caged compound, o-nitrophenyl ethylene glycol tetraacetic acid (NP-EGTA), with an ultraviolet light-emitting diode (LED). Calcium maps (heart loaded with di-4-ANBDQPQ and rhod-2) demonstrate successful NP-EGTA loading and local flash photolysis. All imaging systems were built using only a single camera. In conclusion, using novel 2nd-generation voltage dyes, we developed scalable techniques for multi-parametric optical mapping of the whole heart from one point of view and panoramically. In addition to these parameter imaging approaches, we show that it is possible to use caged compounds and ultraviolet LEDs to locally perturb electrophysiological parameters in the whole heart. © The Author(s) 2012.
In most mammalian cardiomyocytes, the transverse tubular system (t-system) is a major site for electrical signaling and excitation-contraction coupling. The t-system consists of membrane invaginations, which are decorated with various proteins involved in excitation-contraction coupling and mechano-electric feedback. Remodeling of the t-system has been reported for cells in culture and various types of heart disease. In this paper, we provide insights into effects of mechanical strain on the t-system in rabbit left ventricular myocytes. Based on fluorescent labeling, three-dimensional scanning confocal microscopy, and digital image analysis, we studied living and fixed isolated cells in different strain conditions. We extracted geometric features of transverse tubules (t-tubules) and characterized their arrangement with respect to the Z-disk. In addition, we studied the t-system in cells from hearts fixed either at zero left ventricular pressure (slack), at 30 mmHg (volume overload), or during lithium-induced contracture, using transmission electron microscopy. Two-dimensional image analysis was used to extract features of t-tubule cross-sections. Our analyses of confocal microscopic images showed that contracture at the cellular level causes deformation of the t-system, increasing the length and volume of t-tubules, and altering their cross-sectional shape. TEM data reconfirmed the presence of mechanically induced changes in t-tubular cross sections. In summary, our studies suggest that passive longitudinal stretching and active contraction of ventricular cardiomyocytes affect the geometry of t-tubules. This confirms that mechanical changes at cellular levels could promote alterations in partial volumes that would support a convection-assisted mode of exchange between the t-system content and extracellular space. © 2012 Elsevier Ltd.
© 2014 Elsevier Ltd. Computational models have become a fundamental tool in cardiac research. Models are evolving to cover multiple scales and physical mechanisms. They are moving towards mechanistic descriptions of personalised structure and function, including effects of natural variability. These developments are underpinned to a large extent by advances in imaging technologies. This article reviews how novel imaging technologies, or the innovative use and extension of established ones, integrate with computational models and drive novel insights into cardiac biophysics. In terms of structural characterization, we discuss how imaging is allowing a wide range of scales to be considered, from cellular levels to whole organs. We analyse how the evolution from structural to functional imaging is opening new avenues for computational models, and in this respect we review methods for measurement of electrical activity, mechanics and flow. Finally, we consider ways in which combined imaging and modelling research is likely to continue advancing cardiac research, and identify some of the main challenges that remain to be solved.
© 2014 The Authors. This paper reviews the development and application of paired muscle preparations, called duplex, for the investigation of mechanisms and consequences of intra-myocardial electro-mechanical heterogeneity. We illustrate the utility of the underlying combined experimental and computational approach for conceptual development and integration of basic science insight with clinically relevant settings, using previously published and new data. Directions for further study are identified.
Living cardiac tissue slices: An organotypic pseudo two-dimensional model for cardiac biophysics research
© 2014 The Authors. Living cardiac tissue slices, a pseudo two-dimensional (2D) preparation, have received less attention than isolated single cells, cell cultures, or Langendorff-perfused hearts in cardiac biophysics research. This is, in part, due to difficulties associated with sectioning cardiac tissue to obtain live slices. With moderate complexity, native cell-types, and well-preserved cell-cell electrical and mechanical interconnections, cardiac tissue slices have several advantages for studying cardiac electrophysiology. The trans-membrane potential (V m ) has, thus far, mainly been explored using multi-electrode arrays. Here, we combine tissue slices with optical mapping to monitor V m and intracellular Ca 2+ concentration ([Ca 2+ ] i ). This combination opens up the possibility of studying the effects of experimental interventions upon action potential (AP) and calcium transient (CaT) dynamics in 2D, and with relatively high spatio-temporal resolution. As an intervention, we conducted proof-of-principle application of stretch. Mechanical stimulation of cardiac preparations is well-established for membrane patches, single cells and whole heart preparations. For cardiac tissue slices, it is possible to apply stretch perpendicular or parallel to the dominant orientation of cells, while keeping the preparation in a constant focal plane for fluorescent imaging of in-slice functional dynamics. Slice-to-slice comparison furthermore allows one to assess transmural differences in ventricular tissue responses to mechanical challenges. We developed and tested application of axial stretch to cardiac tissue slices, using a manually-controlled stretching device, and recorded V m and [Ca 2+ ] i by optical mapping before, during, and after application of stretch. Living cardiac tissue slices, exposed to axial stretch, show an initial shortening in both AP and CaT duration upon stretch application, followed in most cases by a gradual prolongation of AP and CaT duration during stretch maintained for up to 50min. After release of sustained stretch, AP duration (APD) and CaT duration reverted to shorter values. Living cardiac tissue slices are a promising experimental model for the study of cardiac mechano-electric interactions. The methodology described here can be refined to achieve more accurate control over stretch amplitude and timing (e.g. using a computer-controlled motorised stage, or by synchronising electrical and mechanical events) and through monitoring of regional tissue deformation (e.g. by adding motion tracking).
© 2014. With its conceptualisation nearly fifty years ago, cardiac mechano-electric coupling (MEC) has developed from a collection of anecdotal reports into a field of research that - in spite of early scepticism - is now an accepted part of cardiac structure-function considerations. Throughout this development, MEC studies have been both driver and beneficiary of technological innovation: from sharp electrode recordings for the study of in situ cell responses to cell isolation and patch clamp from early approaches to mechanical stimulation of tissue using photographic diaphragms to modern force-length feedback systems for isolated cells; and from strain gauge force recordings to genetically encodes stress probes. While much is now known about subcellular contributors to cardiac MEC, including stretch-activated ion channels and mechanical modulation of cell calcium handling, their integration at higher levels of structural complexity, and the generation of clinically-translatable knowledge, have remained challenging. This short review provides a brief summary of past achievements, current activities, and potentially rewarding future directions of cardiac MEC research. We highlight challenges and opportunities on the way to an integrated understanding of how external and intrinsic mechanical factors affect the heartbeat in health and disease, and how such understanding may improve the ways in which we prevent and/or treat cardiac pathology.
Single-sensor system for spatially resolved, continuous, and multiparametric optical mapping of cardiac tissue
BACKGROUND Simultaneous optical mapping of multiple electrophysiologically relevant parameters in living myocardium is desirable for integrative exploration of mechanisms underlying heart rhythm generation under normal and pathophysiologic conditions. Current multiparametric methods are technically challenging, usually involving multiple sensors and moving parts, which contributes to high logistic and economic thresholds that prevent easy application of the technique. OBJECTIVE The purpose of this study was to develop a simple, affordable, and effective method for spatially resolved, continuous, simultaneous, and multiparametric optical mapping of the heart, using a single camera. METHODS We present a new method to simultaneously monitor multiple parameters using inexpensive off-the-shelf electronic components and no moving parts. The system comprises a single camera, commercially available optical filters, and light-emitting diodes (LEDs), integrated via microcontroller-based electronics for frame-accurate illumination of the tissue. For proof of principle, we illustrate measurement of four parameters, suitable for ratiometric mapping of membrane potential (di-4-ANBDQPQ) and intracellular free calcium (fura-2), in an isolated Langendorff-perfused rat heart during sinus rhythm and ectopy, induced by local electrical or mechanical stimulation. RESULTS The pilot application demonstrates suitability of this imaging approach for heart rhythm research in the isolated heart. In addition, locally induced excitation, whether stimulated electrically or mechanically, gives rise to similar ventricular propagation patterns. CONCLUSION Combining an affordable camera with suitable optical filters and microprocessor-controlled LEDs, single-sensor multiparametric optical mapping can be practically implemented in a simple yet powerful configuration and applied to heart rhythm research. The moderate system complexity and component cost is destined to lower the threshold to broader application of functional imaging and to ease implementation of more complex optical mapping approaches, such as multiparametric panoramic imaging. A proof-of-principle application confirmed that although electrically and mechanically induced excitation occur by different mechanisms, their electrophysiologic consequences downstream from the point of activation are not dissimilar. © 2011 Heart Rhythm Society.
Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.
COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.
Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): Standardised reporting for model reproducibility, interoperability, and data sharing
Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work. © 2011 Elsevier Ltd.
The development of mathematical models of the heart has been an ongoing concern for many decades. The initial focus of this work was on single cell models that incorporate varyingly detailed descriptions of the mechanisms that give rise to experimentally observed action potential shapes. Clinically relevant heart rhythm disturbances, however, are multicellular phenomena, and there have been many initiatives to develop multidimensional representations of cardiac electromechanical activity. Here, we discuss the merits of dimensionality, from 0D single cell models, to 1D cell strands, 2D planes and 3D volumes, for the simulation of normal and disturbed rhythmicity. We specifically look at models of: (i) the origin and spread of cardiac excitation from the sino-atrial node into atrial tissue, and (ii) stretch-activated channel effects on ventricular cell and tissue activity. Simulation of the spread of normal and disturbed cardiac excitation requires multicellular models. 1D architectures suffer from limitations in neighbouring tissue effects on individual cells, but they can (with some modification) be applied to the simulation of normal spread of excitation or, in ring-like structures, re-entry simulation (colliding wave fronts, tachycardia). 2D models overcome many of the limitations imposed by models of lower dimensionality, and can be applied to the study of complex co-existing re-entry patterns or even fibrillation. 3D implementations are closest to reality, as they allow investigation of scroll waves. Our results suggest that 2D models offer a good compromise between computational resources, complexity of electrophysiological models, and applicability to basic research, and that they should be considered as an important stepping-stone towards anatomically detailed simulations. This highlights the need to identify and use the most appropriate model for any given task. The notion of a single and ultimate model is as useful as the idea of a universal mechanical tool for all possible repairs and servicing requirements in daily life. The ideal model will be as simple as possible and as complex as necessary for the particular question raised. © 2004 Elsevier Ltd. All rights reserved.
Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy.
The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.
Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency.
The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.