Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Various approaches to the search for the producers of bacteriocin-like substances among bacteria of the genus Pseudomonas

Some methods providing rapid and methodically simple study of large microbial collections and detection of cultures producing bacteriocin-like substances perspective for further investigation are described. The use of cellophane disks and study of the proteolytic enzyme effects allowed the authors to detect organisms producing high molecular antibiotics of protein nature.     

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 μmol CH₄ g⁻¹ oil d⁻¹, orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC–MS and FTICR–MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.     

Mapping the Genes for Susceptibility and Response to Leishmania tropica in Mouse

Background

L. tropica can cause both cutaneous and visceral leishmaniasis in humans. Although the L. tropica-induced cutaneous disease has been long known, its potential to visceralize in humans was recognized only recently. As nothing is known about the genetics of host responses to this infection and their clinical impact, we developed an informative animal model. We described previously that the recombinant congenic strain CcS-16 carrying 12.5% genes from the resistant parental strain STS/A and 87.5% genes from the susceptible strain BALB/c is more susceptible to L. tropica than BALB/c. We used these strains to map and functionally characterize the gene-loci regulating the immune responses and pathology.

Methods

We analyzed genetics of response to L. tropica in infected F₂ hybrids between BALB/c×CcS-16. CcS-16 strain carries STS-derived segments on nine chromosomes. We genotyped these segments in the F₂ hybrid mice and tested their linkage with pathological changes and systemic immune responses.

Principal Findings

We mapped 8 Ltr (Leishmania tropica response) loci. Four loci (Ltr2, Ltr3, Ltr6 and Ltr8) exhibit independent responses to L. tropica, while Ltr1, Ltr4, Ltr5 and Ltr7 were detected only in gene-gene interactions with other Ltr loci. Ltr3 exhibits the recently discovered phenomenon of transgenerational parental effect on parasite numbers in spleen. The most precise mapping (4.07 Mb) was achieved for Ltr1 (chr.2), which controls parasite numbers in lymph nodes. Five Ltr loci co-localize with loci controlling susceptibility to L. major, three are likely L. tropica specific. Individual Ltr loci affect different subsets of responses, exhibit organ specific effects and a separate control of parasite load and organ pathology.

Conclusion

We present the first identification of genetic loci controlling susceptibility to L. tropica. The different combinations of alleles controlling various symptoms of the disease likely co-determine different manifestations of disease induced by the same pathogen in individual mice.     

A Complex Interplay between Personality Domains,Marital Status and a Variant in CHRNA5 on the Risks of Cocaine,Nicotine Dependences and Cocaine-Induced Paranoia

Background

Personality correlates highly with both cocaine and nicotine dependencies (CD, ND), and their co-morbid psychopathologies. However, little is known about the nature of these relationships. This study examined if environment (marriage) or genetics (a single SNP, CHRNA5*rs16969968) would moderate the correlation of personality with CD, ND and cocaine-induced paranoia (CIP) in African and European Americans (AAs, EAs).

Methods

1432 EAs and 1513 AAs were examined using logistic regression. Personality was assessed by NEO-PI-R, while CD, ND and CIP were diagnosed according to DSM-IV. ND and CD were examined as binary traits and for the analysis of CIP, subjects were divided into 3 groups: (A) Controls with no CIP; (B) CD cases without CIP; and (C) CD cases with CIP. Multiple testing was Bonferroni-corrected.

Results

For CD and ND in the EA population, marital status proved to be a significant moderator in their relationship with openness only (OR = 1.90, 95%CI = 1.36–2.64, p = 1.54e-04 and OR = 2.12, 95%CI = 1.52–2.90, p = 4.65e-06 respectively). For CIP, marriage was observed to moderate its correlation with openness and neuroticism (OR = 1.39, 95%CI = 1.18–1.63, p = 7.64e-04 and OR = 1.26, 95%CI = 1.12–1.42, p = 1.27e-03 respectively). The correlations moderated by rs16969968 were those of conscientiousness and CD (OR = 1.62, 95%CI: 1.23–2.12, p = 8.94e-04) as well as CIP (OR = 1.21, 95%CI: 1.11–1.32, p = 4.93e-04 when comparing group A versus group C). No significant interactions were observed in AA population. The Bonferroni-corrected significance threshold was set to be 1.67e-03.

Conclusion

The role of personality in CD and CIP may be interceded by both environment and genetics, while in ND by environment only.     

Processes of plant colonization by Methylobacterium strains and some bacterial properties

The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacterium are indespensible inhabitants of the plant phyllosphere. Using maize Zea mays as a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicum APR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicum APR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicum cells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicum or M. extorquens did exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas camprestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense, and Agrobacterium tumifaciens). Neither epiphytic and soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, and M. fujisawaense catalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacterium can protect plants against adverse environmental factors.     

In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.     

Structural studies of tri-functional human GART

Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.     

Recovery of tubulin functions after freeze-drying in the presence of trehalose

Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at −80 °C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assembly-competent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.