Target-enrichment strategies for next-generation sequencing.

We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.

Recent trends in molecular beacon design and applications.

A molecular beacon (MB) is a hairpin-structured oligonucleotide probe containing a photoluminescent species (PLS) and a quencher at different ends of the strand. In a recognition and detection process, the hybridization of MBs with target DNA sequences restores the strong photoluminescence, which is quenched before hybridization. Making better MBs involves reducing the background photoluminescence and increasing the brightness of the PLS, which therefore involves the development of new PLS and quenchers, as well as innovative PLS-quencher systems. Heavy-metal complexes, nanocrystals, pyrene compounds, and other materials with excellent photophysical properties have been applied as PLS of MBs. Nanoparticles, nanowires, graphene, metal films, and many other media have also been introduced to quench photoluminescence. On the basis of their high specificity, selectivity, and sensitivity, MBs are developed as a general platform for sensing, producing, and carrying molecules other than oligonucleotides.

Surface-attached sensors for cation and anion recognition.

The development of surface-attached sensors for cationic and anionic guests is of intense current research interest. In addition to the environmental flexibility, robustness and reusability of such devices, surface-confined sensors typically exhibit an amplified response to target analytes owing to preorganization of the receptor. Whereas redox-active cations may be sensed by studying the cyclic voltammetry of host-guest systems containing ion-selective receptors attached to an appropriate electrode, redox-inactive ionic species require the use of electrochemical impedance spectroscopy, with appropriately functionalized electrodes and redox probes. Alternatively, receptors may be constructed that incorporate an electrochemical or optical reporter group within their structure to provide a macroscopic response to the presence of an ionic guest. This critical review seeks to present an up-to-date, although necessarily selective, account of the progress in the field, and provides insights into possible future developments, including the utilization of receptor-nanoparticle conjugates and mechanically interlocked receptors.

Future lab-on-a-chip technologies for interrogating individual molecules.

Advances in technology have allowed chemical sampling with high spatial resolution and the manipulation and measurement of individual molecules. Adaptation of these approaches to lab-on-a-chip formats is providing a new class of research tools for the investigation of biochemistry and life processes.

Fluorescence correlation spectroscopy: past, present, future.

In recent years fluorescence correlation spectroscopy (FCS) has become a routine method for determining diffusion coefficients, chemical rate constants, molecular concentrations, fluorescence brightness, triplet state lifetimes, and other molecular parameters. FCS measures the spatial and temporal correlation of individual molecules with themselves and so provides a bridge between classical ensemble and contemporary single-molecule measurements. It also provides information on concentration and molecular number fluctuations for nonlinear reaction systems that complement single-molecule measurements. Typically implemented on a fluorescence microscope, FCS samples femtoliter volumes and so is especially useful for characterizing small dynamic systems such as biological cells. In addition to its practical utility, however, FCS provides a window on mesoscopic systems in which fluctuations from steady states not only provide the basis for the measurement but also can have important consequences for the behavior and evolution of the system. For example, a new and potentially interesting field for FCS studies could be the study of nonequilibrium steady states, especially in living cells.

A crowdsourcing evaluation of the NIH chemical probes.

Between 2004 and 2008, the US National Institutes of Health Molecular Libraries and Imaging initiative pilot phase funded 10 high-throughput screening centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the Molecular Libraries and Imaging initiative output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

Response to treatment series: part 2, tumor response assessment--using new and conventional criteria.

OBJECTIVE: Conventional anatomic imaging biomarkers, including World Health Organization (WHO) criteria and Response Evaluation Criteria in Solid Tumors (RECIST), although effective, have limitations. This article will discuss the conventional and newer morphologic imaging biomarkers for the assessment of tumor response to therapy. CONCLUSION: Applying established methods of assessing tumor response to therapy allows consistency in image interpretation and facilitates communication with oncologists. Because of the new methods of treatment, assessment of necrosis and volumetric information will need to be incorporated into size-based criteria.

FRET for lab-on-a-chip devices - current trends and future prospects.

This review focuses on the use of Förster Resonance Energy Transfer (FRET) to monitor intra- and intermolecular reactions occurring in microfluidic reactors. Microfluidic devices have recently been used for performing highly efficient and miniaturised biological assays for the analysis of biological entities such as cells, proteins and nucleic acids. Microfluidic assays are characterised by nanolitre to femtolitre reaction volumes, which necessitates the adoption of a sensitive optical detection scheme. FRET serves as a strong 'spectroscopic ruler' for elucidating the tertiary structure of biomolecules, as the efficiency of the non-radiative energy transfer is extremely sensitive to nanoscale changes in the separation between donor and acceptor markers attached to the biomolecule of interest. In this review, we will review the implementation of various microfluidic assays which employ FRET for diverse applications in the biomedical field, along with the advantages and disadvantages of the various approaches. The future prospects for development of microfluidic devices incorporating FRET detection will be discussed.

Science to practice: the dawn of molecular US imaging for clinical cancer imaging.

Ultrasonography (US) is one of the most commonly used diagnostic modalities in clinical medicine. Gas-filled microbubbles can be used to enhance the contrast of tumors by indicating increased vascularity. Because microbubbles can be detected with high sensitivity and specificity, they fulfill an important precondition for use as a molecular imaging probe. Over the past several years, there have been an increasing number of published studies that showed that markers of angiogenesis and inflammation can be assessed reliably when microbubbles are coupled to antibodies and peptides. Recently, target-specific microbubbles have been developed that are suited for use in humans. Now the identification of the optimal clinical indications for molecular US imaging in clinics is required. In this context, advantages and limitations of US with targeted microbubbles, when compared with other imaging modalities, must be carefully considered. Because US is a transportable, cheap, real-time imaging modality, molecular US imaging may have advantages for initial tumor screening and US-guided interventions; furthermore, it may support therapy monitoring in intervals between whole-body images obtained with positron emission tomography (PET), computed tomography (CT), and magnetic resonance (MR) imaging.

