Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays

In vivo bioluminescence imaging has become a cornerstone technology for preclinical molecular imaging. This imaging method is based on light-emitting enzymes, luciferases, which require specific substrates for light production. When linked to a specific biological process in an animal model of human biology or disease, the enzyme-substrate interactions become biological indicators that can be studied noninvasively in living animals. Signal intensity in these animal models depends on the availability of the substrate for the reaction within living cells in intact organs. The biodistribution and clearance rates of the substrates are therefore directly related to optimal imaging times and signal intensities and ultimately determine the sensitivity of detection and predictability of the model. Modifications of d-luciferin, the substrate for the luciferases obtained from beetle, including fireflies, result in novel properties and offer opportunities for improved bioassays. For this purpose, we have synthesized a conjugate, glycine-d-aminoluciferin, and investigated its properties relative to those of d-aminoluciferin and d-luciferin. The three substrates exhibited different kinetic properties and different intracellular accumulation profiles due to differences in their molecular structure, which in turn influenced their biodistribution in animals. Glycine-d-aminoluciferin had a longer in vivo circulation time than the other two substrates. The ability to assay luciferase in vitro and in vivo using these substrates, which exhibit different pharmacokinetic and pharmacodynamic properties, will provide flexibility and improve current imaging capabilities.     

iRFP is a sensitive marker for cell number and tumor growth in high-throughput systems

GFP and luciferase are used extensively as markers both in vitro and in vivo although both have limitations. The utility of GFP fluorescence is restricted by high background signal and poor tissue penetrance. Luciferase throughput is limited in vitro by the requirement for cell lysis, while in vivo, luciferase readout is complicated by the need for substrate injection and the dependence on endogenous ATP. Here we show that near-infrared fluorescent protein in combination with widely available near-infrared scanners overcomes these obstacles and allows for the accurate determination of cell number in vitro and tumor growth in vivo in a high-throughput manner and at negligible per-well costs. This system represents a significant advance in tracking cell proliferation in tissue culture as well as in animals, with widespread applications in cell biology.     

Designer proteins: applications of genetic code expansion in cell biology

Designer amino acids, beyond the canonical 20 that are normally used by cells, can now be site-specifically encoded into proteins in cells and organisms. This is achieved using 'orthogonal' aminoacyl-tRNA synthetase-tRNA pairs that direct amino acid incorporation in response to an amber stop codon (UAG) placed in a gene of interest. Using this approach, it is now possible to study biology in vitro and in vivo with an increased level of molecular precision. This has allowed new biological insights into protein conformational changes, protein interactions, elementary processes in signal transduction and the role of post-translational modifications.     

Biomedical applications of fluorescence imaging in vivo

Optical imaging can advance knowledge of cellular biology and disease at the molecular level in vitro and, more recently, in vivo. In vivo optical imaging has enabled real-time study to track cell movement, cell growth, and even some cell functions. Thus, it can be used in intact animals for disease detection, screening, diagnosis, drug development, and treatment evaluation. This review includes a brief introduction to fluorescence imaging, fluorescent probes, imaging devices, and in vivo applications in animal models. It also describes a quantitative fluorescence detection method with a reconstruction algorithm for determining the location of fluorophores in tissue and addresses future applications of in vivo fluorescence imaging.     

New perspectives in the differentiation of bone-forming cells.

Bone formation comprises a complex but ordered sequence of events which involves the proliferation and differentiation of chondrogenic and osteoblastic precursor cells ultimately leading to the formation of a calcified extracellular matrix. This process can be observed in vivo but under these conditions is difficult to study at the molecular level. A number of in vitro models have been developed which recapitulate discrete elements of this process. Using these models, detailed information has been obtained regarding the differentiation of bone forming cells and the molecular biology of the mineralization process. It has been shown that, in vitro, osteoblastic precursor cells can form a mineralized matrix similar to that seen in vivo. This calcification process was shown to consist of three interdependent phases: proliferation, matrix maturation and mineralization. Each of these phases was characterized by the expression of particular genes. Osteoblast precursors have been cloned and consequently shown to be able to differentiate in vitro into a number of other mesenchymal cells, supporting the theory that osteoblasts are derived from multipotent mesenchymal cells. It is possible that markers derived from these models could be used in the future to extend our knowledge of bone formation in vivo.     

