Oxidative DNA damage in reconstituting T cells is associated with relapse and inferior survival after allo-SCT.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the only curative treatment option for a number of hematologic malignancies. Its therapeutic potential relies on the potency of donor T cells to eliminate residual malignant cells, the so-called graft-versus-leukemia (GVL) effect. Disease relapse remains the most frequent treatment failure and is associated with poor outcome. Therefore, it is inevitable to decipher mechanisms that weaken GVL. In recent years, studies of tumor biology have revealed that metabolic remodeling of the micromilieu can critically regulate immune responses. Accumulation of reactive oxygen species leads to a metabolic condition known as oxidative stress, which can severely hamper T cells. Currently, only a few studies, mainly using preclinical models, have demonstrated the occurrence of oxidative stress after allo-SCTs. Therefore, we sought to investigate oxidative stress in a well-characterized group of patients who underwent allo-SCT and its impact on reconstituting T cells. We identified high concentrations of serum 8-hydroxydeoxyguanosine (8-OHdG) as an established biomarker for oxidative stress. 8-OHdG is one of the major products of DNA oxidation, which is normally rapidly removed. After allo-SCT, T cells accumulated oxidative DNA damage. High cellular 8-OHdG content (8-OHdGhi) was associated not only with signs of enhanced T-cell activation but also premature exhaustion. The inability of 8-OHdGhi T cells to efficiently target malignant cells or produce cytotoxic granzyme B and interferon gamma was associated with a significantly increased relapse risk and a shorter overall survival. Taken together, our novel findings could give reason to focus on bolstering DNA repair in reconstituting T cells as a means to improve GVL efficacy.

Sexual dimorphism of AMBRA1-related autistic features in human and mouse.

Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1+/- mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation-inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.

Frequency of regulatory T cells determines the outcome of the T-cell-engaging antibody blinatumomab in patients with B-precursor ALL.

Blinatumomab can induce a complete haematological remission in patients in 46.6% with relapsed/refractory B-precursor acute lymphoblastic leukemia (r/r ALL) resulting in a survival benefit when compared with chemotherapy. Only bone marrow blast counts before therapy have shown a weak prediction of response. Here we investigated the role of regulatory T cells (Tregs), measured by CD4/CD25/FOXP3 expression, in predicting the outcome of immunotherapy with the CD19-directed bispecific T-cell engager construct blinatumomab. Blinatumomab responders (n=22) had an average of 4.82% Tregs (confidence interval (CI): 1.79-8.34%) in the peripheral blood, whereas non-responders (n=20) demonstrated 10.25% Tregs (CI: 3.36-65.9%). All other tested markers showed either no prediction value or an inferior prediction level including blast BM counts and the classical enzyme marker lactate dehydrogenase. With a cutoff of 8.525%, Treg enumeration can identify 100% of all blinatumomab responders and exclude 70% of the non-responders. The effect is facilitated by blinatumomab-activated Tregs, leading to interleukin-10 production, resulting in suppression of T-cell proliferation and reduced CD8-mediated lysis of ALL cells. Proliferation of patients' T cells can be restored by upfront removal of Tregs. Thus, enumeration of Treg identifies r/r ALL patients with a high response rate to blinatumomab. Therapeutic removal of Tregs may convert blinatumomab non-responders to responders.

Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus.

Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a 15N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated 15N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.

Analyzing Thiol-Dependent Redox Networks in the Presence of Methylene Blue and Other Antimalarial Agents with RT-PCR-Supported in silico Modeling.

BACKGROUND: In the face of growing resistance in malaria parasites to drugs, pharmacological combination therapies are important. There is accumulating evidence that methylene blue (MB) is an effective drug against malaria. Here we explore the biological effects of both MB alone and in combination therapy using modeling and experimental data. RESULTS: We built a model of the central metabolic pathways in P. falciparum. Metabolic flux modes and their changes under MB were calculated by integrating experimental data (RT-PCR data on mRNAs for redox enzymes) as constraints and results from the YANA software package for metabolic pathway calculations. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, chloroquine resistance based on pfmdr/and pfcrt transporters, as well as pyrimethamine/sulfadoxine resistance (by mutations in DHF/DHPS), were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs. CONCLUSIONS: Theoretical and experimental results support that methylene blue should, because of its resistance-breaking potential, be further tested as a key component in drug combination therapy efforts in holoendemic areas.

G244E in the canine factor IX gene leads to severe haemophilia B in Rhodesian Ridgebacks.

Haemophilia B in Rhodesian Ridgebacks is currently the most important canine haemophilia in Germany. The aim of this study was to define the underlying genetic defect. Genetic studies were performed including six phenotypically affected male dogs (factor IX activity: approximately 1%), four suspected carriers (factor IX activity 48-69%, one confirmed by affected offspring), and 12 healthy dogs. Comparison of the entire coding region of the canine factor IX DNA sequences and exon-intron junctions from affected dogs with the wild type canine factor IX DNA revealed a G-A missense mutation in exon 7. This mutation results in a glycine (GGA) to glutamic acid (GAA) exchange in the catalytic domain of the haemophilic factor IX. All affected dogs were hemizygous for the detected mutation and carriers were heterozygous, whereas none of the Rhodesian Ridgebacks with normal factor IX activity showed the mutation. No further alterations in the sequences between affected dogs and the healthy control group could be observed. None of the Rhodesian Ridgebacks with undefined haemophilia B status (n=30) and no individual of three other dog breeds (Doberman Pinscher: n=20; German Wire haired Pointer: n=20; Labrador: n=25) showed the presence of the mutation. Amino acid sequence alignment and protein structural modelling analysis indicate that the detected mutation causes a relevant functional defect. The results of this study suggest that the detected mutation is responsible for a severe form of haemophilia B in Rhodesian Ridgebacks.

Germinal center B cell-like (GCB) and activated B cell-like (ABC) type of diffuse large B cell lymphoma (DLBCL): analysis of molecular predictors, signatures, cell cycle state and patient survival.

Aiming to find key genes and events, we analyze a large data set on diffuse large B-cell lymphoma (DLBCL) gene-expression (248 patients, 12196 spots). Applying the loess normalization method on these raw data yields improved survival predictions, in particular for the clinical important group of patients with medium survival time. Furthermore, we identify a simplified prognosis predictor, which stratifies different risk groups similarly well as complex signatures. We identify specific, activated B cell-like (ABC) and germinal center B cell-like (GCB) distinguishing genes. These include early (e.g. CDKN3) and late (e.g. CDKN2C) cell cycle genes. Independently from previous classification by marker genes we confirm a clear binary class distinction between the ABC and GCB subgroups. An earlier suggested third entity is not supported. A key regulatory network, distinguishing marked over-expression in ABC from that in GCB, is built by: ASB13, BCL2, BCL6, BCL7A, CCND2, COL3A1, CTGF, FN1, FOXP1, IGHM, IRF4, LMO2, LRMP, MAPK10, MME, MYBL1, NEIL1 and SH3BP5. It predicts and supports the aggressive behaviour of the ABC subgroup. These results help to understand target interactions, improve subgroup diagnosis, risk prognosis as well as therapy in the ABC and GCB DLBCL subgroups.

Exploring the pathway structure of metabolism: decomposition into subnetworks and application to Mycoplasma pneumoniae.

MOTIVATION: Reconstructing and analyzing the metabolic map of microorganisms is an important challenge in bioinformatics. Pathway analysis of large metabolic networks meets with the problem of combinatorial explosion of pathways. Therefore, appropriate algorithms for an automated decomposition of these networks into smaller subsystems are needed. RESULTS: A decomposition algorithm for metabolic networks based on the local connectivity of metabolites is presented. Interrelations of this algorithm with alternative methods proposed in the literature and the theory of small world networks are discussed. The applicability of our method is illustrated by an analysis of the metabolism of Mycoplasma pneumoniae, which is an organism of considerable medical interest. The decomposition gives rise to 19 subnetworks. Three of these are here discussed in biochemical terms: arginine degradation, the tetrahydrofolate system, and nucleotide metabolism. The interrelations of pathway analysis of biochemical networks with Petri net theory are outlined.

When genetic distance matters: measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species.

Genetic distance measurements are an important tool to differentiate field populations of disease vectors such as the mosquito vectors of malaria. Here, we have measured the genetic differentiation between Anopheles arabiensis and Anopheles gambiae, as well as between proposed emerging species of the latter taxon, in whole genome scans by using 23-25 microsatellite loci. In doing so, we have reviewed and evaluated the advantages and disadvantages of standard parameters of genetic distance, F(ST), R(ST), (delta mu)(2), and D. Further, we have introduced new parameters, D' and D(K), which have well defined statistical significance tests and complement the standard parameters to advantage. D' is a modification of D, whereas D(K) is a measure of covariance based on Pearson's correlation coefficient. We find that A. gambiae and A. arabiensis are closely related at most autosomal loci but appear to be distantly related on the basis of X-linked chromosomal loci within the chromosomal Xag inversion. The M and S molecular forms of A. gambiae are practically indistinguishable but differ significantly at two microsatellite loci from the proximal region of the X, outside the Xag inversion. At one of these loci, both M and S molecular forms differ significantly from A. arabiensis, but remarkably, at the other locus, A. arabiensis is indistinguishable from the M molecular form of A. gambiae. These data support the recent proposal of genetically differentiated M and S molecular forms of A. gambiae.

Solvent entropy-driven searching for protein modeling examined and tested in simplified models.

Solvent entropy is a force to consider in protein folding and protein design but is difficult to model. It is investigated here in the context of the hp model: Two types of residues, hydrophobic and hydrophilic, are modeled on a lattice. Nine chains and two- and three-dimensional simulations are compared. We show that considering solvent entropy alone, efficient folding of lattice chains (identification of the native fold) can be achieved by an entropy-driven simulation on its own. Moreover, in a detailed comparison over a wide range of parameters, entropy-guided searching outperforms an energy-driven search in the model. The combination of energy- and entropy-driven search yields the most efficient searching. It is compared in detail with the above results, indicating also how this solvent shell model may advantageously be implemented in more complex protein modeling simulations.

Structure--function characterization of cellulose synthase: relationship to other glycosyltransferases.

A combined structural and functional model of the catalytic region of cellulose synthase is presented as a prototype for the action of processive beta-glycosyltransferases and other glycosyltransferases. A 285 amino acid segment of the Acetobacter xylinum cellulose synthase containing all the conserved residues in the globular region was subjected to protein modeling using the genetic algorithm. This region folds into a single large domain with a topology exhibiting a mixed alpha/beta structure. The predicted structure serves as a topological outline for the structure of this processive beta-glycosyltransferase. By incorporating new site-directed mutagenesis data and comparative analysis of the conserved aspartic acid residues and the QXXRW motif we deduce a number of functional implications based on the structure. This includes location of the UDP--glucose substrate-binding cavity, suggestions for the catalytic processing including positions of conserved and catalytic residues, secondary structure arrangement and domain organization. Comparisons to cellulose synthases from higher plants (genetic algorithm based model for cotton CelA1), data from neural network predictions (PHD), and to the recently experimentally determined structures of the non-processive SpsA and beta 4-galactosyltransferase retest and further validate our structure-function description of this glycosyltransferase.

Medical target prediction from genome sequence: combining different sequence analysis algorithms with expert knowledge and input from artificial intelligence approaches.

By exploiting the rapid increase in available sequence data, the definition of medically relevant protein targets has been improved by a combination of: (i) differential genome analysis (target list): and (ii) analysis of individual proteins (target analysis). Fast sequence comparisons, data mining, and genetic algorithms further promote these procedures. Mycobacterium tuberculosis proteins were chosen as applied examples.

Re-annotating the Mycoplasma pneumoniae genome sequence: adding value, function and reading frames.

Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.

The 3'-untranslated region of the human estrogen receptor alpha gene mediates rapid messenger ribonucleic acid turnover.

Human estrogen receptor-alpha messenger RNA (hERalpha mRNA) has a relatively short half-life, which was determined to be approximately 5 h in MCF-7 cell line after actinomycin D treatment. The 3'-untranslated region (3'UTR) of hERalpha mRNA was previously shown to completely down-regulate chloramphenicol acetyltransferase activity when present at the 3'-end of chloramphenicol acetyltransferase transcripts, suggesting a destabilizing function of the hERalpha 3'UTR sequence. Chimeric genes composed of a serum-inducible Fos promoter, GH-coding sequences, and different segments of the hERalpha complementary DNA 3'UTR sequence were used to confirm this hypothesis and to localize the RNA region responsible for the destabilizing effect. The presence of the complete hERalpha 3'UTR reduced the half-life of the reporter mRNA from more than 24 to 3 h. When the hERalpha 3'UTR was subdivided into four fragments (UTR1-4), one fragment, UTR2, retained the most ability to down-regulate the reporter mRNA (t1/2 = 4 h). A stretch of four AUUUA motifs within UTR2 was shown not to mediate mRNA destabilization. In contrast, further subdivision of the UTR2 into three parts (UTR2a-c) resulted in the loss of the destabilizing activity. Finally, recombination of two UTR2 subfragments (UTR2a and -b) partially restored this function, indicating a cooperative role among the three UTR2a-c subfragments in the process that leads to destabilization of the hERalpha transcript.

DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination.

We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.

A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks.

A set of linear pathways often does not capture the full range of behaviors of a metabolic network. The concept of 'elementary flux modes' provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. We have used this concept to analyze the interplay between the pentose phosphate pathway (PPP) and glycolysis. The set of elementary modes for this system involves conventional glycolysis, a futile cycle, all the modes of PPP function described in biochemistry textbooks, and additional modes that are a priori equally entitled to pathway status. Applications include maximizing product yield in amino acid and antibiotic synthesis, reconstruction and consistency checks of metabolism from genome data, analysis of enzyme deficiencies, and drug target identification in metabolic networks.

Quality-of-life assessment in the old using the WHOQOL 100: differences between patients with senile dementia and patients with cancer.

INTRODUCTION: The measurement of quality of life is an increasingly important issue, particularly in regard to treatment of severe and chronic diseases. The aim of this pilot study was to assess potentially divergent profiles of quality of life in persons with two different pathologies: moderate dementia and cancer. METHOD: This pilot study was carried out in the neurology and cancer services of the medical school in Montpellier, France (Hôpital Gui de Chaulliac and CRLC Val d'Aurelle). The cumulative self-reporting test WHOQOL 100 (World Health Organization Quality of Life with 100 questions) was administered in 57 patients with either moderate senile dementia (27 cases with a Mini-Mental State Examination score >15; mean age of 73) or cancer (30 cases, mainly women with breast cancer; mean age of 53). The stability of responses was tested in a 2-week period. RESULTS: Results of the study showed clear and significant differences between the two groups in the domains of mobility and psychology. Further, eight questions and six facets with a significant difference in responses were found. Responses seemed more stable in the domains of autonomy, social relationship, and religion for the cancer group, and in autonomy and psychology for the dementia group. The age difference may be an important factor in the different quality of life measured but did not significantly influence responses to the test questions. CONCLUSION: The WHOQOL 100 seems a powerful instrument to assess quality of life in diseases such as cancer and moderate dementia. In this study, interesting differences in responses to the test questions between the two pathologic conditions were identified. Items that were unreliable on retesting are singled out. These results will be applied and reevaluated in the development of future, illness-specific and shorter versions of the WHOQOL 100.

Pore-forming peptides of Entamoeba dispar. Similarity and divergence to amoebapores in structure, expression and activity.

Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.

Pathway alignment: application to the comparative analysis of glycolytic enzymes.

Comparative analysis of metabolic pathways in different genomes yields important information on their evolution, on pharmacological targets and on biotechnological applications. In this study on glycolysis, three alternative ways of comparing biochemical pathways are combined: (1) analysis and comparison of biochemical data, (2) pathway analysis based on the concept of elementary modes, and (3) a comparative genome analysis of 17 completely sequenced genomes. The analysis reveals a surprising plasticity of the glycolytic pathway. Isoenzymes in different species are identified and compared; deviations from the textbook standard are detailed. Several potential pharmacological targets and by-passes (such as the Entner-Doudoroff pathway) to glycolysis are examined and compared in the different species. Archaean, bacterial and parasite specific adaptations are identified and described.

Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster.

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site-specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha-helical structure for the main contact area.