Proximal Cluster Research Articles

The Mössbauer Parameters of the Proximal Cluster of Membrane-Bound Hydrogenase Revisited: A Density Functional Theory Study.

An unprecedented [4Fe-3S] cluster proximal to the regular [NiFe] active site has recently been found to be responsible for the ability of membrane-bound hydrogenases (MBHs) to oxidize dihydrogen in the presence of ambient levels of oxygen. Starting from proximal cluster models of a recent DFT study on the redox-dependent structural transformation of the [4Fe-3S] cluster, 57Fe Mössbauer parameters (electric field gradients, isomer shifts, and nuclear hyperfine couplings) were calculated using DFT. Our results revise the previously reported correspondence of Mössbauer signals and iron centers in the [4Fe-3S]3+ reduced-state proximal cluster. Similar conflicting assignments are also resolved for the [4Fe-3S]5+ superoxidized state with particular regard to spin-coupling in the broken-symmetry DFT calculations. Calculated 57Fe hyperfine coupling (HFC) tensors expose discrepancies in the experimental set of HFC tensors and substantiate the need for additional experimental work on the magnetic properties of the MBH proximal cluster in its reduced and superoxidized redox states.

Defining Engraftment Patterns and Interactions Between Hematopoietic Stem Cells and Stromal Cells after in Utero Transplantation

In utero hematopoietic stem cell transplantation (IUHSCT) has only been fully successful in recipients with severe combined immunodeficiency disorders. Because IUHSCT must be performed without myeloablation or immunosuppression, immunologic barriers and absence of stress-induced signaling have been considered significant contributors to the limited success of this procedure. Other potential barriers to successful engraftment of allogeneic adult donor hematopoietic stem/progenitor cells (HSC) in a fetal recipient result from the unique characteristics of fetal HSC biology and the fetal microenvironment. Transplanted adult cells could be out-competed by fetal HSC, and the fetal microenvironment might not support engraftment and/or expansion of HSC derived from ontogenically disparate sources, as differences in membrane composition and response to cytokines exist between fetal and adult cells. We have previously shown that increased levels of HSC engraftment can be achieved in a fetal setting, if bone marrow (BM)-derived mesenchymal stromal cells (MSC) or (BM)-derived endothelial progenitor cells (EPC) are administered prior to, or at the time of, HSC transplantation. Using a fetal sheep model of IUHSCT, we interrogated mechanisms by which transplanting sheep EPC or MSC prior to, or concurrently with, sheep HSC leads to statistically significant higher levels of HSC engraftment when compared to HSC alone. Using confocal microscopy and fluorescently-labeled cells, we first determined the sites of engraftment/localization of donor EPC, MSC, and HSC at 2-2.5 months after IUHSCT when transplanted at the does of 7.1 x 106 /Kg, 2.5 x 106 /kg, and 2.1 x 106 /kg, respectively, at 60-65 days of gestation (human equivalent of 16 gestational weeks). We demonstrated that EPC and MSC engraft at different sites after IUHSCT than the transplanted HSC. MSC and EPC engrafted in the diaphysis, where they integrated into the vascular and perivascular niches and produced SCF and CXCL12. Confocal analysis demonstrated that transplanted EPC produced higher levels of CXCL12 than endogenous cells, explaining, at least in part, the higher levels of hematopoietic engraftment seen in the BM and PB of animals transplanted with EPC 3 days prior to HSC. HSC engrafted mainly in the metaphysis, a metabolically active tissue in the fetus, which is comprised of large sinusoidal veins expressing high levels of hyaluronan (HA) synthase. In addition, donor HSC in the metaphysis were actively cycling (>90%) as determined by Ki67 staining. Fewer donor HSC were found in the diaphysis, and only a small percentage of these cells were dividing, but they localized in clusters in close proximity to donor MSC or EPC. Moreover, a direct correlation was found between the levels of donor-derived perivascular cells engrafted within the vasculature, and the numbers of donor-derived HSC present in the perivascular areas (defined as being within a distance of 5 cell nuclei from vessel), suggesting that, in the diaphysis, adult MSC and EPC provided support/attracted adult HSC. In summary, we have shown that, in a non-myeloablative fetal setting without stress-induced signaling, allogeneic adult donor HSC engraft and proliferate efficiently along with endogenous hematopoietic cells within the metaphysis, and that the high levels of CXCL12 produced by adult EPC were potentially responsible for their ability to significantly enhance HSC engraftment after IUHSCT. DisclosuresNo relevant conflicts of interest to declare.

Cadmium exposure affects on the expression of metallothionein 2 gene in grass carp (Ctenopharyngodon idellus)

The metallothionein 2 (mt2) genomic gene with 667 bp was cloned from the grass carp (Ctenopharyngodon idellus). The structure of mt2 gene was similar with the reported vertebrae mt gene, containing three exons interrupted by two introns. Only one MRE mortify was found in the 5′ flanking region at proximal cluster which was quite different from the previous reports. The number of deduced amino acid was 60, characterized by cysteine-rich. The basal levels and the transcriptional responses of mt2 gene in different organs to cadmium (Cd) were measured using the quantitative real-time PCR (qRT-PCR). In the kidney, the constructive level of the mt2 mRNA was the highest, while the lowest expression level was detected in the skin. The induced impacts of Cd on mt2 mRNA expression were various from tissues. In the liver, gill and skin, the gene was quickly induced at 6 h. After a transient suppression, the expression levels of mt2 mRNA were increased in the intestine and spleen at 12 h, and mt2 mRNA expressed upwards in the bone at 24 h. In the kidney and bone, the expressions elevated only after 48 h Cd induction. The inducibility of mt2 gene to Cd demonstrated that it played a significant role during the metal toxicant detoxification in grass carp and the mt2 gene in the liver was most sensitive to Cd induction.

Synergistic Effects in the Coupling of Plasmon Resonance of Metal Nanoparticles with Excited Gold Clusters.

When molecules or clusters are within the proximity of metal particles, their electronic transitions can be drastically enhanced. We have now probed the off-resonance excitation of molecule-like, glutathione-capped gold clusters (Au-GSH) in the close proximity of larger (plasmonic) Au and Ag nanoparticles. The excited state absorption spectrum of Au-GSH* is obtained with monophotonic excitation. The characteristic absorption of Au-GSH* allows us to probe the influence of excited plasmonic nanoparticles coupled with the clusters. Although infrared (775 nm) lasers pulses do not produce Au-GSH*, the excited states of these clusters are formed when coupled with metal (Au, Ag) nanoparticles. Interestingly, the coupled excitation of Au-GSH/AgNP with 775 nm laser pulses also results in an enhanced field effect, as seen from increased plasmon response of the metal nanoparticles. Transient absorption measurements confirm the synergy between these two inherently different nanomaterials, causing them to display greater excitation features. Better understanding of metal cluster-metal nanoparticle interactions will have important implications in designing light harvesting systems, and optoelectronic devices.

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

Impact of the iron-sulfur cluster proximal to the active site on the catalytic function of an O2-tolerant NAD(+)-reducing [NiFe]-hydrogenase.

The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O2 sensitivity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H2/D(+) exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.

NiFe]-hydrogenases revisited: nickel-carboxamido bond formation in a variant with accrued O2-tolerance and a tentative re-interpretation of Ni-SI states.

[NiFe]-hydrogenases are well-studied enzymes capable of oxidizing molecular hydrogen and reducing protons. EPR and FTIR spectroscopic studies have shown that these enzymes can be isolated in several redox states that include paramagnetic oxidized inactive Ni-A and Ni-B species and a reduced Ni-C form. The latter and the diamagnetic respectively more oxidized Ni-SI and more reduced Ni-R forms are generally thought to be involved in the catalytic cycle of [NiFe]-hydrogenases. With the exception of Ni-SI, these different stable states have been well characterized. Here, based on the crystal structure of a partially reduced Desulfovibrio fructosovorans (Df) enzyme and data from the literature we propose that at least one of the Ni-SI sub-states contains an unexpected combination of hydride and sulfenic acid moieties. We have also determined the structure of the less oxygen-sensitive Df [NiFe]-hydrogenase V74C mutant and found that more than half of the active site nickel occupies a novel position, called Ni'. In this new position, the metal ion is coordinated by two cysteine thiolates, a bridging species modeled as SH(-) and a main chain carboxamido N atom. The Ni' coordination is similar to the one found in Ni superoxide dismutase, an enzyme that operates at significantly more positive potentials than [NiFe]-hydrogenases. We propose that the oxygen-tolerance of the V74C variant results from a high potential stabilization of a Ni'(iii) species induced by the change in the metal ion coordination sphere. We also propose that transient Ni'(iii) species can rapidly attract successive electrons from the Fe4S4 proximal cluster accelerating the reduction of oxygen to water and hydroxide. The naturally occurring oxygen-tolerant [NiFe]-hydrogenases have an unusual proximal cluster that has been shown to be exceptionally plastic and capable of undergoing two successive one-electron oxidations. This double oxidation is modulated by the migration of one of the iron atoms in the cluster to the main chain where, as Fe(iii), it forms a bond with a carboxamido N ligand. Like in the Df V74C variant the electrons from the proximal cluster help reducing O2 to H2O and OH(-). In conclusion, in both cases a metal-carboxamido bond may explain, at least partially, the observed oxygen tolerance.

Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity.

Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel IGLV alleles, one novel IGLC allele, and an 11.9-kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH.Supplementary informationThe online version of this article (doi:10.1038/gene.2014.56) contains supplementary material, which is available to authorized users.

Structural differences of oxidized iron–sulfur and nickel–iron cofactors in O2-tolerant and O2-sensitive hydrogenases studied by X-ray absorption spectroscopy

The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel–iron active site of hydrogen turnover and of the iron–sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7Å Fe–Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron–oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni–A state included a ~0.05Å longer Ni-O bond, a two times larger spread of the Ni–S bond lengths, and a ~0.1Å shorter Ni–Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni–Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.

Control of the transition between Ni-C and Ni-SI(a) states by the redox state of the proximal Fe-S cluster in the catalytic cycle of [NiFe] hydrogenase.

[NiFe] hydrogenase catalyzes the reversible cleavage of H2. The electrons produced by the H2 cleavage pass through three Fe-S clusters in [NiFe] hydrogenase to its redox partner. It has been reported that the Ni-SI(a), Ni-C, and Ni-R states of [NiFe] hydrogenase are involved in the catalytic cycle, although the mechanism and regulation of the transition between the Ni-C and Ni-SI(a) states remain unrevealed. In this study, the FT-IR spectra under light irradiation at 138-198 K show that the Ni-L state of [NiFe] hydrogenase is an intermediate between the transition of the Ni-C and Ni-SI(a) states. The transition of the Ni-C state to the Ni-SI(a) state occurred when the proximal [Fe4S4]p(2+/+) cluster was oxidized, but not when it was reduced. These results show that the catalytic cycle of [NiFe] hydrogenase is controlled by the redox state of its [Fe4S4]p(2+/+) cluster, which may function as a gate for the electron flow from the NiFe active site to the redox partner.

VITAL: Structured and clustered super-peer network for similarity search

VITAL is a novel P2P indexing structure that provides on top of exact search a similarity search of multidimensional vectors. It is designed to scale to millions of peers and billions of shared documents and to adapt to high network dynamics. To exploit peer heterogeneity, VITAL is a super-peer (SP) network where every peer is an SP candidate and a simple election protocol is run to select SPs. On the other hand, every SP locally monitors its “vital” signs of memory, processing, and bandwidth and initiates the SP election protocol based on its capacity and load limits. In addition, the SP overlay is structured as CAN distributed hash table to guarantee both the correctness and responsiveness of the query protocol. A novel data replication model is introduced, where every peer clusters its shared documents to local clusters (LCs) and each LC summary is published at few SPs to achieve content-based clustering and firework query propagation. Every peer establishes TCP connections with many SPs that maintain its LC summaries. VITAL has no central component and does not require global knowledge, however it requires identifying a set of global cluster (GC) centriods to be disjointly managed by the elected SPs. In addition, CAN zone overloading is seamlessly applied to relief overwhelmed SPs and it provided an extra layer of physical proximity clustering. The scalability analysis shows that peer index requires less than 3 % of extra storage and a query (on average) can be satisfied by visiting 1.6 % of the number of established TCP connections.

Atypical breakpoint in a t(6;17) translocation case of acampomelic campomelic dysplasia

Campomelic dysplasia (CD) is a skeletal dysplasia characterized by Pierre Robin sequence (PRS), shortened and bowed long bones, airway instability, and the potential for sex reversal. A subtype of CD, acampomelic CD (ACD), is seen in approximately 10% of cases and preserves long bone straightness. Both syndromes are caused by alterations in SOX9, with translocations and missense mutations being overrepresented in ACD cases. We report a term infant with PRS, severe cervical spine abnormalities, eleven rib pairs, hypoplastic scapulae, and female genitalia. Chromosome analysis identified a 46,XY,t(6;17)(q25;q24) karyotype. FISH analysis with a series of BAC probes localized the translocation breakpoints to 6q27 and a region at 17q24.3 in the range of 459–379 kb upstream of SOX9. Therefore, this case extends the region classified as the proximal breakpoint cluster. In addition, the comorbidity of acampomelia, complete sex reversal, and severe spinal anomalies in our patient underscores the variability in the level of malformation in the CD/ACD family of disorders.

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Aerobic Damage to [FeFe]‐Hydrogenases: Activation Barriers for the Chemical Attachment of O2

Abstract[FeFe]‐hydrogenases are the best natural hydrogen‐producing enzymes but their biotechnological exploitation is hampered by their extreme oxygen sensitivity. The free energy profile for the chemical attachment of O2 to the enzyme active site was investigated by using a range‐separated density functional re‐parametrized to reproduce high‐level ab initio data. An activation free‐energy barrier of 13 kcal mol−1 was obtained for chemical bond formation between the di‐iron active site and O2, a value in good agreement with experimental inactivation rates. The oxygen binding can be viewed as an inner‐sphere electron‐transfer process that is strongly influenced by Coulombic interactions with the proximal cubane cluster and the protein environment. The implications of these results for future mutation studies with the aim of increasing the oxygen tolerance of this enzyme are discussed.

Aerobic damage to [FeFe]-hydrogenases: activation barriers for the chemical attachment of O2.

[FeFe]-hydrogenases are the best natural hydrogen-producing enzymes but their biotechnological exploitation is hampered by their extreme oxygen sensitivity. The free energy profile for the chemical attachment of O2 to the enzyme active site was investigated by using a range-separated density functional re-parametrized to reproduce high-level ab initio data. An activation free-energy barrier of 13 kcal mol−1 was obtained for chemical bond formation between the di-iron active site and O2, a value in good agreement with experimental inactivation rates. The oxygen binding can be viewed as an inner-sphere electron-transfer process that is strongly influenced by Coulombic interactions with the proximal cubane cluster and the protein environment. The implications of these results for future mutation studies with the aim of increasing the oxygen tolerance of this enzyme are discussed.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.

Identification of an Endogenous Allosteric Modulator's Binding Site at the Human Cannabinoid-1 Receptor, using the Forced-Biased Metropolis Monte Carlo Simulated Annealing Method (Mmc)

The CB1 endogenous, positive allosteric modulator, lipoxin A4, increases the equilibrium binding and efficacy of CP55,940 and anandamide (orthosteric agonists), yet has no significant effect when applied alone. Unlike ORG27569 (a negative allosteric modulator of CB1), lipoxin A4 does not significantly displace SR141716A (an orthosteric inverse agonist) in equilibrium binding assays. In addition, we have previously reported that ORG27569's binds in the THM3/TMH6/TMH7 region (Shore et al., ICRS, 2012); at this site, ORG27569 sterically blocks important movements of the second and third extracellular loops, as well as those of TMH6, that are necessary for G protein-mediated signaling. Because lipoxin A4 is a positive allosteric modulator, one would not expect it to sterically block these functionally-important conformational changes. Together, these results may suggest that lipoxin A4 binds in a topographically different region of CB1 than ORG27569. To identify lipoxin A4's binding site(s) at CB1, we are using the Forced-Biased Metropolis Monte Carlo simulated annealing program, MMC. In this method, lipoxin A4 was separated into 4 fragments. Four MMC runs are currently being performed, in which our in silico CB1 receptor model (with CP55,940 docked) was immersed in a box filled with copies of one of these fragments. The system chemical potential is then systematically annealed, causing only those fragment copies with the best free energy of binding to the protein surface to remain. Ultimately, analysis of these four MMC runs will suggest region(s) in which all four fragments cluster in the correct spatial proximity, thus suggesting a possible binding site(s). [Support: RO1 DA003934 and KO5 DA021358 (PHR)]

Modelling innovation support systems for regional development – analysis of cluster structures in innovation in Portugal

The present article offers a concise theoretical conceptualization and operational analysis of the contribution of innovation to regional development. The latter concepts are closely related to geographical proximity, knowledge diffusion and filters and clustering. Institutional innovation profiles and regional patterns of innovation are two mutually linked, novel conceptual elements in this article. Next to a theoretical framing, the article employs the regional innovation systems concept as a vehicle to analyse institutional innovation profiles. Our case study addresses three Portuguese regions and their institutions, included in a web-based inventory of innovation agencies which offered the foundation for an extensive database. This data-set was analysed by means of a recently developed principal coordinates analysis followed by a Logistic Biplot approach (leading to a Voronoi mapping) to design a systemic typology of innovation structures where each institution is individually represented. There appears to be a significant difference in the regional innovation patterns resulting from the diverse institutional innovation profiles concerned. These profiles appear to be region specific. Our conclusion highlights the main advantages in the use of the method used for policy-makers and business companies.

Polyelectrolyte Microcapsule Based Assay for Monitoring Biotechnological Processes In Vitro and In Vivo

The development of non-invasive techniques for monitoring diverse biotechnological processes under production conditions is a challenging and fascinating task. It requires a novel tool that specifically detects final or intermediate products of biotechnological processes, e.g. antibodies or carbohydrates in cell cultures, or moreover any impurities, such as lipopolysaccharides, while eliminating the risk of contamination. The proposed assay should be universally adaptable to many analytes at technologically relevant concentrations and should provide real-time monitoring, high sensitivity, specificity, and robustness under production conditions.Polyelectrolyte (PE) microcapsules produced by Layer-by-Layer (LbL) assembly can serve as a perfect platform for multiple sensing, because their surface chemistry can be broadly adapted to detect various biomarkers. The measurement principle is based on the induced proximity (clustering) of two different populations of PE-microcapsules carrying detectors for specific recognition sites of an analyte. Due to the relatively large surface area and large number of binding sites, the analyte can be detected over a wide range of concentrations. As a future prospective, the methods developed on the basis of this measurement principle will provide the opportunity to determine the analytes that cannot yet be detected (e.g. intracellular proteins, nucleic acids or metabolites) with high temporal and spatial resolution.

2013 Late-Breaking Basic Science Abstracts

### 21998 #### Enhanced Myocardial Repair with CardioChimeras Pearl J Quijada, Jonathan D Cubillo, Claudio Staub, Mark A Sussman; San Diego State Univ, San Diego, CA Dual stem cell transplantation of c-kit positive cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction support modest improvements in cardiac function, however there are currently no reports substantiating a single stem cell type supporting both direct and indirect mechanisms of myocardial repair. Therefore we created a CardioChimera, a stem/progenitor cell formed by fusion between CPCs and MSCs, resulting in a unique progeny superior to either individual precursor. CardioChimeras were purified after cell fusion with Hemagglutinating virus of Japan and expanded clonally based on dual expression of fluorescent proteins mCherry and eGFP from CPCs and MSCs respectively. CardioChimeras are mono-nucleated, have comparable growth kinetics to parental CPCs and MSCs and display increased cellular size unrelated to cell cycle arrest and/or senescence. CardioChimeras have increased expression of cardiomyogenic lineage markers cardiac troponin T (2.5-fold), smooth muscle 22 (9.3-fold), and CD31 (10.7-fold) concomitantly associated with decreased c-kit protein expression (50%) relative to parent CPCs. Lineage commitment of CardioChimeras is bolstered by dexamethasone treatment measured by mRNA levels of cardiogenic genes and increases in active mitochondria (2.2-fold) after labeling with MitoTracker. Induction of apoptosis is blunted in cardiomyocytes co-cultured with CardioChimeras compared to co-culturing with CPCs and MSCs alone, or combination of non-fused parent cells. CardioChimeras enhance cardiomyocyte growth similar to parent MSCs owing to an increased propensity to secrete pro-growth factors. Collectively, CardioChimeras represent an adaptable cell therapy combining the beneficial properties of CPCs to undergo cardiac specific commitment as well as MSCs that foster an improved microenvironment with protective paracrine secretion. Clinically, CardioChimeras merge the application of distinct cell types into a single entity for increased engraftment, mitigation of inflammation and blunting the progression of heart failure by promoting myocardial regeneration. …