Death Mechanism of Breast Adenocarcinoma Cells Caused by BRET-Induced Cytotoxicity of miniSOG Depends on the Intracellular Localization of the NanoLuc–miniSOG Fusion Protein

Photodynamic therapy (PDT) is widely used in clinical practice to influence neoplasms in the presence of a photosensitizer, oxygen, and light source. The main problem of PDT of deep tumors is the problem of delivering excitation light (without lost of its intensity) inside the body. An alternative to the external light sources can be the internal light sources based on luciferase–substrate bioluminescent systems. In our work, we used the NanoLuc–furimazine system as an internal light source. This system can be successfully used to excite the protein photosensitizer miniSOG and to induce the phototoxicity of this flavoprotein in cancer cells during bioluminescent resonance energy transfer (BRET). It was shown that the mechanism of cell death caused by BRET-induced phototoxicity of mimiSOG in the presence of furimazine depends on the intracellular localization of the NanoLuc–miniSOG fusion protein: BRET-mediated activation of miniSOG in mitochondrial localization causes apoptosis, while the membrane localization of PS causes necrosis of cancer cells.     

Functional reconstitution of bacterial Tat translocation in vitro

The Tat (twin-arginine translocation) pathway is a Sec-independent mechanism for translocating folded preproteins across or into the inner membrane of Escherichia coli. To study Tat translocation, we sought an in vitro translocation assay using purified inner membrane vesicles and in vitro synthesized substrate protein. While membrane vesicles derived from wild-type cells translocate the Sec-dependent substrate proOmpA, translocation of a Tat-dependent substrate, SufI, was not detected. We established that in vivo overexpression of SufI can saturate the Tat translocase, and that simultaneous overexpression of TatA, B and C relieves this SufI saturation. Using membrane vesicles derived from cells overexpressing TatABC, in vitro translocation of SufI was detected. Like translocation in vivo, translocation of SufI in vitro requires TatABC, an intact membrane potential and the twin-arginine targeting motif within the signal peptide of SUFI: In contrast to Sec translocase, we find that Tat translocase does not require ATP. The development of an in vitro translocation assay is a prerequisite for further biochemical investigations of the mechanism of translocation, substrate recognition and translocase structure.     

Understanding bone cell biology requires an integrated approach: Reliable opportunities to study osteoclast biology in vivo

The relative simplicity of all in vitro methods to study bone cell biology will at best result in oversimplification of the development and functional capacity of the skeleton in vivo. We have shown this to be true for selected aspects of bone cell biology, but numerous other examples are available. One alternative is to undertake skeletal research in vivo. It is important that those in bone research be willing to move increasingly in this direction not only to understand the true complexitities of skeletal versatility, but also to avoid repetition and perpetuation of erroneous or irrelevant conclusions which waste resources. Toward this end we have described two situations, osteopetrosis and tooth eruption, in which reproducible abrogations or local activations of bone resorption can be examined in vivo. The application of emerging molecular and morphological techniques that permit the subcellular dissection of metabolic pathways and their precise cellular localization, such as a combination of the variety of in situ hybridzation technologies with PCR, antisense probes, and antibody blockase, will allow the investigator greater control of variables in vivo. We expect that these technologies, largely worked out in vitro, combined with highly selected, appropriate models, as we have oulined here for osteoclast biology worked out in vitro, combined with highly selected, appropriate models, as we have ourlined here for osteoclast biology, will make research in vivo less intimidating and increase the frequency with which the real biology is studied directly.     

Senescent phenotype achieved in vitro is indistinguishable, with the exception of Bcl-2 content, from that attained during the in vivo aging process

Senescent phenotype can be attained by diverse agents, thus suggesting that there might be molecular differences between the senescence achieved in vivo and the senescence-like state attained in vitro under culture conditions. In this study we compare the senescent phenotype reached by cells derived from young animals when cultured in vitro with the one associated with the in vivo aging process. Several in vitro senescence parameters, including MTT reduction, proliferation rate, DNA synthesis, SA-beta-gal staining, and both in vivo and in vitro Bcl-2 content, were determined. Alterations in DNA electrophoretic mobility were evaluated to test differences in bulk chromatin structure. Our results indicate that although it is possible to achieve a senescent phenotype with cells derived from young animals aged in culture, this phenotype differs from the one observed in older animals, due to lack of in vivo damage inducers to which cells are being exposed during natural aging.     

Neuronal-glial interactions in central nervous system neurogenesis: the neural stem cell perspective

Essentially, three neuroectodermal-derived cell types make up the complex architecture of the adult CNS: neurons, astrocytes and oligodendrocytes. These elements are endowed with remarkable morphological, molecular and functional heterogeneity that reaches its maximal expression during development when stem/progenitor cells undergo progressive changes that drive them to a fully differentiated state. During this period the transient expression of molecular markers hampers precise identification of cell categories, even in neuronal and glial domains. These issues of developmental biology are recapitulated partially during the neurogenic processes that persist in discrete regions of the adult brain. The recent hypothesis that adult neural stem cells (NSCs) show a glial identity and derive directly from radial glia raises questions concerning the neuronal-glial relationships during pre- and post-natal brain development. The fact that NSCs isolated in vitro differentiate mainly into astrocytes, whereas in vivo they produce mainly neurons highlights the importance of epigenetic signals in the neurogenic niches, where glial cells and neurons exert mutual influences. Unravelling the mechanisms that underlie NSC plasticity in vivo and in vitro is crucial to understanding adult neurogenesis and exploiting this physiological process for brain repair. In this review we address the issues of neuronal/glial cell identity and neuronal-glial interactions in the context of NSC biology and NSC-driven neurogenesis during development and adulthood in vivo, focusing mainly on the CNS. We also discuss the peculiarities of neuronal-glial relationships for NSCs and their progeny in the context of in vitro systems.     

Complete genome sequence of Bifidobacterium bifidum S17

Here, we report on the first completely annotated genome sequence of a Bifidobacterium bifidum strain. B. bifidum S17, isolated from feces of a breast-fed infant, was shown to strongly adhere to intestinal epithelial cells and has potent anti-inflammatory activity in vitro and in vivo. The genome sequence will provide new insights into the biology of this potential probiotic organism and allow for the characterization of the molecular mechanisms underlying its beneficial properties.     

基因打靶技术的原理及操作

基因打靶是近几年发展起来的一种通过同源重组定点改变小鼠基因组特定位点的技术,其诞生是分子生物学与实验胚胎学方法相结合的产物,它的出现又导致了体内研究与体外研究、分子生物学与临床病理学的有机结合,为研究基因的体内功能和疾病的致病机理提供了一种有力的实验手段。本文以基因打靶的实验过程为主线,介绍该技术的原理、操作、进展和应用。     

Chromosome variation in the embryos of domestic animals

Chromosome abnormalities in the embryos of domestic animals are mostly eliminated during development. De novo chromosome abnormalities in the embryos of domestic animals have been detected in a larger proportion of embryos produced by in vitro fertilization and somatic cell nuclear transfer than in those produced by natural mating or artificial insemination. The increased incidence of abnormalities in embryos produced in vitro provides evidence for an influence of the embryo production procedures on chromosome stability. Research strategies involving cytogenetics, molecular biology and reproductive biotechnologies hold the promise of yielding insight into the mechanisms underlying chromosome instability in embryos and the impact of the in vitro environment on the chromosome make-up of embryos.     

