Population structure of invasive Streptococcus pneumoniae in The Netherlands in the pre-vaccination era assessed by MLVA and capsular sequence typing

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA).To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in The Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination.     

Targeting human glioblastoma cells: comparison of nine viruses with oncolytic potential

Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma.     

Analysis of ionizing radiation-induced foci of DNA damage repair proteins

Repair of DNA double-strand breaks by homologous recombination requires an extensive set of proteins. Among these proteins are Rad51 and Mre11, which are known to re-localize to sites of DNA damage into nuclear foci. Ionizing radiation-induced foci can be visualized by immuno-staining. Published data show a large variation in the number of foci-positive cells and number of foci per nucleus for specific DNA repair proteins. The experiments described here demonstrate that the time after induction of DNA damage influenced not only the number of foci-positive cells, but also the size of the individual foci. The dose of ionizing radiation influenced both the number of foci-positive cells and the number of foci per nucleus. Furthermore, ionizing radiation-induced foci formation depended on the cell cycle stage of the cells and the protein of interest that was investigated. Rad51 and Mre11 foci seemed to be mutually exclusive, though a small subset of cells did show co-localization of these proteins, which suggests a possible cooperation between the proteins at a specific moment during DNA repair.     

Dynamics of relative chromosome position during the cell cycle

The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photobleaching of GFP-labeled histone H2B, local UV-marked DNA, and photobleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers, suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods.     

Modelling viral impact on bacterioplankton in the North Sea using artificial neural networks

The temporal variability of the viral impact on bacterioplankton during the summer-winter transition in the North Sea was determined and artificial neural networks (ANNs) were developed to predict viral production and the frequency of infected bacterial cells (FIC). Viral production and FIC were estimated using a virus-dilution approach during four cruises in the southern North Sea between July and December 2000 and an additional cruise in June 2001. Supplementary data such as bacterial production, and bacterial and viral abundance were collected to relate changes in FIC and viral production to the dynamics of other biotic parameters. Average viral abundance varied between 4.4 x 10(6) ml(-1) in December and 29.8 x 10(6) ml(-1) in July. Over the seasonal cycle, viral abundance correlated best with bacterial production. Average bacterial abundance varied between 0.5 x 10(6) ml(-1) in December and 1.3 x 10(6) ml(-1) in July. Monthly average values of FIC ranged from 9% in September to 39% in June and the average viral production from 11 x 10(4) ml(-1) h(-1) in December to 35 x 10(4) ml(-1) h(-1) in July. The data set was used to develop ANN-based models of viral production and FIC. Viral production was modelled best using sampling time, and bacterial and viral abundance as input parameters to an ANN with two hidden neurons. Modelling of FIC was performed using bacterial production as an additional input parameter for an ANN with three hidden neurons. The models can be used to simulate viral production and FIC based on regularly recorded and easily obtainable parameters such as bacterial production, bacterial and viral abundance.     

Platelet factor 4 and interleukin-8 CXC chemokine heterodimer formation modulates function at the quaternary structural level

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.     

Spectroscopic properties of the carotenoid 3'-hydroxyechinenone in the orange carotenoid protein from the cyanobacterium Arthrospira maxima

The cyanobacterial water-soluble orange carotenoid binding protein (OCP) is an ideal system for study of the effects of protein environment on photophysical properties of carotenoids. It contains a single pigment, the carotenoid 3'-hydoxyechinenone (hECN). In this study, we focus on spectroscopic properties of hECN in solution and in the OCP, aiming to elucidate the spectroscopic effects of the carotenoid-protein interaction in the context of the function(s) of the OCP. The noncovalent binding of hECN to the OCP causes a conformational change in the hECN, leading to a prolongation of the effective conjugation length. This change is responsible for shortening of the S(1) lifetime from 6.5 ps in solution to 3.3 ps in the OCP. The conformational change and the hydrogen bonding via the carbonyl group of hECN result in stabilization of an intramolecular charge-transfer (ICT) state. No signs of the ICT state were found in hECN in solution, regardless of the solvent polarity; spectral bands in transient absorption spectra of OCP-bound hECN exhibit features typical for the ICT state. Application of global fitting analysis revealed further effects of binding hECN in the OCP. The S(1) state of hECN in the OCP decays with two time constants of 0.9 and 3.3 ps. Modeling of the excited-state processes suggests that these two components are due to two populations of hECN in the OCP that differ in the hydrogen bonding via the carbonyl group. These results support the hypothesis that the OCP functions as a photoprotective shield under excess light. Mechanistically, the broadening of the hECN absorption spectrum upon binding to OCP enhances filtering effect of hECN. Furthermore, the binding-induced conformational change and activation of the ICT state that leads to a shortening of hECN lifetime effectively makes the protein-bound hECN a more effective energy dissipator.     

The unfolding action of GroEL on a protein substrate

A molecular dynamics simulation of the active unfolding of denatured rhodanese by the chaperone GroEL is presented. The compact denatured protein is bound initially to the cis cavity and forms stable contacts with several of the subunits. As the cis ring apical domains of GroEL undergo the transition from the closed to the more open (ATP-bound) state, they exert a force on rhodanese that leads to the increased unfolding of certain loops. The contacts between GroEL and rhodanese are analyzed and their variation during the GroEL transition is shown. The major contacts, which give rise to the stretching force, are found to be similar to those observed in crystal structures of peptides bound to the apical domains. The results of the simulation show that multidomain interactions play an essential role, in accord with experiments. Implications of the results for mutation experiments and for the action of GroEL are discussed.     

ZNF674: a new kruppel-associated box-containing zinc-finger gene involved in nonsyndromic X-linked mental retardation

Array-based comparative genomic hybridization has proven to be successful in the identification of genetic defects in disorders involving mental retardation. Here, we studied a patient with learning disabilities, retinal dystrophy, and short stature. The family history was suggestive of an X-linked contiguous gene syndrome. Hybridization of full-coverage X-chromosomal bacterial artificial chromosome arrays revealed a deletion of ~1 Mb in Xp11.3, which harbors RP2, SLC9A7, CHST7, and two hypothetical zinc-finger genes, ZNF673 and ZNF674. These genes were analyzed in 28 families with nonsyndromic X-linked mental retardation (XLMR) that show linkage to Xp11.3; the analysis revealed a nonsense mutation, p.E118X, in the coding sequence of ZNF674 in one family. This mutation is predicted to result in a truncated protein containing the Kruppel-associated box domains but lacking the zinc-finger domains, which are crucial for DNA binding. We characterized the complete ZNF674 gene structure and subsequently tested an additional 306 patients with XLMR for mutations by direct sequencing. Two amino acid substitutions, p.T343M and p.P412L, were identified that were not found in unaffected individuals. The proline at position 412 is conserved between species and is predicted by molecular modeling to reduce the DNA-binding properties of ZNF674. The p.T343M transition is probably a polymorphism, because the homologous ZNF674 gene in chimpanzee has a methionine at that position. ZNF674 belongs to a cluster of seven highly related zinc-finger genes in Xp11, two of which (ZNF41 and ZNF81) were implicated previously in XLMR. Identification of ZNF674 as the third XLMR gene in this cluster may indicate a common role for these zinc-finger genes that is crucial to human cognitive functioning.