Modern medicine will unequivocally include regenerative medicine as a major breakthrough in the re-establishment of damaged or lost tissues due to degenerative diseases or injury. In this scenario, millions of patients worldwide can have their quality of life improved by stem cell implantation coupled with endogenous secretion or administration of survival and differentiation promoting factors. Large efforts, relying mostly on flow cytometry and imaging techniques, have been put into cell isolation, immunophenotyping, and studies of differentiation properties of stem cells of diverse origins. Mesenchymal stem cells (MSCs) are particularly relevant for therapy due to their simplicity of isolation. A minimal phenotypic pattern for the identification of MSCs cells requires them to be immunopositive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, and HLA-DR and other surface markers. MSCs identified by their cell surface marker expression pattern can be readily purified from patient's bone marrow and adipose tissues. Following expansion and/or predifferentiation into a desired tissue type, stem cells can be reimplanted for tissue repair in the same patient, virtually eliminating rejection problems. Transplantation of MSCs is subject of almost 200 clinical trials to cure and treat a very broad range of conditions, including bone, heart, and neurodegenerative diseases. Immediate or medium term improvements of clinical symptoms have been reported as results of many clinical studies. ' 2012 International Society for Advancement of Cytometry

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid ␤ peptide (A␤) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by A␤ has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking A␤-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to A␤. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to A␤ and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that A␤ inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by A␤. These results indicate that the A␤ binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for A␤ neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble A␤ in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD. Alzheimer disease (AD)1 is the most common age-related neurodegenerative disorder. This irreversible disease is caused by extensive synaptic dysfunction, resulting in the impairment of cognitive and other cerebral functions. Clinical investigations as well as studies on animal models suggest that the primary agent of neurodegeneration is a peptide of 39 -43 amino acid residues known as amyloid ␤ peptide (A␤) (1, 2). Although the majority of secreted A␤ is 40 amino acids long (A␤40), the smaller fraction of longer, 42-amino acid species (A␤42) has received greater attention because of its high propensity to nucleate and drive the formation of amyloid aggregates and fibrils (3). Amyloid fibrils found in the senile plaques present in the brains of AD patients were originally considered to be primarily responsible for the clinical progression of AD. However, more recent evidence indicates that the cognitive decline correlates better with the amount of soluble A␤ and with the loss of synaptic proteins than with the formation and deposition of amyloid plaques (e.g. Refs. 4 and 5).Multiple neural systems are affected in AD, and a key feature of the neurodegenerative process is the loss of cholinergic neurons and nicotinic ACh receptors (nAChRs) throughout the brain (6). High affinity association of A␤42 with ␣7 and ␣4␤2 nAChRs has recently been observed in amyloid plaques and neurons of AD patients (7,8). Partial functional inhibition of ␣4␤2 and ␣7 nAChRs by A␤ has also been demonstrated (9 -12). This, however, is still a somewhat controversial issue, as a recent report has described that picomolar concentrations of A␤ activate, rather than inhibit, whole-cell current responses of ␣7-nAChRs (13).In this study, we have used a phage display approac...

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins b3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional Nmethyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain. ' 2011 International Society for Advancement of Cytometry

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono-and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. ' 2011 International Society for Advancement of Cytometry Key termsPlasmodium falciparum; melatonin; cell cycle; flow cytometry MALARIA is caused by the Apicomplexan parasite Plasmodium falciparum. Its asexual replicative cycle inside red blood cell (RBC) is responsible for pathogenesis and induces several structural and biochemical changes within the host cell (1,2). One striking feature regarding P. falciparum infection is associated with 48-h fever peaks intervals, as a result of from synchronous release of merozoites into the blood stream (3).Melatonin, a hormone produced by the pineal gland of vertebrates, transmit the darkness signal based on circadian and seasonal time measurements (4). The presence of melatonin, however, is not restricted to vertebrates but is also found in phylogenetically divergent species including bacteria, plants and protozoa (4). We have previously shown that this hormone is able to synchronize Plasmodium falciparum and P. chabaudi cell infections (5-7). The synchronicity was lost in vitro when parasites had been incubated with the melatonin antagonist luzindole; the same effect was obtained in vivo in pinealectomized mice and after the injection of luzindole (5). In P. falciparum, melatonin induces a complex signaling pathway with the participation of IP3 generation (8) and subsequent transients in cytosolic calcium concentration, cAMP production and protein kinase A (PKA) (9,10) and protease activation (11).Flow cytometry (FCM) is a powerful method for evaluation of human erythrocyte infection rates as well as for discrimination of Plasmodium falciparum developmental stages (12-16). Several methods of detection of malaria parasite by flow cytometry have been developed taking advantage of the absence of DNA in erythrocytes. Different dyes such as acridine orange (17,18), hydroethidine (19,20), SYTO 16 (21) and thiazole orange (22) were already employed for the determination of parasitemia

P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X(2) receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [alpha-32P]ATP, whereas suramin did not interfere with [alpha-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X(2) receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X(2) receptor inhibition.

RNA and DNA aptamers developed by systematic evolution of ligands by exponential enrichment (SELEX) have turned into important tools in diagnostics, research, and therapeutics. Unlike antibodies, high-affinity and specific aptamers identified through an in vitro selection process can be chemically modified to gain nuclease resistances in biological fluids and to extend their bioavailability in animals. Aptamers can be raised against virtually any target including those which are toxic or do not elicit any immune response in animals. They can be developed in automated processes against various protein targets and then easily modified by attaching fluorescence reporters, nanoparticles or biotin moieties, rival antibodies in high-throughput proteomics and cell separations. In this review, we will discuss the high competence of aptamers in recognizing biomarkers and molecular signatures of cell surfaces, and how these unique features can be exploited for the identification and isolation of cancer, stem cells and even detection of parasite-infected cells.

Studies have confirmed that bone marrow-derived mesenchymal stem cells (MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs (BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs (ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham (only sciatic nerve exposed), Matrigel (MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs (sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs (sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios (axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment.