Stem Cell and Regenerative Medicine Global Conference (SCRGC) 2016 (August 23-24, 2016 - Gyeonggi-do, Korea).

In its third edition, the Stem Cell and Regenerative Medicine Global Conference (SCRGC) organized by the Global Stem Cell & Regenerative Medicine Acceleration Center (GSRAC) was focused on breaking barriers to accelerate the pace of innovation and development of the regenerative medicine industry. GSRAC is both a think tank and a global network of key opinion leaders from the public and the private sectors. GSRAC was commissioned in 2011 by the Ministry of Health and Welfare (MOHW) of Korea. GSRAC's primary mission is to enable and accelerate the delivery of innovative technologies to patients who are affected by currently untreatable diseases. This goal is notably achieved by resolving hurdles in the field of regenerative medicine. With a total of 30 speakers and panelists from 8 different countries and more than 400 attendees from an array of institutions including hospitals, clinics, biotechnology companies, pharmaceutical companies, scientists, as well as policy makers, the 2-day SCRGC highlighted critical challenges and paths to resolving them in policy and regulatory, and industrial-scale manufacturing of gene-based and cell-based therapies, comprising plenary lectures and sessions covering strategic policy, regulatory, reimbursement and business development, and business of manufacturing, and production technologies. Several of these presentations are summarized in this report.

Desorption/ionization on silicon nanowires.

Dense arrays of single-crystal silicon nanowires (SiNWs) have been used as a platform for laser desorption/ionization mass spectrometry of small molecules, peptides, protein digests, and endogenous and xenobiotic metabolites in biofluids. Sensitivity down to the attomole level has been achieved on the nanowire surfaces by optimizing laser energy, surface chemistry, nanowire diameter, length, and growth orientation. An interesting feature of the nanowire surface is that it requires lower laser energy as compared to porous silicon and MALDI to desorb/ionize small molecules, therefore reducing background ion interference. Taking advantage of their high surface area and fluid wicking capabilities, SiNWs were used to perform chromatographic separation followed by mass analysis of the separated molecules providing a unique platform that can integrate separation and mass spectrometric detection on a single surface.

Magnetic and Mössbauer Studies of Magnetite-Loaded Polyvinyl Alcohol Hydrogels.

Materials producing strain in a magnetic field are known as magnetoelastic or magnetostrictive materials. A new type of material that is able to produce giant strain in a nonhomogeneous magnetic field has been developed. In these magnetic-field-sensitive gels (ferrogels) fine colloidal particles having superparamagnetic behavior are incorporated into a highly swollen elastic polymer network. Magnetic properties of ferrogels have been investigated using electron microscopy, static magnetization measurements, and Mössbauer spectroscopy. Analysis of the data yielded information on the superparamagnetic behavior of ferrogels and made it possible to estimate the size distribution of the magnetic cores of magnetite particles made by chemical precipitation and built into a chemically cross-linked polyvinyl alcohol matrix. The results are interpreted on the basis of a core-shell model. Copyright 2000 Academic Press.

[Preparation and spectroscopic studies of diorganotin(IV) complexes with adenosine and related compounds]. / Az adenozin és rokonvegyületei di-n-butilón(IV) komplexeinek elóállítása és spektroszkópiás vizsgálata.

Nine complexes of adenosine and related compounds (adenosine-5'-monophosphate, adenosine-5'-triphosphate, 1-methyl-adenosine, pyridoxal-5-phosphate and beta-nicotinamide-adenine-dinucleotide-phosphoric acid) with di-n-butyltin(IV) oxide and/or di-n-butytin(IV) dichloride were prepared in the solid state. The compositions of the complexes were determined by standard analytical methods. It was found that the complexes contain organotin(IV) moiety and the ligand in a ratio of 1:1 or 2:1. The FTIR spectra demonstrated that di-n-butyltin(IV) oxide reacts with the D-ribose moiety of the ligands, while di-n-butyltin(IV) dichloride is co-ordinated to the deprotonated phosphate group. The basic part of the ligands does not participate directly in complex formation. Comparison of the experimental Mössbauer delta E values with those calculated on the basis of the PQS concept revealed that the organotin(IV) moiety has trigonal-bipyramidal, octahedral and in some cases tetrahedral geometry also. Some of the complexes contain the organotin(IV) cation in two different surroundings.