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies.

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-alpha, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with (99m)Tc or (111)In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and follow-up.

Labelling of mammalian cells for visualisation by MRI.

Through labelling of cells with magnetic contrast agents it is possible to follow the fate of transplanted cells in vivo with magnetic resonance imaging (MRI) as has been demonstrated in animal studies as well as in a clinical setting. A large variety of labelling strategies are available that allow for prolonged and sensitive detection of the labelled cells with MRI. The various protocols each harbour specific advantages and disadvantages. In choosing a particular labelling strategy it is also important to ascertain that the labelling procedure does not negatively influence cell functionality, for which a large variety of assays are available. In order to overcome the challenges still faced in fully exploiting the benefits of in vivo cell tracking by MRI a good understanding and standardisation of the procedures and assays used will be crucial.

Optical molecular imaging in atherosclerosis.

Current imaging techniques focus on evaluating the anatomical structure of blood vessel wall and atherosclerotic plaque. These techniques fail to evaluate the biological processes which take place in the vessel wall and inside the plaque. Novel imaging techniques like optical imaging can evaluate the biological and cellular processes inside the plaque and provide information which can be vital for better patient risk stratification. This review highlights the various optical imaging techniques and their application in assessing biological processes in atherosclerosis.

Advances in radionuclide molecular imaging in myocardial biology.

Molecular imaging is a new and evolving field that employs a targeted approach to noninvasively assess biologic processes in vivo. By assessing key elements in specific cellular processes prior to irreversible end-organ damage, molecular tools will allow for earlier detection and intervention, improving management and outcomes associated with cardiovascular diseases. The goal of those working to expand this field is not just to provide diagnostic and prognostic information, but rather to guide an individual's pharmacological, cell-based, or genetic therapeutic regimen. This article will review molecular imaging tools in the context of our current understanding of biological processes of the myocardium, including angiogenesis, ventricular remodeling, inflammation, and apoptosis. The focus will be on radiotracer-based molecular imaging modalities with an emphasis on clinical application. Though this field is still in its infancy and may not be fully ready for widespread use, molecular imaging of myocardial biology has begun to show promise of clinical utility in acute and chronic ischemia, acute myocardial infarction, congestive heart failure, as well as in more global inflammatory and immune-mediated responses in the heart-like myocarditis and allogeneic cardiac transplant rejection. With continued research and development, molecular imaging promises to be an important tool for the optimization of cardiovascular care.

[Molecular tests in modern medicine - concepts, technical aspects and future direction]. / Molekulare Früherkennung in der Medizin - Konzept, Techniken, Perspektiven.

Early diagnosis by molecular tests has increasingly catched attention after deciphering the complete human genome sequence in 2001. Meanwhile, complete genome sequencing will soon become available for each individual. Molecular testing is standard of care in certain infectious or malignant diseases. Novel biomarkers are emerging as a result of modern comprehensive procedures (transcriptomics, proteomics, metabolomics etc). However, hype and hope are particularly close in this field and responsible conduct by clinicians will ensure beneficial use of new tests.

Will molecular MR imaging play a role in identification and treatment of patients with vulnerable atherosclerotic plaques?

Although the translation of experimental molecular MR imaging agents is highly compelling, it is substantially more complex than the translation of radiolabeled imaging agents. The prognostic value, safety, and cost-effectiveness of molecular MR imaging for the detection of plaque inflammation will need to be demonstrated and will require a robust and collaborative effort among basic scientists, clinicians, epidemiologists, and industry. The well-conducted and valuable study reported by Amirbekian et al in this issue of Radiology adds further momentum to this important endeavor.

Vessel growth and function: depiction with contrast-enhanced MR imaging.

Magnetic resonance (MR) imaging is a versatile noninvasive diagnostic tool that can be applied to the entire human body to revealing morphologic, functional, and metabolic information. The authors review how MR imaging can depict both the established and the developing vasculature with techniques involving intravenously administered contrast agents. In addition to macrovascular morphology and flow, MR imaging is able to exploit microvascular properties, including vessel size distribution, hyperpermeability, flow heterogeneity, and possibly also upregulation of endothelial biomarkers. For each MR method, the basic principles, potential acquisition and interpretation pitfalls, solutions, and applications are described. Furthermore, discussion includes current shortcomings and the impact of future developments (eg, higher magnetic field strength systems, targeted macromolecular contrast agents) on the visualization of blood vessel growth and function with contrast-enhanced MR imaging.

Nanoparticles for optical molecular imaging of atherosclerosis.

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

Quantum dots for in vivo small-animal imaging.

Nanotechnology is poised to transform research, prevention, and treatment of cancer through the development of novel diagnostic imaging methods and targeted therapies. In particular, the use of nanoparticles for imaging has gained considerable momentum in recent years. This review focuses on the growing contribution of quantum dots (QDs) for in vivo imaging in small-animal models. Fluorescent QDs, which are small nanocrystals (1-10 nm) made of inorganic semiconductor materials, possess several unique optical properties best suited for in vivo imaging. Because of quantum confinement effects, the emission color of QDs can be precisely tuned by size from the ultraviolet to the near-infrared. QDs are extremely bright and photostable. They are also characterized by a wide absorption band and a narrow emission band, which makes them ideal for multiplexing. Finally, the large surface area of QDs permits the assembly of various contrast agents to design multimodality imaging probes. To date, biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Here we consider these novel breakthroughs in light of their potential clinical applications and discuss how QDs might offer a suitable platform to unite disparate imaging modalities and provide information along a continuum of length scales.