Nigrostriatal reduction of aromatic L-amino acid decarboxylase activity in MPTP-treated squirrel monkeys: in vivo and in vitro investigations

Aromatic L-amino acid decarboxylase (AAAD) activity was examined in vivo with positron emission tomography (PET) using 6-[18F]fluoro-L-DOPA (FDOPA) in squirrel monkeys lesioned with graded doses of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In vitro biochemical determinations of AAAD activity in caudate, putamen, substantia nigra, and nucleus accumbens were performed in the same animals to establish a direct comparison of in vivo and in vitro measurements. In vivo and in vitro AAAD activities in caudate/ putamen were substantially reduced in animals treated with the highest dose of MPTP (2.0 mg/kg). The percent change in the striatal FDOPA uptake (K(i)) and decarboxylation rate constant (k3) values resulting from MPTP treatment showed highly significant correlations with in vitro-determined AAAD activities. However, decarboxylase rates within individual animals presented as approximately 10-fold difference between in vivo and in vitro values. Lower in vivo k3 measurements may be attributed to several possibilities, including transport restrictions limiting substrate availability to AAAD within the neuron. In addition, reductions in AAAD activity in the substantia nigra did not parallel reductions in AAAD activity within the striatum, supporting the notion of a nonlinear relationship between nigrostriatal cell degeneration and terminal losses. This work further explores the role of AAAD in Parkinson's disease, a more important factor than previously thought.     

Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes

In vitro drug metabolism by cultured rat, rabbit and human adult hepatocytes has been studied, using ketotifen (ZADITEN) as a model substrate because it is biotransformed in vivo by various metabolic pathways in man and animals. The major in vivo pathways were demonstrated in vitro, namely oxidation in rat hepatocytes, oxidation, glucuronidation and sulfation in rabbit hepatocytes, reduction and glucuronidation in human hepatocytes. Human hepatocytes were the most stable in culture, displaying ketotifen biotransformation for at least one week. These results clearly demonstrated that cultured hepatocytes retain their in vivo specific drug metabolizing activities, including inter-species polymorphism, for a few days. Therefore, pure hepatocyte cultures represent a useful system suitable for drug metabolism studies.     

Bacteriophage endolysins--current state of research and applications

Endolysins are phage-encoded enzymes that break down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle. Their action is tightly regulated by holins, by membrane arrest, and by conversion from their inactive to active state. Recent research has not only revealed the unexpected diversity of these highly specific hydrolases but has also yielded insights into their modular organization and their three-dimensional structures. Their N-terminal catalytic domains are able to target almost every possible bond in the peptidoglycan network, and their corresponding C-terminal cell wall binding domains target the enzymes to their substrate. Owing to their specificity and high activity, endolysins have been employed for various in vitro and in vivo aims, in food science, in microbial diagnostics, and for treatment of experimental infections. Clearly, phage endolysins represent great tools for use in molecular biology, biotechnology and in medicine, and we are just beginning to tap this potential.     

Stem cell-biomaterial interactions for regenerative medicine

The synergism of stem cell biology and biomaterial technology promises to have a profound impact on stem-cell-based clinical applications for tissue regeneration. Biomaterials development is rapidly advancing to display properties that, in a precise and physiological fashion, could drive stem-cell fate both in vitro and in vivo. Thus, the design of novel materials is trying to recapitulate the molecular events involved in the production, clearance and interaction of molecules within tissue in pathologic conditions and regeneration of tissue/organs. In this review we will report on the challenges behind translating stem cell biology and biomaterial innovations into novel clinical therapeutic applications for tissue and organ replacements (graphical abstract).     

A steady-state modeling approach to validate an in vivo mechanism of the GAL regulatory network in Saccharomyces cerevisiae.

Cellular regulation is a result of complex interactions arising from DNA-protein and protein-protein binding, autoregulation, and compartmentalization and shuttling of regulatory proteins. Experiments in molecular biology have identified these mechanisms recruited by a regulatory network. Mathematical models may be used to complement the knowledge-base provided by in vitro experimental methods. Interactions identified by in vitro experiments can lead to the hypothesis of multiple candidate models explaining the in vivo mechanism. The equilibrium dissociation constants for the various interactions and the total component concentration constitute constraints on the candidate models. In this work, we identify the most plausible in vivo network by comparing the output response to the experimental data. We demonstrate the methodology using the GAL system of Saccharomyces cerevisiae for which the steady-state analysis reveals that Gal3p neither dimerizes nor shuttles between the cytoplasm and the nucleus.     

Profiling protease activities by dynamic proteomics workflows

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.