[Preparation, X-ray structural and spectroscopic studies of some D-lactobionic acid complexes with Cs(I), Fe(III) and di-n-butyltin(IV)]. / A Cs(I)-, a Fe(III) és a di-n-butilón(IV)2+ kationok D-laktobionsavval alkotott komplexeinek elóállítása, illetve röntgendiffrakciós és spektroszkópiás vizsgálata.

D-Lactobionic acid (4-O-beta-D-galactopyranosyl-D-gluconic acid) complexes of Cs(I), Fe(III) and di-n-butyltin(IV)2+ ions were prepared in the solid state. The bonding sites of the ligands were verified by means of FTIR, Raman and 13C NMR spectroscopic measurements. The Cs(I)-D-lactobionate was obtained in single-crystal form. The X-ray crystallographic results on Cs(I)-D-lactobionate demonstarted that each Cs(I) ion is bonded to four D-lactobionate ions, forming an intricate 3D network. The asymmetric unit consists of one Cs(I), one D-lactobionate ion and one water molecule. For the di-n-butyltin(IV) complex, Mössbauer pqs calculations indicated octahedral and trigonalbipyramidal stereochemistry around the central tin atom in the oligomeric compound. In DMSO solution, the polymeric structure does not remain as shown by 13C NMR measurement. One solvent molecule is coordinated additionally to the tin center, and the carboxylate group has become monodentate. According to the EPR measurement, the Fe(III) complexes obtained at different pH have at least dimeric or oligomeric structure.

[Positron lifetime studies of sodium- and zinc-hyaluronates]. / Nátrium- és cink-hialuronát vizsgálata pozitronélettartam-spektroszkópiával.

The aim of this work was to test the positron lifetime technique (PLT) as a tool of the structure study of sodium- and zinc-hyaluronates. The information based on the PLT measurements (outlined as follows) proved that this method can be useful in this field as well. The lifetime of ortho-positronium (o-Ps) significantly increased and its intensity decreased in the samples containing Zn2+, comparing to Na-hyaluronates. It showed that the electronic orbitals are more closed in the case of Zn2+ cation that means the overlapping between the wave functions of the positron and of the electrons decreased. (See equation No 1.) The study of the effect of water content suggested that the hydrogen-bridge-bonds "localised" the free electron pairs. The increasing pressure increased the lifetime and it is an evidence that the effect of the cations (Na+ and Zn2+) can be explained rather with the change of the electronic structure than with the altering of the free volumes of the samples.

A novel scheme for the time-of-flight analysis of extended ion packets

A new scheme is proposed for providing enhanced time-of-flight focusing of spatially extended ion packets moving with close to uniform velocity, sufficiently high for reliable ion detection. The arrangement consists of two decelerating regions with homogeneous electric fields similar to the two-stage ion reflector. The decelerating field in the first decelerating stage is created in a pulsed fashion after the ion packet has entered the first region. The effect of fringe fields produced by shielding rings and the microchannel plate detector is discussed. Copyright 1999 John Wiley & Sons, Ltd.

[Unusual coordination behavior of D-fructose towards dimethyltin(IV)2+: metal-promoted deprotonation of alcoholic hydroxyl groups in aqueous solutions at low pH]. / A D-fruktóz szokatlan viselkedése dimetilón(IV)2+ kation jelenlétében: az alkoholos hidroxilcsoport fémion által indukált deprotonálódása vizes közegben, alacsony pH-n.

To study the effect of the conformation of sugar hydroxy groups on metal complexation processes, complex formation of eight saccharides (D-fructose, L-sorbose, L-arabinose, D-arabinose, D-glucose, D-sorbitol, 2-deoxy-D-glucose and D-saccharose) with dimethyltin(IV)2+ cations was investigated in aqueous solution by potentiometric equilibrium measurements, 13C NMR, polarimetric and Mössbauer spectroscopic methods. The experimental results proved that deprotonation of D-fructose and L-sorbose is caused by the coordination of dimethyltin(IV)2+ in the unusual low pH interval 4-6 in contrast to the other saccharides deprotonated in analogous way at pH > 8. Increasing the pH of the solution resulted in the formation of further complexes. Stability and composition of the species was determined by potentiometric studies. 13C NMR measurements led to the assignment of the sugar OH groups participating in the processes. Mössbauer investigations in the quick-frozen solutions permitted the determination of the stereochemistry of tin(IV) in the complexes.

Peptide mapping and disulfide bond analysis of myeloid progenitor inhibitory chemokine and keratinocyte growth factor by matrix-assisted laser desorption ionization mass spectrometry.

Peptide mapping and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) were conducted to characterize the human genome-based recombinant proteins. Accurate mass values for the deleted forms of the myeloid progenitor inhibitory factor chemokine (MPIF-1d23), and the keratinocyte growth factor (KGF-2d33) were measured as 8848.55 +/- 0.25 and 16,175.87 +/- 0.89 Da, respectively. The mass accuracy of delayed ion extraction MALDI-MS measurements was within 20 ppm of the cDNA predicted value. Reduction and alkylation of the chemokine showed the presence of six cysteine residues and three disulfide bonds. Additional confirmation of disulfide bonding among the cysteine residues of the chemokine was demonstrated by identifying the RP-HPLC separated tryptic and endoprotease Glu-C peptides. Three methionine residues of the chemokine were identified by MALDI-MS of its cyanogen bromide (CNBr) cleavage products. The KGF-2d33 growth factor, however, lacked the disulfide bonding between the two-cysteine residues and contained two free sulfhydryl groups. Direct analysis of the growth factor CNBr digest showed 7542.99, 4993.4, and 3107.7 Da peptides, which identified the methionine residues. Peptide mapping mass spectrometry indicated that host-specific posttranslational modifications had not influenced the gene expression work.

[Use of an internal mammary artery graft in the revascularization of the myocardium. A ten-year follow up study]. / Az arteria mammaria interna graftok alkalmazása a szívizom revascularisatiójában. Tízéves nyomon követési vizsgálat.

Between September 1986 and August 1996, 233 patients (187 males, 46 females) ranging in age from 32 to 81 (mean 54.7) years received at least one internal mammary artery (IMA) graft for coronary artery bypass procedures. The mean left ventricular ejection fraction was 55.1% (range, 17 to 75%). An average of 3.1 distal anastomoses per patient was constructed. Hospital mortality was 2.1%. Perioperative myocardial infraction was seen in 2.1%. The mean follow-up of hospital survivors was 39.2 (range, 4 to 123) months. Ten-year actuarial survival for patients discharged from the hospital was 96%. Recurrence of angina occurred in 18 patients and reoperation or PTCA was performed in 3 patients in the late follow-up period. These results support the continuing use of IMA grafts for myocardial revascularization.

Primary structure of ovine fibroblast growth factor-1 deduced by protein and cDNA analysis.

The amino acid sequence of full-length ovine fibroblast growth factor-1 (FGF-1) was determined by a combination of protein and cDNA sequencing. FGF-1 cDNA analysis indicated that ovine kidney cells express mRNAs encoding both full-length FGF-1 and a truncated FGF-1 variant. An overall comparison of the ovine FGF-1 primary sequence to the eight species studied to date revealed a high degree of conservation, with ovine FGF-1 sharing 90 and 95% sequence identity with human FGF-1 and bovine FGF-1, respectively. Additionally, the FGF-1 proteins from the various species have conserved cysteine residues at positions 30 and 97 and contain acetylated amino-terminal alanine residues. Mass spectrometry analysis confirmed that the blocking group of ovine FGF-1 is also consistent with that of an acetyl-moiety. In contrast to the other FGF-1 proteins, the 154 residue primary sequence of ovine FGF-1 contains three unique amino acid differences: Arg9, Arg44, and Ile123. Ovine FGF-1, unlike human FGF-1, is a potent mitogenic factor for NIH 3T3 fibroblasts in the absence of heparin. In the presence of exogenous heparin, the mitogenic activity of ovine FGF-1 is potentiated slightly.

Compact tunable Cr:LiSAF laser for infrared matrix-assisted laser desorption/ionization.

A tunable Cr:LiSAF laser-pumped optical parametric oscillator was used for mid-infrared matrix-assisted laser desorption/ionization (MALDI) mass spectrometry experiments. The mass spectra of substance P, bovine insulin and pd(T)10 in the 2.88-2.96 microns range showed excellent single shot signal quality (signal-to-noise-ratio, resolution) but poor shot-to-shot reproducibility. The reproducibility is expected to improve with more stable laser design. No correlation was found between the absorption spectrum of the matrix and the MALDI response.

Detection and quantitation of beta-2-microglobulin glycosylated end products in human serum by matrix-assisted laser desorption/ionization mass spectrometry.

beta-2-Microglobulin (beta 2M) is a major protein component found in the amyloid deposits of dialysis-related amyloidosis (DRA) patients. Evidence has been shown that the advanced glycosylated end-products (AGEs) of beta 2M present in sera were related to DRA. We demonstrated that matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a useful tool to investigate the nature of glycosylation of beta 2M and detection of beta 2M or beta 2M-AGEs in human serum. The high-mass end of beta 2M-AGE distribution was found to extend to the neighborhood of 12,868 Da, corresponding to condensations with seven glucose molecules. We also have shown that both beta 2M and beta 2M-AGEs can be detected at low picomole levels directly in bovine serum. Based on these findings, the sera of DRA patients were studied to determine whether beta 2M-AGEs can be detected by MALDI-MS. In an attempt to investigate the possibility of quantitation with MALDI, human sera samples with different concentrations of beta 2M-AGE were examined. We were able to correlate the concentration of beta 2M-AGE with the number of detected AGE products, pointing to the feasibility of MALDI as a quantitative tool.

Noncovalent protein--oligonucleotide interactions monitored by matrix-assisted laser desorption/ionization mass spectrometry.

Positive ion mode matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to explore nonspecific interactions between proteins and oligonucleotides. The formation of noncovalent complexes showed correlation with the type of oligonucleotide bases and with the amino acid composition of the proteins. Among the four DNA homooligomers, abundant protein-nucleic acid complexes were detected for pd(T)n, whereas negligible attachment was evident for pd(A)n, pd(C)n, and pd(G)n. Mixed base sequence nucleic acids (pd(AGCTCAGCTT) and d(TTAGCAGCTT) also showed affinity to Arg-Lys. The protein affinity of pd(T)n turned out to be nonspecific and produced a larger variety of complexes when the number of basic residues in the protein was increased. Complexation of pd(T)n with small basic dipeptides (Arg-Lys or His-His) led to significant improvement in the mass resolution for positive ions. For example, the mass resolution of the pd(T)20/Arg-Lys complex exhibited about 4 times improvement over pd(T)20 alone. The protein--oligonucleotide interactions were also pH and matrix dependent. Lowering the pH from its original value (pH = 1.7) led to diminishing complex related signal, whereas increasing the pH resulted in the appearance of a larger variety of complexes. 2,5-Dihydroxybenzoic acid matrix demonstrated much greater tendency to produce complex ions than did the three other matrix materials we tested. A possible explanation of the observed phenomena was based on pH-controlled ion pair formation between oligonucleotides and proteins.

Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli.

A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength.

Cloning and sequence determination of the aspartase-encoding gene from Brevibacterium flavum MJ233.

A 2.5-kb EcoRI fragment containing the aspartase-encoding gene (aspA) of Brevibacterium flavum MJ233 was cloned into plasmid pUC18 using Southern hybridization with the Escherichia coli aspA gene as a probe. The complete nucleotide (nt) sequence of the cloned DNA indicated that the deduced gene product of the Br. flavum aspA is composed of 526 amino acids (aa). Comparison of the aa sequence to the corresponding sequences from E. coli, Bacillus subtilis and Pseudomonas fluorescens revealed 63, 47 and 57% homology, respectively. The aspA product was determined to have a size of approx. 57 kDa by SDS-PAGE.

Transposon mutagenesis of coryneform bacteria.

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.