Principal component analysis and artificial neural network analysis of oral tissue fluorescence spectra: classification of normal premalignant and malignant pathological conditions

Pulsed laser-induced autofluorescence spectroscopic studies of pathologically certified normal, premalignant, and malignant oral tissues were carried out at 325 nm excitation. The spectral analysis and classification for discrimination among normal, premalignant, and malignant conditions were performed using principal component analysis (PCA) and artificial neural network (ANN) separately on the same set of spectral data. In case of PCA, spectral residuals, Mahalanobis distance, and scores of factors were used for discrimination among normal, premalignant, and malignant cases. In ANN, parameters like mean, spectral residual, standard deviation, and total energy were used to train the network. The ANN used in this study is a classical multiplayer feed-forward type with a back-propagation algorithm for the training of the network. The specificity and sensitivity were determined in both classification schemes. In the case of PCA, they are 100 and 92.9%, respectively, whereas for ANN they are 100 and 96.5% for the data set considered.     

One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.     

Causes of fluorescence in bidistilled water after electrochemical treatment

Fluorescence spectroscopy was used to study the shortwave fluorescence of bidistilled water treated in the cathode and anode chambers of two types of electrolyzers made of different materials. Electrochemical treatment in the quartz glass electrolyzer did not induce intrinsic fluorescence of the anolyte or catholyte. An increase in the shortwave fluorescence of the anolyte and catholyte was observed in the electrolyzer made of Plexiglass, which was probably due to the release of microcontaminants from components of the electrolyzer.     

Ordering in a glycine-rich peptide conjugate: microscopic, fluorescence, and metalation studies

Glycine residues play an intriguing role in peptide/protein structure where they can act as tightly packing amino acids with flexible bond angles. For example, structural role of glycines is highlighted in natural silk fibers where different structural polymorphs have been reported. This study deals with a glycine-rich segment from the conserved octarepeat (PHGGGWGQ) in prion protein. We have synthesized a bis-conjugate 3, containing a truncated pentapeptide segment (GGGWG), to study its time-dependent solution phase aggregation by a combination of microscopic methods and fluorescence. This discontinuous peptide conjugate 3 exhibited interesting photophysical properties upon self-assembly allowing us to propose a possible model of peptide filament formation. Taking note of the fact that prion octarepeats bind copper, we also demonstrate the ability of this conjugate to bind copper and the growth and ultrastructure of metallized fibers formed upon incubation. Enforcing peptide fiber formation in metal binding motifs offers an entry into metal impregnated fibers for possible nanobiotechnological applications.     

Design and performance of a 24-station high throughput microbioreactor

Two prototype 24-unit microbioreactors are presented and reviewed for their relative merits. The first used a standard 24-well plate as the template, while the second consisted of 24-discrete units. Both systems used non-invasive optical sensors to monitor pH and dissolved oxygen. The systems were used to cultivate Escherichia coli. Both designs had their merits and the results obtained are presented. In addition, dissolved oxygen control was demonstrated at the milliliter scale and 24 simultaneously monitored fermentations were successfully carried out. These results demonstrated high quality high throughput bioprocessing and provide important insights into operational parameters at small scale.     

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.     

Toxoplasma gondii: comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7x10(7) viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10=2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as "gold standard" and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISAxIFAT (kappa = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 +/- 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs.     

Differences in the regulation of microtubule stability by the pro-rich region variants of microtubule-associated protein 4

We have recently reported a neural variant of microtubule-associated protein 4 with a short pro-rich region (MAP4-SP). Here, we show that the neural MAP4 has reduced microtubule-stabilizing activity, compared to the ubiquitous MAP4 with a long pro-rich region (MAP4-LP), both in vitro and in vivo. Fluorescence recovery after photobleaching analyses revealed that the interaction of MAP4-SP with the microtubules is very rapid, with a half-time of fluorescence recovery of 7 +/- 2.36 s, compared to 19.5 +/- 3.03 s in case of MAP4-LP. The dynamic interaction of MAP4-SP with microtubules in neural cells may contribute to the dynamic behaviors of extending neurites.     

Molecular dynamics study of major urinary protein-pheromone interactions: a structural model for ligand-induced flexibility increase

Recently, two independent (15)N NMR relaxation studies indicated that in contrast to the decreased flexibility expected for induced-fit interactions, the backbone flexibility of major urinary protein isoform I (MUP-I) slightly increased upon complex formation with its natural pheromone 2-sec-butyl-4,5-dihydrothiazol. We have investigated the subtle details of molecular interactions by molecular dynamics simulations in explicit solvent. The calculated order parameters S(2) for a free- and ligand-bound protein supply evidence that mobility in various regions of MUP-I can be directly related to small conformational changes of the free- and complexed protein resulting from modifications of the hydrogen bonding network.     

Nuclear envelope localization of human UNC84A does not require nuclear lamins

The SUN proteins are a conserved family of proteins in eukaryotes. Human UNC84A (Sun1) is a homolog of Caenorhabditis elegans UNC-84, a protein involved in nuclear anchorage and migration. We have analyzed targeting of UNC84A to the nuclear envelope (NE) and show that the N-terminal 300 amino acids are crucial for efficient NE localization of UNC84A whereas the conserved C-terminal SUN domain is not required. Furthermore, we demonstrate by combining RNA interference with immunofluorescence and fluorescence recovery after photobleaching analysis that localization and anchoring of UNC84A is not dependent on the lamin proteins, in contrast to what had been observed for C. elegans UNC-84.