An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).

The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 microm cells with large nuclei)

Intramolecular Vibrational Energy Redistribution in 2‑Thiocytosine: SH Rotamerization Induced by Near-IR Selective Excitation of NH₂ Stretching Overtone

Near-IR-induced transformations, converting one amino-thiol conformer of 2-thiocytosine into another, were observed for monomers of the compound isolated in Ne, Ar, and N₂ low-temperature matrixes. The two conformers involved in this phototransformation differ from each other by 180° rotation of the SH group. To induce the conversion, conformers of 2-thiocytosine were selectively excited to the overtone (or combination) NH₂ stretching vibrational states, using very narrowband (fwhm <1 MHz) near-IR light generated in a tunable diode laser. The conformational changes were monitored by IR spectroscopy. The conformational transformation observed in the current work provides a clear evidence of the vibrational energy redistribution from the initially excited NH₂ moiety to the remote SH group that changes its orientation

Tunable Diode Lasers as a Tool for Conformational Control: The Case of Matrix-Isolated Oxamic Acid

A tunable diode laser was applied as a source of narrowband near-infrared light used to manipulate the structure of the molecule of oxamic acid. Monomers of the most stable conformer <b>I</b> of the molecule, with the trans orientation of the OCOH group and the trans orientation of the OCCO fragment, were trapped from the gas phase in low-temperature argon, neon, and nitrogen matrixes. Monomers of oxamic acid, isolated in argon or neon matrixes, were then irradiated with narrowband near-IR light from the diode laser tuned at 6833 (Ar) or 6840 cm^–1 (Ne). Upon such irradiation another conformer, <b>II</b>, of oxamic acid was generated, with cis orientation of the OCOH group and trans orientation of the OCCO fragment. Both forms were identified by comparison of their experimental mid-IR spectra with the spectra theoretically calculated for <b>I</b> and <b>II</b>. Subsequent irradiation of the matrix at 6940 (Ar) or 6991 cm^–1 (Ne), where absorption appeared in the near-IR spectrum of the photoproduct, led to photoconversion of conformer <b>II</b> into form <b>I</b>. In a series of subsequent irradiations at 6833­(Ar)/6840­(Ne) cm^–1 and at 6940­(Ar)/6991­(Ne) cm^–1, the population of oxamic acid molecules was selectively shifted several times from <b>I</b> to <b>II</b> and vice versa. As far as we know, this is the first reported study where a tunable diode laser source of narrowband near-IR light was used to manipulate the structure of a molecule. Spontaneous <b>II</b> → <b>I</b> transformation was observed for Ne and Ar matrixes kept in the dark and at cryogenic temperature

Conformational Changes in Thiazole-2-carboxylic Acid Selectively Induced by Excitation with Narrowband Near-IR and UV Light

Conformers and photoinduced conformational transformations were studied for monomers of thiazole-2-carboxylic acid (TCA). The matrix-isolation technique and excitations with narrowband near-IR and UV light, tuned in an optical parametric oscillator, were used for this purpose. Form <b>I</b>, with the carboxylic moiety in the trans orientation and with the hydrogen atom of the OH group directed toward the nitrogen atom of the ring, was the most abundant in low-temperature argon or nitrogen matrixes. Conformer <b>II</b>, differing from <b>I</b> by 180° rotation of the OH group around the C–O bond, was also trapped in the matrixes, but in much smaller amount. The abundance of form <b>II</b> was experimentally determined as ∼6% of the total amount of TCA molecules. Selective excitation of <b>I</b> with narrowband near-IR laser light resulted in <b>I</b> → <b>II</b> transformation. This near-IR-induced conformational change was photoreversible: form <b>II</b> converted back to <b>I</b> upon selective excitation of <b>II</b> with near-IR light of different wavelength. Conformational conversions of <b>I</b> into <b>II</b>, or vice versa, were also induced in TCA monomers by narrowband UV excitations at 300 nm (for <b>I</b> → <b>II</b> transformation) and at 305 nm (for <b>II</b> → <b>I</b> transformation). A spontaneous conversion of photogenerated <b>II</b> into the most stable form <b>I</b> was observed for the compound trapped in the matrix at 15 K and kept in the dark. This process was very slow; the estimated half-life time of conformer <b>II</b> was longer than 50 h. Finally, TCA was shown to thermally decompose at room temperature, yielding CO₂ and thiazole

Conformers of Kojic Acid and Their Near-IR-Induced Conversions: Long-Range Intramolecular Vibrational Energy Transfer

Conformational transformations were investigated for molecules of kojic acid trapped in low-temperature argon and nitrogen matrixes. Two conformers, differing from each other by 120° rotation of the hydroxymethyl (−CH₂OH) moiety, were found to be populated in freshly deposited matrixes, prior to any irradiation. Matrixes containing isolated monomers of kojic acid were irradiated with narrowband, tunable near-infrared (near-IR) laser light. Excitations at wavenumbers corresponding to the overtone of the stretching vibration of the OH bond of the hydroxymethyl group led to conversion of one of the observed conformers into another. The direction of this conformational transformation depended on the wavenumber (within the 7126–7115 cm^–1 range) used for irradiation. The same conformational photoconversion was also observed to occur upon narrowband irradiation at much lower wavenumbers (from the 6468–6447 cm^–1 range). Near-IR light from this range selectively excites overtone vibrations of the OH group directly attached to the heterocyclic ring. Such an observation provides a convincing evidence of a long-range vibrational energy transfer from the initially excited OH group (directly attached to the ring) to the remote hydroxymethyl fragment which changes its orientation. Structural changes, occurring in matrix-isolated molecules of kojic acid upon near-IR excitation, were monitored by FTIR spectroscopy

Conformational Transformation in Squaric Acid Induced by Near-IR Laser Light

Two conformers of monomeric squaric acid (3,4-dihydroxy-3-cyclobutene-1,2-dione) were studied using the matrix-isolation method. Both forms of the compound, differing in rotation of one of the OH groups by 180°, were trapped from the gas phase into a low-temperature nitrogen matrix, whereas only the lowest-energy conformer was trapped in solid argon and in solid neon. Narrowband near-infrared laser light was used to induce transformation of the most stable form of squaric acid (having <i>C</i>_2<i>v</i> symmetry) into the higher-energy conformer (<i>C_s</i> symmetry). Effective stabilization of the photogenerated species occurred only for the compound isolated in a nitrogen matrix. Moreover, the stabilization of the higher-energy <i>C</i>_<i>s</i> conformer of squaric acid by the solid nitrogen environment was found to strongly depend on the trapping site in the matrix. The spectroscopic characterization of the higher energy <i>C_s</i> conformer is reported here for the first time

Three Conformers of 2‑Furoic Acid: Structure Changes Induced with Near-IR Laser Light

Conformers of 2-furoic acid were studied using the matrix-isolation technique combined with narrow-band near-IR excitations with tunable laser light. Two conformers of the compound were trapped from the gas phase into low-temperature Ar or Ne matrixes with the population ratio of nearly 1:1. The two forms differ from each other by 180° rotation of the carboxylic group with respect to the furan ring. In both structures, the OH group adopts the cis orientation, with its H atom directed toward the CO bond of the OC–O–H group. Narrow-band near-IR excitations of the OH stretching overtone vibrations resulted in transformation of one of the initially observed conformers into a third conformational structure. This near-IR-induced isomerization concerned rotation of the OH group from the initial cis orientation to the trans conformation with the hydrogen atom directed toward the oxygen atom of the furan ring. In the photoproduced conformer, the hydrogen-bond-like O–H···O interaction (between O–H and the oxygen atom of the furan ring) is rather weak. Nevertheless, this interaction stabilized the structure so that it was present in the matrix for several hours after the near-IR-induced generation. The spontaneous conversion of the photogenerated, higher-energy form back into the more stable conformer with the carboxylic group in cis orientation was monitored for 2-furoic acid isolated in Ar and Ne matrixes. The speed of this process was found to be dependent on temperature and on the matrix material. The experimentally determined half-life times of this conformational conversion occurring in the dark are <i>t</i>_1/2 = 1390 min (Ar, 5.5 K); <i>t</i>_1/2 = 630 min (Ar, 15 K); <i>t</i>_1/2 = 240 min (Ne, 5.5 K). The three conformers of 2-furoic acid observed in the present work were identified by comparison of their infrared spectra with the spectra theoretically calculated for the candidate structures

Into Peripheral Blood in Patients After Stroke Clinical Evidence That Very Small Embryonic-Like Stem Cells Are Mobilized Clinical Evidence That Very Small Embryonic-Like Stem Cells Are Mobilized Into Peripheral Blood in Patients After Stroke

Background and Purpose-In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. Methods-We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22 age-matched controls. After each patient&apos;s stroke, PB samples were harvested during the first 24 hours, on day ϩ3, and on day ϩ7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. Results-In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR