A Hybrid Least Squares and Principal Component Analysis Algorithm for Raman Spectroscopy

Raman spectroscopy is a powerful technique for detecting and quantifying analytes in chemical mixtures. A critical part of Raman spectroscopy is the use of a computer algorithm to analyze the measured Raman spectra. The most commonly used algorithm is the classical least squares method, which is popular due to its speed and ease of implementation. However, it is sensitive to inaccuracies or variations in the reference spectra of the analytes (compounds of interest) and the background. Many algorithms, primarily multivariate calibration methods, have been proposed that increase robustness to such variations. In this study, we propose a novel method that improves robustness even further by explicitly modeling variations in both the background and analyte signals. More specifically, it extends the classical least squares model by allowing the declared reference spectra to vary in accordance with the principal components obtained from training sets of spectra measured in prior characterization experiments. The amount of variation allowed is constrained by the eigenvalues of this principal component analysis. We compare the novel algorithm to the least squares method with a low-order polynomial residual model, as well as a state-of-the-art hybrid linear analysis method. The latter is a multivariate calibration method designed specifically to improve robustness to background variability in cases where training spectra of the background, as well as the mean spectrum of the analyte, are available. We demonstrate the novel algorithm’s superior performance by comparing quantitative error metrics generated by each method. The experiments consider both simulated data and experimental data acquired from in vitro solutions of Raman-enhanced gold-silica nanoparticles

Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization

Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders. © 2013 Stelling et al

Biogenic and Synthetic Polyamines Bind Cationic Dendrimers

Biogenic polyamines are essential for cell growth and differentiation, while polyamine analogues exert antitumor activity in multiple experimental model systems, including breast and lung cancer. Dendrimers are widely used for drug delivery in vitro and in vivo. We report the bindings of biogenic polyamines, spermine (spm), and spermidine (spmd), and their synthetic analogues, 3,7,11,15-tetrazaheptadecane.4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333) to dendrimers of different compositions, mPEG-PAMAM (G3), mPEG-PAMAM (G4) and PAMAM (G4). FTIR and UV-visible spectroscopic methods as well as molecular modeling were used to analyze polyamine binding mode, the binding constant and the effects of polyamine complexation on dendrimer stability and conformation. Structural analysis showed that polyamines bound dendrimers through both hydrophobic and hydrophilic contacts with overall binding constants of Kspm-mPEG-G3 = 7.6×104 M−1, Kspm-mPEG-PAMAM-G4 = 4.6×104 M−1, Kspm-PAMAM-G4 = 6.6×104 M−1, Kspmd-mPEG-G3 = 1.0×105 M−1, Kspmd-mPEG-PAMAM-G4 = 5.5×104 M−1, Kspmd-PAMAM-G4 = 9.2×104 M−1, KBE-333-mPEG-G3 = 4.2×104 M−1, KBe-333-mPEG-PAMAM-G4 = 3.2×104 M−1, KBE-333-PAMAM-G4 = 3.6×104 M−1, KBE-3333-mPEG-G3 = 2.2×104 M−1, KBe-3333-mPEG-PAMAM-G4 = 2.4×104 M−1, KBE-3333-PAMAM-G4 = 2.3×104 M−1. Biogenic polyamines showed stronger affinity toward dendrimers than those of synthetic polyamines, while weaker interaction was observed as polyamine cationic charges increased. The free binding energies calculated from docking studies were: −3.2 (spermine), −3.5 (spermidine) and −3.03 (BE-3333) kcal/mol, with the following order of binding affinity: spermidine-PAMAM-G-4>spermine-PAMMAM-G4>BE-3333-PAMAM-G4 consistent with spectroscopic data. Our results suggest that dendrimers can act as carrier vehicles for delivering antitumor polyamine analogues to target tissues

A fractal nature for polymerized laminin

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.This work was supported by a grant from the Brazilian National Research Council (CNPq; 476772/2008-7) to TCS. MSS acknowledges support from the European Research Council through ERC - 306990. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Hochman Méndez, C.; Cantini ., M.; Moratal Pérez, D.; Salmerón Sánchez, M.; Coelho-Sampaio, T. (2014). A fractal nature for polymerized laminin. PLoS ONE. 9(10):109388-1-109388-11. https://doi.org/10.1371/journal.pone.0109388S109388-1109388-11910Durbeej, M. (2009). Laminins. Cell and Tissue Research, 339(1), 259-268. doi:10.1007/s00441-009-0838-2Miner, J. H., & Yurchenco, P. D. (2004). LAMININ FUNCTIONS IN TISSUE MORPHOGENESIS. Annual Review of Cell and Developmental Biology, 20(1), 255-284. doi:10.1146/annurev.cellbio.20.010403.094555Yurchenco, P. D. (2010). Basement Membranes: Cell Scaffoldings and Signaling Platforms. Cold Spring Harbor Perspectives in Biology, 3(2), a004911-a004911. doi:10.1101/cshperspect.a004911Hohenester, E., & Yurchenco, P. D. (2013). Laminins in basement membrane assembly. Cell Adhesion & Migration, 7(1), 56-63. doi:10.4161/cam.21831Freire, E., & Coelho-Sampaio, T. (2000). Self-assembly of Laminin Induced by Acidic pH. Journal of Biological Chemistry, 275(2), 817-822. doi:10.1074/jbc.275.2.817Freire, E., Sant’Ana Barroso, M. M., Klier, R. N., & Coelho-Sampaio, T. (2011). Biocompatibility and Structural Stability of a Laminin Biopolymer. Macromolecular Bioscience, 12(1), 67-74. doi:10.1002/mabi.201100125Freire, E. (2002). Structure of laminin substrate modulates cellular signaling for neuritogenesis. Journal of Cell Science, 115(24), 4867-4876. doi:10.1242/jcs.00173Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., & Coelho-Sampaio, T. (2013). Polylaminin recognition by retinal cells. Journal of Neuroscience Research, 92(1), 24-34. doi:10.1002/jnr.23298Menezes, K., Ricardo Lacerda de Menezes, J., Assis Nascimento, M., de Siqueira Santos, R., & Coelho-Sampaio, T. (2010). Polylaminin, a polymeric form of laminin, promotes regeneration after spinal cord injury. The FASEB Journal, 24(11), 4513-4522. doi:10.1096/fj.10-157628Barroso, M. M. S., Freire, E., Limaverde, G. S. C. S., Rocha, G. M., Batista, E. J. O., Weissmüller, G., … Coelho-Sampaio, T. (2008). Artificial Laminin Polymers Assembled in Acidic pH Mimic Basement Membrane Organization. Journal of Biological Chemistry, 283(17), 11714-11720. doi:10.1074/jbc.m709301200Freire, E. (2004). Sialic acid residues on astrocytes regulate neuritogenesis by controlling the assembly of laminin matrices. Journal of Cell Science, 117(18), 4067-4076. doi:10.1242/jcs.01276Hausdorff, F. (1918). Dimension und �u�eres Ma�. Mathematische Annalen, 79(1-2), 157-179. doi:10.1007/bf01457179Soille, P., & Rivest, J.-F. (1996). On the Validity of Fractal Dimension Measurements in Image Analysis. Journal of Visual Communication and Image Representation, 7(3), 217-229. doi:10.1006/jvci.1996.0020Theiler, J. (1990). Estimating fractal dimension. Journal of the Optical Society of America A, 7(6), 1055. doi:10.1364/josaa.7.001055Otsu, N. (1979). A Threshold Selection Method from Gray-Level Histograms. IEEE Transactions on Systems, Man, and Cybernetics, 9(1), 62-66. doi:10.1109/tsmc.1979.4310076Iranfar, H., Rajabi, O., Salari, R., & Chamani, J. (2012). Probing the Interaction of Human Serum Albumin with Ciprofloxacin in the Presence of Silver Nanoparticles of Three Sizes: Multispectroscopic and ζ Potential Investigation. The Journal of Physical Chemistry B, 116(6), 1951-1964. doi:10.1021/jp210685qPalmero, C. Y., Miranda-Alves, L., Sant’Ana Barroso, M. M., Souza, E. C. L., Machado, D. E., Palumbo-Junior, A., … Nasciutti, L. E. (2013). The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH. Molecular and Cellular Endocrinology, 376(1-2), 12-22. doi:10.1016/j.mce.2013.05.020Behrens, D. T., Villone, D., Koch, M., Brunner, G., Sorokin, L., Robenek, H., … Hansen, U. (2012). The Epidermal Basement Membrane Is a Composite of Separate Laminin- or Collagen IV-containing Networks Connected by Aggregated Perlecan, but Not by Nidogens. Journal of Biological Chemistry, 287(22), 18700-18709. doi:10.1074/jbc.m111.336073Colognato, H., Winkelmann, D. A., & Yurchenco, P. D. (1999). Laminin Polymerization Induces a Receptor–Cytoskeleton Network. The Journal of Cell Biology, 145(3), 619-631. doi:10.1083/jcb.145.3.619Liesi, P., & Silver, J. (1988). Is astrocyte laminin involved in axon guidance in the mammalian CNS? Developmental Biology, 130(2), 774-785. doi:10.1016/0012-1606(88)90366-1Zhou, F. C. (1990). Four patterns of laminin-immunoreactive structure in developing rat brain. Developmental Brain Research, 55(2), 191-201. doi:10.1016/0165-3806(90)90200-iGarcia-Abreu, J., Cavalcante, L. A., & Neto, V. M. (1995). Differential patterns of laminin expression in lateral and medial midbrain glia. NeuroReport, 6(5), 761-764. doi:10.1097/00001756-199503270-00014Kazanis, I., & ffrench-Constant, C. (2011). Extracellular matrix and the neural stem cell niche. Developmental Neurobiology, 71(11), 1006-1017. doi:10.1002/dneu.20970Mercier F, Schnack J, Chaumet MSG (2011) Chapter 4 Fractones: home and conductors of the neural stem cell niche. In: Seki, T., Sawamoto, K., Parent, J. M., Alvarez-Buylla, A., (Eds.) Neurogenesis in the adult brain I: neurobiology. Springer. pp 109–133.CAVALCANTIADAM, E., MICOULET, A., BLUMMEL, J., AUERNHEIMER, J., KESSLER, H., & SPATZ, J. (2006). Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly. European Journal of Cell Biology, 85(3-4), 219-224. doi:10.1016/j.ejcb.2005.09.011Frith, J. E., Mills, R. J., & Cooper-White, J. J. (2012). Lateral spacing of adhesion peptides influences human mesenchymal stem cell behaviour. Journal of Cell Science, 125(2), 317-327. doi:10.1242/jcs.087916Hernández, J. C. R., Salmerón Sánchez, M., Soria, J. M., Gómez Ribelles, J. L., & Monleón Pradas, M. (2007). Substrate Chemistry-Dependent Conformations of Single Laminin Molecules on Polymer Surfaces are Revealed by the Phase Signal of Atomic Force Microscopy. Biophysical Journal, 93(1), 202-207. doi:10.1529/biophysj.106.102491Douet, V., Kerever, A., Arikawa-Hirasawa, E., & Mercier, F. (2013). Fractone-heparan sulphates mediate FGF-2 stimulation of cell proliferation in the adult subventricular zone. Cell Proliferation, 46(2), 137-145. doi:10.1111/cpr.12023Nikolova, G., Strilic, B., & Lammert, E. (2007). The vascular niche and its basement membrane. Trends in Cell Biology, 17(1), 19-25. doi:10.1016/j.tcb.2006.11.005Yurchenco, P. D., Amenta, P. S., & Patton, B. L. (2004). Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biology, 22(7), 521-538. doi:10.1016/j.matbio.2003.10.006Nikolova, G., Jabs, N., Konstantinova, I., Domogatskaya, A., Tryggvason, K., Sorokin, L., … Lammert, E. (2006). The Vascular Basement Membrane: A Niche for Insulin Gene Expression and β Cell Proliferation. Developmental Cell, 10(3), 397-405. doi:10.1016/j.devcel.2006.01.015Qu, H., Liu, X., Ni, Y., Jiang, Y., Feng, X., Xiao, J., … Zheng, C. (2014). Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells. Journal of Translational Medicine, 12(1), 135. doi:10.1186/1479-5876-12-135Kanatsu-Shinohara, M., & Shinohara, T. (2013). Spermatogonial Stem Cell Self-Renewal and Development. Annual Review of Cell and Developmental Biology, 29(1), 163-187. doi:10.1146/annurev-cellbio-101512-122353Lander, A. D., Kimble, J., Clevers, H., Fuchs, E., Montarras, D., Buckingham, M., … Oskarsson, T. (2012). What does the concept of the stem cell niche really mean today? BMC Biology, 10(1). doi:10.1186/1741-7007-10-19Loulier, K., Lathia, J. D., Marthiens, V., Relucio, J., Mughal, M. R., Tang, S.-C., … ffrench-Constant, C. (2009). β1 Integrin Maintains Integrity of the Embryonic Neocortical Stem Cell Niche. PLoS Biology, 7(8), e1000176. doi:10.1371/journal.pbio.100017

Probing tRNA interaction with biogenic polyamines

Biogenic polyamines are found to modulate protein synthesis at different levels. This effect may be explained by the ability of polyamines to bind and influence the secondary structure of tRNA, mRNA, and rRNA. We report the interaction between tRNA and the three biogenic polyamines putrescine, spermidine, spermine, and cobalt(III)hexamine at physiological conditions, using FTIR spectroscopy, capillary electrophoresis, and molecular modeling. The results indicated that tRNA was stabilized at low biogenic polyamine concentration, as a consequence of polyamine interaction with the backbone phosphate group. The main tRNA reactive sites for biogenic polyamine at low concentration were guanine-N7/O6, uracil-O2/O4, adenine-N3, and 2′OH of the ribose. At high polyamine concentration, the interaction involves guanine-N7/O6, adenine-N7, uracil-O2 reactive sites, and the backbone phosphate group. The participation of the polycation primary amino group, in the interaction and the presence of the hydrophobic contact, are also shown. The binding affinity of biogenic polyamine to tRNA molecule was in the order of spermine > spermidine > putrescine with KSpm = 8.7 × 105 M−1, KSpd = 6.1 × 105 M−1, and KPut = 1.0 × 105 M−1, which correlates with their positively charged amino group content. Hill analysis showed positive cooperativity for the biogenic polyamines and negative cooperativity for cobalt-hexamine. Cobalt(III)hexamine contains high- and low-affinity sites in tRNA with K1 = 3.2 × 105 M−1 and K2 = 1.7 × 105 M−1, that have been attributed to the interactions with guanine-N7 sites and the backbone PO2 group, respectively. This mechanism of tRNA binding could explain the condensation phenomenon observed at high Co(III) content, as previously shown in the Co(III)–DNA complexes

Transporting antitumor drug tamoxifen and its metabolites, 4-hydroxytamoxifen and endoxifen by chitosan nanoparticles.

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K(tam-ch-15) = 8.7 ( ± 0.5) × 10(3) M(-1), K(tam-ch-100) = 5.9 (± 0.4) × 10(5) M(-1), K(tam-ch-200) = 2.4 (± 0.4) × 10(5) M(-1) and K(hydroxytam-ch-15) = 2.6(± 0.3) × 10(4) M(-1), K(hydroxytam - ch-100) = 5.2 ( ± 0.7) × 10(6) M(-1) and K(hydroxytam-ch-200) = 5.1 (± 0.5) × 10(5) M(-1), K(endox-ch-15) = 4.1 (± 0.4) × 10(3) M(-1), K(endox-ch-100) = 1.2 (± 0.3) × 10(6) M(-1) and K(endox-ch-200) = 4.7 (± 0.5) × 10(5) M(-1) with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH2 residues with drug OH and NH2 groups in the drug-polymer adducts. The free binding energies of -3.46 kcal/mol for tamoxifen, -3.54 kcal/mol for 4-hydroxytamoxifen and -3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD