Bioluminescent indicators for in vivo measurements of gene expression

Recent developments in in vivo imaging using optical, radionuclide and paramagnetic reporter probes now enables continuous measurements of gene expression in living animals. In vivo bioluminescence imaging (BLI) is a sensitive, versatile and accessible imaging strategy that has been applied to a variety of small-animal models of human biology and disease. We discuss current strategies in BLI and the potential of combining BLI with other in vivo and ex vivo techniques. BLI will have a significant role in in vivo cellular and molecular imaging, a field that will help reveal the molecular basis of biology and disease.     

The Facts and Fancy of Microgravity Protein Crystallization

In vivo molecular imaging enables non-invasive visualization of biological processes within living subjects, and holds great promise for diagnosis and monitoring of disease. The ability to create new agents that bind to molecular targets and deliver imaging probes to desired locations in the body is critically important to further advance this field. To address this need, phage display, an established technology for the discovery and development of novel binding agents, is increasingly becoming a key component of many molecular imaging research programs. This review discusses the expanding role played by phage display in the field of molecular imaging with a focus on in vivo applications. Furthermore, new methodological advances in phage display that can be directly applied to the discovery and development of molecular imaging agents are described. Various phage library selection strategies are summarized and compared, including selections against purified target, intact cells, and ex vivo tissue, plus in vivo homing strategies. An outline of the process for converting polypeptides obtained from phage display library selections into successful in vivo imaging agents is provided, including strategies to optimize in vivo performance. Additionally, the use selections are performed against pre-defined targets, the use of cell lines, tissue, and in vivo homing selections have also been valuable. These latter strategies avoid the need to identify a specific target at the outset, allow library selections under conditions potentially more relevant to a clinical setting, and can lead to the discovery of unanticipated and interesting targets. The full potential of phage display is far from being completely explored; many library formats and selection strategies have not been fully exploited for the production of molecular imaging agents. The successful and rapid translation of phage-derived molecular imaging agents into the clinic remains a challenge, but new methods and tools are becoming available for optimizing in vivo performance. In conclusion, phage display will continue to be a significant driving force and a key player in enabling in vivo molecular imaging to deliver on its promise for both basic science and clinical applications.     

Treatment of Mitochondrial Disease

Defects of the mitochondrial genome are widely recognized as important causes of disease in man. Patients may present at any age with clinical symptoms that vary from acute episodes of lactic acidosis in infancy to severe neurodegenerative illness in adulthood. While modern molecular genetic techniques have facilitated major advances in the diagnosis and characterization of specific molecular defects, treatment for the majority of patients remains supportive in the absence of definitive biochemical therapies. As a consequence, the possibilities for mitochondrial DNA gene therapy must be considered. In this review, we will evaluate the current biochemical strategies available to clinicians for the management of patients with mitochondrial disease and examine the possible approaches to the gene therapy of mitochondrial DNA defects.     

Abdominal magnetic resonance imaging in small rodents using a clinical 1.5 T MR scanner

Because of superior soft-tissue contrast compared to other imaging techniques, non-invasive abdominal magnetic resonance imaging (MRI) is ideal for monitoring organ regeneration, tissue repair, cancer stage, and treatment effects in a wide variety of experimental animal models. Currently, sophisticated MR protocols, including technically demanding procedures for motion artefact compensation, achieve an MRI resolution limit of < 100 microm under ideal conditions. However, such a high spatial resolution is not required for most experimental rodent studies. This article describes both a detailed imaging protocol for MR data acquisition in a ubiquitously and commercially available 1.5 T MR unit and 3-dimensional volumetry of organs, tissue components, or tumors. Future developments in MR technology will allow in vivo investigation of physiological and pathological processes at the cellular and even the molecular levels. Experimental MRI is crucial for non-invasive monitoring of a broad range of biological processes and will further our general understanding of physiology and disease.     

Airway imaging in disease: Gimmick or useful tool?

Airway remodeling is an important pathophysiological mechanism in a variety of chronic airway diseases. Historically investigators have had to use invasive techniques such as histological examination of excised tissue to study airway wall structure. The last several years has seen a proliferation of relatively noninvasive techniques to assess the airway branching pattern, wall thickness, and more recently, airway wall tissue components. These methods include computed tomography, magnetic resonance imaging, and optical coherence tomography. These new imaging technologies have become popular because to understand the physiology of lung disease it is important we understand the underlying anatomy. However, these new approaches are not standardized or available in all centers so a review of their validity and clinical utility is appropriate. This review documents how investigators are working hard to correct for inconsistencies between techniques so that they become more accepted and utilized in clinical settings. These new imaging techniques are very likely to play a frontline role in the study of lung disease and will, hopefully, allow clinicians and investigators to better understand disease pathogenesis and to design and assess new therapeutic interventions.     

Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and F?rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.     

Strategies for dealing with incomplete information in the modeling of molecular interaction networks

Modelers of molecular interaction networks encounter the paradoxical situation that while large amounts of data are available, these are often insufficient for the formulation and analysis of mathematical models describing the network dynamics. In particular, information on the reaction mechanisms and numerical values of kinetic parameters are usually not available for all but a few well-studied model systems. In this article we review two strategies that have been proposed for dealing with incomplete information in the study of molecular interaction networks: parameter sensitivity analysis and model simplification. These strategies are based on the biologically justified intuition that essential properties of the system dynamics are robust against moderate changes in the value of kinetic parameters or even in the rate laws describing the interactions. Although advanced measurement techniques can be expected to relieve the problem of incomplete information to some extent, the strategies discussed in this article will retain their interest as tools providing an initial characterization of essential properties of the network dynamics.     

Molecular imaging of gliomas

Gliomas are the most common types of brain tumors. Although sophisticated regimens of conventional therapies are being carried out to treat patients with gliomas, the disease invariably leads to death over months or years. Before new and potentially more effective treatment strategies, such as gene- and cell-based therapies, can be effectively implemented in the clinical application, certain prerequisites have to be established. First of all, the exact localization, extent, and metabolic activity of the glioma must be determined to identify the biologically active target tissue for a biological treatment regimen; this is usually performed by imaging the expression of up-regulated endogenous genes coding for glucose or amino acid transporters and cellular hexokinase and thymidine kinase genes, respectively. Second, neuronal function and functional changes within the surrounding brain tissue have to be assessed in order to save this tissue from therapy-induced damage. Third, pathognomonic genetic changes leading to disease have to be explored on the molecular level to serve as specific targets for patient-tailored therapies. Last, a concerted noninvasive analysis of both endogenous and exogenous gene expression in animal models as well as the clinical setting is desirable to effectively translate new treatment strategies from experimental into clinical application. All of these issues can be addressed by multi-modal radionuclide and magnetic resonance imaging techniques and fall into the exciting and fast growing field of molecular and functional imaging. Noninvasive imaging of endogenous gene expression by means of positron emission tomography (PET) may reveal insight into the molecular basis of pathogenesis and metabolic activity of the glioma and the extent of treatment response. When exogenous genes are introduced to serve for a therapeutic function, PET imaging may reveal the assessment of the "location," "magnitude," and "duration" of therapeutic gene expression and its relation to the therapeutic effect. Detailed reviews on molecular imaging have been published from the perspective of radionuclide imaging (Gambhir et al., 2000; Blasberg and Tjuvajev, 2002) as well as magnetic resonance and optical imaging (Weissleder, 2002). The present review focuses on molecular imaging of gliomas with special reference on the status and perspectives of imaging of endogenous and exogenously introduced gene expression in order to develop improved diagnostics and more effective treatment strategies of gliomas and, in that, to eventually improve the grim prognosis of this devastating disease.     

Advancing molecular therapies through in vivo bioluminescent imaging

Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI) is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLi is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.     

Molecular-genetic imaging: a nuclear medicine-based perspective

Molecular imaging is a relatively new discipline, which developed over the past decade, initially driven by in situ reporter imaging technology. Noninvasive in vivo molecular-genetic imaging developed more recently and is based on nuclear (positron emission tomography [PET], gamma camera, autoradiography) imaging as well as magnetic resonance (MR) and in vivo optical imaging. Molecular-genetic imaging has its roots in both molecular biology and cell biology, as well as in new imaging technologies. The focus of this presentation will be nuclear-based molecular-genetic imaging, but it will comment on the value and utility of combining different imaging modalities. Nuclear-based molecular imaging can be viewed in terms of three different imaging strategies: (1) "indirect" reporter gene imaging; (2) "direct" imaging of endogenous molecules; or (3) "surrogate" or "bio-marker" imaging. Examples of each imaging strategy will be presented and discussed. The rapid growth of in vivo molecular imaging is due to the established base of in vivo imaging technologies, the established programs in molecular and cell biology, and the convergence of these disciplines. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases and disease processes.     

High-density single-particle tracking: quantifying molecule organization and dynamics at the nanoscale

The organization and dynamics of proteins are fundamental parameters for cellular function. Their study, at the single-molecule level, provides precise information on molecular interactions. Over the last 30 years, the single-particle tracking imaging technique has proven its capability to efficiently quantify such parameters in many biological systems, with nanometric accuracy and millisecond temporal resolutions. Nevertheless, the low concentration of labeling required for single-molecule imaging usually prevents the extraction of large statistics. The advent of high-density single-molecule-based super-resolution techniques has revolutionized the field, allowing monitoring of thousands of biomolecules in the minute timescale and providing unprecedented insight into the molecular organization and dynamics of cellular compounds. In this issue, I will review the main principles of single-particle tracking, a highly interdisciplinary technique at the interface between microscopy, image analysis and labeling strategies. I will point out the advantages brought by high-density single-particle tracking which will be illustrated with a few recent biological results.     

Protein-based tumor molecular imaging probes

Molecular imaging is an emerging discipline which plays critical roles in diagnosis and therapeutics. It visualizes and quantifies markers that are aberrantly expressed during the disease origin and development. Protein molecules remain to be one major class of imaging probes, and the option has been widely diversified due to the recent advances in protein engineering techniques. Antibodies are part of the immunosystem which interact with target antigens with high specificity and affinity. They have long been investigated as imaging probes and were coupled with imaging motifs such as radioisotopes for that purpose. However, the relatively large size of antibodies leads to a half-life that is too long for common imaging purposes. Besides, it may also cause a poor tissue penetration rate and thus compromise some medical applications. It is under this context that various engineered protein probes, essentially antibody fragments, protein scaffolds, and natural ligands have been developed. Compared to intact antibodies, they possess more compact size, shorter clearance time, and better tumor penetration. One major challenge of using protein probes in molecular imaging is the affected biological activity resulted from random labeling. Site-specific modification, however, allows conjugation happening in a stoichiometric fashion with little perturbation of protein activity. The present review will discuss protein-based probes with focus on their application and related site-specific conjugation strategies in tumor imaging.     

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza <i>Closterovirus</i>

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards. The citrus species grafted on sour orange rootstock are affected by this disease. Predominantly, the sweet orange, grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline, vein clearing, pin holing, bark scaling and degeneration leading to variable symptoms. Symptomatic expression of Citrus tristeza virus (CTV) in different hosts has been attributed to virus isolates which are from severe to mild. Different serological and molecular assays have been deployed to differentiate the strains of CTV. Citrus tristeza virus is diversified towards its strains on the basis of biological, serological and molecular characterization. Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation. This review will give a brief idea about the different CTV isolates, their characterization based on nucleic acid and serological assays. Different methods along with salient features for strain characterization has also been reviewed. This review will also open the new aspects towards formulation of management strategies through different detection techniques.     

Imaging angiogenesis

Angiogenesis represents the formation of new capillaries by cellular outgrowth from existing microvessels and plays a critical role in the response to ischemia associated with peripheral arterial disease and myocardial infarction. Imaging of angiogenesis would be valuable in risk stratification of patients with arterial occlusive disease. The progress in noninvasive imaging strategies to assess angiogenesis has been made possible with the availability of many technological advances, which include dedicated hybrid SPECT-CT and PET-CT systems and agents targeted at molecular markers of the angiogenic process, involving both receptor-probe interactions and reporter gene technology. These novel targeted approaches for imaging angiogenesis will complement standard imaging of physiological parameters and will play a crucial role for evaluation of therapeutic interventions to promote angiogenesis.     

Chirurgie de l’hyperparathyroïdie primaire en 2008

In recent years, different minimally invasive techniques of parathyroidectomy have been described. The concept of these limited explorations is based on the fact that 85% of patients will have a single-gland disease. Minimally invasive techniques are targeted on one specific parathyroid gland and in most cases the exploration of other glands is not performed. These interventions are today possible for three main reasons: the available imaging techniques permit to localize the diseased gland, the use of rapid intraoperative PTH assay can confirm the successful extirpation, new instrumentation and miniaturized cameras have been adapted for this kind of surgery. Not all patients presenting with primary hyperparathyroidism are candidate for this surgery. Contraindications are mainly due to a large goiter, previous surgery in the parathyroid vicinity, suspicious multiglandular disease and equivocal preoperative localization studies. Currently 60% of patients with primary hyperparathyroidism can benefit of these techniques. Studies comparing conventional parathyroid surgery to minimally-invasive techniques have shown a diminution of postoperative pain and better cosmetic results with minimally-invasive techniques. If early results are similar to those obtained with conventional parathyroidectomies, it is still too soon to evaluate what will be the recurrence rate of these new techniques. One can expect that minimally invasive and conventional parathyroid surgery will probably turn out to be complementary in the near future.     

Dissecting the molecular mechanisms of cancer through bioinformatics-based experimental approaches

Cancer is a disease of aberrant gene expression characterized by inappropriate (temporal or quantitative) expression of positive mediators of cell proliferation in conjunction with diminished expression of negative mediators of cell growth. Alteration of the normal balance of these positive and negative mediators leads to the abnormal growth of cells and tissues that typify neoplastic disease. Development of a better understanding of the genetic and epigenetic mechanisms that induce neoplastic transformation and drive the cancer phenotype is essential for continued progress towards the design of practical molecular diagnostics and effective treatment strategies. Over the past decades, molecular techniques that facilitate the assessment of gene expression, identification of gene mutations, and characterization of chromosome abnormalities (numeric and structural) have been established and applied to cancer research. However, many of these techniques are slow and labor-intensive. More recently, high-throughput technologies have emerged that generate large volumes of data related to the genetics and epigenetics of cancer (or other disorders). These advances in molecular genetic technology required the development of sophisticated bioinformatic tools to manage the large datasets generated. The combination of high-throughput molecular assays and bioinformatic-based data mining strategies has significantly impacted our understanding of the molecular pathogenesis of cancer, classification of tumors, and now the management of cancer patients in the clinic. This article will review basic molecular techniques and bioinformatic-based experimental approaches used to dissect the molecular mechanisms of carcinogenesis.     

Molecular mechanisms for Alzheimer's disease: implications for neuroimaging and therapeutics

Alzheimer's disease is a progressive neurodegenerative disorder characterised by the gradual onset of dementia. The pathological hallmarks of the disease are beta-amyloid (Abeta) plaques, neurofibrillary tangles, synaptic loss and reactive gliosis. The current therapeutic effort is directed towards developing drugs that reduce Abeta burden or toxicity by inhibiting secretase cleavage, Abeta aggregation, Abeta toxicity, Abeta metal interactions or by promoting Abeta clearance. A number of clinical trials are currently in progress based on these different therapeutic strategies and they should indicate which, if any, of these approaches will be efficacious. Current diagnosis of Alzheimer's disease is made by clinical, neuropsychologic and neuroimaging assessments. Routine structural neuroimaging evaluation with computed tomography and magnetic resonance imaging is based on non-specific features such as atrophy, a late feature in the progression of the disease, hence the crucial importance of developing new approaches for early and specific recognition at the prodromal stages of Alzheimer's disease. Functional neuroimaging techniques such as functional magnetic resonance imaging, magnetic resonance spectroscopy, positron emission tomography and single photon emission computed tomography, possibly in conjunction with other related Abeta biomarkers in plasma and CSF, could prove to be valuable in the differential diagnosis of Alzheimer's disease, as well as in assessing prognosis. With the advent of new therapeutic strategies there is increasing interest in the development of magnetic resonance imaging contrast agents and positron emission tomography and single photon emission computed tomography radioligands that will permit the assessment of Abeta burden in vivo.     

Tailoring the size distribution of ultrasound contrast agents: possible method for improving sensitivity in molecular imaging

Encapsulated microbubble contrast agents incorporating an adhesion ligand in the microbubble shell are used for molecular imaging with ultrasound. Currently available microbubble agents are produced with techniques that result in a large size variance. Detection of these contrast agents depends on properties related to the microbubble diameter such as resonant frequency, and current ultrasound imaging systems have bandwidth limits that reduce their sensitivity to a polydisperse contrast agent population. For ultrasonic molecular imaging, in which only a limited number of targeted contrast agents may be retained at the site of pathology, it is important to optimize the sensitivity of the imaging system to the entire population of contrast agent. This article presents contrast agents with a narrow size distribution that are targeted for molecular imaging applications. The production of a functionalized, lipid-encapsulated, microbubble contrast agent with a monodisperse population is demonstrated, and we evaluate parameters that influence the size distribution and demonstrate initial acoustic testing.