RNAi knock-down mice: an emerging technology for post-genomic functional genetics

RNA interference (RNAi) has been extensively used for sequence-specific silencing of gene function in mammalian cells. The latest major breakthrough in the application of RNAi technology came from experiments demonstrating RNAi-mediated gene repression in mice and rats. After more than two decades of functional mouse research aimed at developing and continuously improving transgenic and knock-out technology, the advent of RNAi knock-down mice represents a valuable new alternative for studying gene function in vivo. In this review we provide some basic insight as to how RNAi can induce gene silencing to then focus on recent findings concerning the applicability of RNAi for regulating gene function in the mouse. Reviewed topics will include delivery methods for RNAi-mediating molecules, a comparison between traditional knock-out and innovative transgenic RNAi technology and the generation of graded RNAi knock-down phenotypes. Apart from the exciting possibilities RNAi provides for studying gene function in mice, we discuss several caveats and limitations to be considered. Finally, we present prospective strategies as to how RNAi technology might be applied for generating conditional and tissue-restricted knock-down mice.     

Cell microarrays: an emerging technology for the characterization of antibodies

The possibility to miniaturize and parallelize biological assays has a great impact on the development of biomedical technologies. Here, we describe a simple, miniaturized, and parallelized method employing entire cells from different cell lines displaying a protein of interest on their surface, which were immobilized on a microarray slide. Antibodies were added to these cellular microarrays, and their specific binding to the cell surface proteins was monitored using appropriate fluorescently labeled detection molecules. This new method is applicable for rapidly screening cell surface-specific antibodies with respect to selectivity and cross-reactivity.     

Mass spectrometry imaging: An emerging technology for the analysis of metabolites in insects

Mass spectrometry imaging (MSI) can visualize the composition, abundance, and spatial distribution of molecules in tissues or cells, which has been widely used in the research of life science. Insects, especially the agricultural pests, have received a great deal of interests from the scientists in biodiversity and food security. This review introduces the major characteristics of MSI, summarizes its application to the investigation of insect endogenous metabolites, exogenous metabolites, and the spatiotemporal changes of metabolites between insects and plants, and discusses its shortfalls and perspectives. The significance of these concerns is beneficial for future insect research such as physiology and metabolism.     

A rapid ex vivo tissue model for optimising drug detection and ionisation in MALDI imaging studies

The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.     

Molecular imaging of rheumatoid arthritis: emerging markers,tools, and techniques

Early diagnosis and effective monitoring of rheumatoid arthritis (RA) are important for a positive outcome. Instant treatment often results in faster reduction of inflammation and, as a consequence, less structural damage. Anatomical imaging techniques have been in use for a long time, facilitating diagnosis and monitoring of RA. However, mere imaging of anatomical structures provides little information on the processes preceding changes in synovial tissue, cartilage, and bone. Molecular imaging might facilitate more effective diagnosis and monitoring in addition to providing new information on the disease pathogenesis. A limiting factor in the development of new molecular imaging techniques is the availability of suitable probes. Here, we review which cells and molecules can be targeted in the RA joint and discuss the advances that have been made in imaging of arthritis with a focus on such molecular targets as folate receptor, F4/80, macrophage mannose receptor, E-selectin, intercellular adhesion molecule-1, phosphatidylserine, and matrix metalloproteinases. In addition, we discuss a new tool that is being introduced in the field, namely the use of nanobodies as tracers. Finally, we describe additional molecules displaying specific features in joint inflammation and propose these as potential new molecular imaging targets, more specifically receptor activator of nuclear factor κB and its ligand, chemokine receptors, vascular cell adhesion molecule-1, α_Vβ₃ integrin, P2X7 receptor, suppression of tumorigenicity 2, dendritic cell-specific transmembrane protein, and osteoclast-stimulatory transmembrane protein.     

Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids

Lipids are a major component of cells and play a variety of roles in organisms. In general, they play a key role in the structural composition of membranes. Some lipids, such as sphingoglycolipids, however, are also mediators of different biological processes, including protein transport, regulation of cell growth, cellular morphogenesis, neuronal plasticity, and regulation of the immune response. With the advent of MALDI mass spectrometry imaging (MALDI MSI), lipids have begun to be intensively investigated by several groups. Here we present a novel development in the detection and study of lipids using an automatic microspotter coupled to specific liquid ionic matrixes based on a 2,5-DHB matrix (i.e., 2,5-DHB/ANI, 2,5-DHB/Pyr, and 2,5-DHB/3-AP). This development allows to decrease the time of the sample preparation by comparison with crystalline 2,5-DHB as matrix and was validated on human ovarian cancer biopsies to demonstrate its use as a precise procedure that is particularly useful for specific diagnoses.     

MALDI imaging mass spectrometry: molecular snapshots of biochemical systems

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.     

Partial cellular reprogramming: A deep dive into an emerging rejuvenation technology

Aging and age-associated disease are a major medical and societal burden in need of effective treatments. Cellular reprogramming is a biological process capable of modulating cell fate and cellular age. Harnessing the rejuvenating benefits without altering cell identity via partial cellular reprogramming has emerged as a novel translational strategy with therapeutic potential and strong commercial interests. Here, we explore the aging-related benefits of partial cellular reprogramming while examining limitations and future directions for the field.     

MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.