Advancing Aptamers as Molecular Probes for Cancer Theranostic Applications—The Role of Molecular Dynamics Simulation

Theranostics cover emerging technologies for cell biomarking for disease diagnosis and targeted introduction of drug ingredients to specific malignant sites. Theranostics development has become a significant biomedical research endeavor for effective diagnosis and treatment of diseases, especially cancer. An efficient biomarking and targeted delivery strategy for theranostic applications requires effective molecular coupling of binding ligands with high affinities to specific receptors on the cancer cell surface. Bioaffinity offers a unique mechanism to bind specific target and receptor molecules from a range of non‐targets. The binding efficacy depends on the specificity of the affinity ligand toward the target molecule even at low concentrations. Aptamers are fragments of genetic materials, peptides, or oligonucleotides which possess enhanced specificity in targeting desired cell surface receptor molecules. Aptamer–target binding results from several inter‐molecular interactions including hydrogen bond formation, aromatic stacking of flat moieties, hydrophobic interaction, electrostatic, and van der Waals interactions. Advancements in Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has created the opportunity to artificially generate aptamers that specifically bind to desired cancer and tumor surface receptors with high affinities. This article discusses the potential application of molecular dynamics (MD) simulation to advance aptamer‐mediated receptor targeting in targeted cancer therapy. MD simulation offers real‐time analysis of the molecular drivers of the aptamer‐receptor binding and generate optimal receptor binding conditions for theranostic applications. The article also provides an overview of different cancer types with focus on receptor biomarking and targeted treatment approaches, conventional molecular probes, and aptamers that have been explored for cancer cells targeting.     

Bone morphogenetic proteins regulate neural tube closure by interacting with the apicobasal polarity pathway

During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.     

Imaging neural crest cell dynamics during formation of dorsal root ganglia and sympathetic ganglia

The neural crest is a migratory population of cells that produces many diverse structures within the embryo. Trunk neural crest cells give rise to such structures as the dorsal root ganglia (DRG) and sympathetic ganglia (SG), which form in a metameric pattern along the anterior-posterior axis of the embryo. While static analyses have provided invaluable information concerning the development of these structures, time-lapse imaging of neural crest cells navigating through their normal environment could potentially reveal previously unidentified cellular and molecular interactions integral to DRG and SG development. In this study, we follow fluorescently labeled trunk neural crest cells using a novel sagittal explant and time-lapse confocal microscopy. We show that along their dorsoventral migratory route, trunk neural crest cells are highly motile and interact extensively with neighboring cells and the environment, with many cells migrating in chain-like formations. Surprisingly, the segregated pattern of crest cell streams through the rostral somite is not maintained once these cells arrive alongside the dorsal aorta. Instead, neural crest cells disperse along the ventral outer border of the somite, interacting extensively with each other and their environment via dynamic extension and retraction of filopodia. Discrete sympathetic ganglia arise as a consequence of intermixing and selective reorganization of neural crest cells at the target site. The diverse cell migratory behaviors and active reorganization at the target suggest that cell-cell and cell-environment interactions are coordinated with dynamic molecular processes.     

Electrophysiological properties of embryonic stem cell-derived neurons

In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP(+) neurons in culture display functional neuronal properties even at early stages of differentiation.     

Strategies to dissect parasite proteomes

Proteomes are complex and dynamic entities that are still poorly understood, but the application of proteomic technologies has become invaluable in many areas of biology, including parasitology. These technologies can be exploited to identify proteins in both complex or relatively simple samples, that formerly could only be characterized by targeted approaches such as Western blotting. Quantitative proteomic approaches can reveal modulations in protein expression that accompany phenotypes of interest. Proteomic approaches have been exploited to understand some of the molecular basis for host:parasite interactions and to elucidate phenotypes such as virulence, antigenicity and drug resistance. Many of the same technologies can also be more easily applied to targeted sub-proteomes. Examples from several studies on pathogen proteomes and sub-proteomes, from bacteria to helminths, are presented to illustrate the potential and limitations of proteomic technologies.     

Integrity of developing spinal motor columns is regulated by neural crest derivatives at motor exit points

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.     

The neural cell adhesion molecule NCAM regulates neuritogenesis by multiple mechanisms of interaction

The neural cell adhesion molecule NCAM and its glycosylation with polysialic acid (polySia) are crucially involved in proliferation, migration and differentiation of neural progenitors. Modification with polySia, homophilic and heterophilic interactions set the function of NCAM, but little is known on their interplay. We have shown recently that removal of polySia induces neuronal differentiation via heterophilic NCAM interactions at cell contacts between SH-SY5Y neuroblastoma cells. Here we analyze the additional impact of NCAM-positive fibroblasts as a ligand-presenting cellular environment, a model often used to demonstrate the neuritogenic effect of homophilic NCAM interactions. Native SH-SY5Y cells did not respond to interactions with fibroblast NCAM. However, after induction of neuronal differentiation by retinoic acid the previously ineffective NCAM signals activated extracellular signal-regulated kinase (ERK) and promoted neuritogenesis. Removal of polySia increased neuritogenesis in retinoic acid-treated cells additive to the NCAM substrate effect. The change in responsiveness to substrate NCAM was associated with a rearrangement of polysialylated NCAM away from its enrichment at homotypic cell-cell contacts and with the appearance of non-polysialylated NCAM, i.e. changes facilitating NCAM interactions with the substrate. Thus, heterophilic and homophilic NCAM interactions are integrated into the cell's response yet they have the capacity to independently trigger neuritogenesis. The actual occurrence of each of these interactions, however, depends on the cellular context, targeted cell surface presentation of NCAM and the dynamic regulation of its modification by polysialic acid. In summary, this study reveals how the complex interplay of NCAM interactions and polysialylation provides an elaborate system to regulate neuritogenesis.     

Isolation of an adhesion-mediating protein from chick neural retina adherons

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.     

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.     

Non‐neural adult stem cells: tools for brain repair?

Stem cells isolated from adult mammalian tissues may provide new approaches for the autologous treatment of disease and tissue repair. Although the potential of adult stem cells has received much attention, it has also recently been brought into question. This article reviews the recent work describing the ability of non-hematopoietic stem cells derived from adult bone marrow to form neural derivatives and their potential for brain repair. Earlier transplantation experiments imply that grafted adult stem cells can differentiate into neural derivatives. Recent reports suggest, however, that such findings may be misleading and grafted cells acquiring different identities may merely be explained by their fusion with host cells and not the result of radical changes to their program of cellular differentiation. Nonetheless, in vitro studies have shown that neural development by bone-marrow-derived stem cells also appears possible. Understanding the molecular mechanisms that specify the neural lineage will lead to the development of tools for the targeted production of neural cell types in vitro that may ultimately provide a source of material to treat specific neurological deficits.     

Induction of the neural crest: a multigene process

In the embryo, the neural crest is an important population of cells that gives rise to diverse derivatives, including the peripheral nervous system and the craniofacial skeleton. Evolutionarily, the neural crest is of interest as an important innovation in vertebrates. Experimentally, it represents an excellent system for studying fundamental developmental processes, such as tissue induction. Classical embryologists have identified interactions between tissues that lead to neural crest formation. More recently, geneticists and molecular biologists have identified the genes that are involved in these interactions; this recent work has revealed that induction of the neural crest is a complex multistep process that involves many genes.     

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.     

Live cell imaging: approaches for studying protein dynamics in living cells

In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions. In this special issue of review articles, we review various imaging technologies of microscope hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging.     

In vivo evidence for short- and long-range cell communication in cranial neural crest cells

The proper assembly of craniofacial structures and the peripheral nervous system requires neural crest cells to emerge from the neural tube and navigate over long distances to the branchial arches. Cell and molecular studies have shed light on potential intrinsic and extrinsic cues, which, in combination, are thought to ensure the induction and specification of cranial neural crest cells. However, much less is known about how migrating neural crest cells interpret and integrate signals from the microenvironment and other neural crest cells to sort into and maintain the stereotypical pattern of three spatially segregated streams. Here, we explore the extent to which cranial neural crest cells use cell-to-cell and cell-environment interactions to pathfind. The cell membrane and cytoskeletal elements in chick premigratory neural crest cells were labeled in vivo. Three-dimensional reconstructions of migrating neural crest cells were then obtained using confocal static and time-lapse imaging. It was found that neural crest cells maintained nearly constant contact with other migrating neural crest cells, in addition to the microenvironment. Cells used lamellipodia or short, thin filopodia (1-2 microm wide) for local contacts (<20 microm). Non-local, long distance contact (up to 100 microm) was initiated by filopodia that extended and retracted, extended and tracked, or tethered two non-neighboring cells. Intriguingly, the cell-to-cell contacts often stimulated a cell to change direction in favor of a neighboring cell's trajectory. In summary, our results present in vivo evidence for local and long-range neural crest cell interactions, suggesting a possible role for these contacts in directional guidance.     

The effect of neuronal cells on kidney differentiation

During embryonic growth, tissue interactions between dissimilar cells are the driving forces of morphogenesis. Although their importance has been well known for over the past 50 years, the molecular background of these interactions has remained unelucidated. The unrecognized heterogeneity of those mesenchymal cells that are involved in the epithelio-mesenchymal tissue interactions may be one reason for this. For example, studies of kidney differentiation show that the metanephric organ rudiment contains more cell-lines than previously thought. Identification of both neural crest- and mesoderm-derived cells in the nephrogenic mesenchyme helps in re-evaluating the biology of the tubule induction. The neural crest-derived cells of the nephric rudiment differentiate into neuronal cells, and later during differentiation some of them are found in the stroma. There is also experimental evidence for the role of these neuronal cells in the morphogenetic tissue interaction.