Evidence for a role of RUNX1 as recombinase cofactor for TCRß rearrangements and pathological deletions in ETV6-RUNX1 ALL.

T-cell receptor gene beta (TCRß) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRß sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRß rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRß gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRß gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRß rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.

Differences in the Pharmacokinetics of Gentamicin between Oncology and Nononcology Pediatric Patients.

Dosing gentamicin in pediatric patients can be difficult due to its narrow therapeutic index. A significantly higher percentage of fat mass has been observed in children receiving oncology treatment than in those who are not. Differences in the pharmacokinetics of gentamicin between oncology and nononcology pediatric patients and individual dosage requirements were evaluated in this study, using normal fat mass (NFM) as a body size descriptor. Data from 423 oncology and 115 nononcology patients were analyzed. Differences in drug disposition were observed between the oncology and nononcology patients, with oncology patients having a 15% lower central volume of distribution and 32% lower intercompartmental clearance. Simulations based on the population pharmacokinetic model demonstrated low exposure target attainment in all individuals at the current clinical recommended starting dose of 7.5 mg/kg of body weight once daily, with 57.4% of oncology and 35.7% of nononcology subjects achieving a peak concentration (Cmax) of ≥25 mg/liter and 64.3% of oncology and 65.6% of nononcology subjects achieving an area under the concentration-time curve at 24 h postdose (AUC24) of ≥70 mg · h/liter after the first dose. Based on simulations, the extent of the impact of differences in drug disposition between the two cohorts appeared to be dependent on the exposure target under examination. Greater differences in achieving a Cmax target of >25 mg/liter than an AUC24 target of ≥70 mg · h/liter between the cohorts was observed. Further investigation into whether differences in the pharmacokinetics of gentamicin between oncology and nononcology patients are a consequence of changes in body composition is required.

Identification of novel PANDAR protein interaction partners involved in splicing regulation.

Interactions of long non-coding RNAs (lncRNA) with proteins play important roles in the regulation of many cellular processes. PANDAR (Promotor of CDKN1A Antisense DNA damage Activated RNA) is a lncRNA that is transcribed in a p53-dependent manner from the CDKN1A promoter and is involved in the regulation of proliferation and senescence. Overexpression of PANDAR has been observed in several tumor species and correlated with a poor prognosis for patient survival rate. Depending on the cellular state, PANDAR is known to interact with proteins such as the nuclear transcription factor Y subunit A (NF-YA) and the scaffold attachment factor A (SAF-A). However, a comprehensive analysis of the PANDAR interactome was missing so far. Therefore, we applied peptide nucleic acid (PNA)-based pull-downs combined with quantitative mass spectrometry to identify new protein binding partners. We confirmed potential candidates like U2AF65 and PTBP1, known to be involved in RNA processing. Furthermore, we observed that overexpression of PANDAR leads to a reduced level of the short pro-apoptotic BCL-X splice variant (BCL-XS) which is regulated by PTBP1. Simultaneous overexpression of PTBP1 was able to rescue this effect. Overall, our data suggest a role for PANDAR in the regulation of splicing events via its interaction partner PTBP1.

A Population Pharmacokinetic Model of Gentamicin in Pediatric Oncology Patients To Facilitate Personalized Dosing.

To ensure the safe and effective dosing of gentamicin in children, therapeutic drug monitoring (TDM) is recommended. TDM utilizing Bayesian forecasting software is recommended but is unavailable, as no population model that describes the pharmacokinetics of gentamicin in pediatric oncology patients exists. This study aimed to develop and externally evaluate a population pharmacokinetic model of gentamicin to support personalized dosing in pediatric oncology patients. A nonlinear mixed-effect population pharmacokinetic model was developed from retrospective data. Data were collected from 423 patients for model building and a further 52 patients for external evaluation. A two-compartment model with first-order elimination best described the gentamicin disposition. The final model included renal function (described by fat-free mass and postmenstrual age) and the serum creatinine concentration as covariates influencing gentamicin clearance (CL). Final parameter estimates were as follow CL, 5.77 liters/h/70 kg; central volume of distribution, 21.6 liters/70 kg; peripheral volume of distribution, 13.8 liters/70 kg; and intercompartmental clearance, 0.62 liter/h/70 kg. External evaluation suggested that current models developed in other pediatric cohorts may not be suitable for use in pediatric oncology patients, as they showed a tendency to overpredict the observations in this population. The final model developed in this study displayed good predictive performance during external evaluation (root mean square error, 46.0%; mean relative prediction error, -3.40%) and may therefore be useful for the personalization of gentamicin dosing in this cohort. Further investigations should focus on evaluating the clinical application of this model.

Donor CD4 T Cell Diversity Determines Virus Reactivation in Patients After HLA-Matched Allogeneic Stem Cell Transplantation.

Delayed reconstitution of the T cell compartment in recipients of allogeneic stem cell grafts is associated with an increase of reactivation of latent viruses. Thereby, the transplanted T cell repertoire appears to be one of the factors that affect T cell reconstitution. Therefore, we studied the T cell receptor beta (TCRß) gene rearrangements of flow cytometry-sorted CD4(+) and CD8(+) T cells from the peripheral blood of 23 allogeneic donors before G-CSF administration and on the day of apheresis. For this purpose, TCRß rearrangements were amplified by multiplex PCR followed by high-throughput amplicon sequencing. Overall, CD4(+) T cells displayed a significantly higher TCRß diversity compared to CD8(+) T cells irrespective of G-CSF administration. In line, no significant impact of G-CSF treatment on the TCR Vß repertoire usage was found. However, correlation of the donor T cell repertoire with clinical outcomes of the recipient revealed that a higher CD4(+) TCRß diversity after G-CSF treatment is associated with lower reactivation of cytomegalovirus and Epstein-Barr virus. By contrast, no protecting correlation was observed for CD8(+) T cells. In essence, our deep TCRß analysis identifies the importance of the CD4(+) T cell compartment for the control of latent viruses after allogeneic stem cell transplantation.

Short Psychological Intervention as a Perioperative Pain Reduction Treatment in Spinal Neurosurgery.

STUDY AIMS: The aim of the present pilot study was to test the feasibility of an innovative Short Psychological Intervention (SPI) for back pain patients as part of an acute inpatient neurosurgical treatment. Fear and fear-avoidance beliefs have been shown to influence the functional outcome in chronic back pain (CBP) patients. Therefore, a reduction of fear and fear-avoidance beliefs should improve the functional outcome and reduce pain in the acute neurosurgical setting. PATIENTS AND METHODS: 39 patients were studied in a randomized prospective longitudinal study. The patients had severe degenerative spinal disease and had undergone posterior lumbar interbody fusion. RESULTS: All patients enrolled in the study were investigated in the immediate preoperative period and 6 weeks postoperatively using a package of standardized questionnaires in which pain intensity, fear-avoidance beliefs, and physical fitness were recorded. In 19 of the patients, the surgical procedure was supplemented by a SPI based on methods to increase self-efficacy by reducing fear-avoidance beliefs. While the intervention group reported a significantly greater reduction in the highest pain intensity and a better physical fitness compared to the control group, we did not find a significant decrease in fear-avoidance beliefs in the intervention group at the second time of assessment, possibly due to the relatively small sample size. CONCLUSIONS: The study confirmed that psychological interventions can offer significant benefits when used in the acute inpatient setting as the outcome of surgery can be positively influenced. Future studies should focus on cost savings related to improved postoperative recovery and a possible reduction of chronic postoperative pain.

RAI1 is a novel polyglutamine encoding gene that is deleted in Smith-Magenis syndrome patients.

The human chromosomal band 17p11.2 is a genetically unstable interval. It has been shown to be deleted in patients suffering from Smith-Magenis syndrome. Previous efforts of physical and transcriptional mapping in 17p11.2 and subsequent genomic sequencing of the candidate interval allowed the identification of new genes that might be responsible for the Smith-Magenis syndrome. In this report, one of these genes named RAI1, the human homologue of the mouse Rai1 gene, has been investigated for its contribution to the syndrome. Expression analysis on different human adult and fetal tissues has shown the existence of at least three splice variants. Moreover, the most interesting feature of the gene is the presence of a polymorphic CAG repeat coding for a polyglutamine stretch in the amino terminal domain of the protein.

Triple A syndrome is caused by mutations in AAAS, a new WD-repeat protein gene.

The triple A syndrome (MIM 231550) is a rare autosomal recessive disorder characterized by adrenal insufficiency, achalasia and alacrima. The frequent association with a variety of neurological features may result in a severely disabling disease. We previously mapped the syndrome to a 6 cM interval on chromosome 12q13 and have now refined the critical region to 0 cM between KRT8 and D12S1651. Overlapping bacterial artificial chromosome (BAC) sequences of a high resolution BAC/P1-derived artificial chromosome (PAC) contig were screened for gene content and a novel gene encoding a 546 amino acid polypeptide was identified. In nine triple A syndrome patients eight different homozygous and compound heterozygous mutations were found in this gene, most of them leading to a truncated protein suggesting loss of function. RNA blotting experiments revealed marked expression in neuroendocrine and gastrointestinal structures, which are predominantly affected in triple A syndrome, supporting the hypothesis that mutations in this triple A syndrome gene (AAAS) are responsible for the disease. The predicted protein belongs to the family of WD repeat-containing proteins which exhibit a high degree of functional diversity including regulation of signal transduction, RNA processing and transcription.

The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration.

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, we replaced the endogenous gene by cDNAs encoding either carboxyterminally truncated (ephrinB2(DeltaC)) or, as a control, full-length ligand (ephrinB2(WT)). While homozygous ephrinB2(WT/WT) animals were viable and fertile, loss of the ephrinB2 cytoplasmic domain resulted in midgestation lethality similar to ephrinB2 null mutants (ephrinB2(KO)). The truncated ligand was sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2(DeltaC/DeltaC) embryos showed defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2(KO/KO) animals. Our results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo.

Analysis of the spermine synthase gene region in Fugu rubripes, Tetraodon fluviatilis, and Danio rerio.

A prerequisite to understanding the evolution of the human X chromosome is the analysis of synteny of X-linked genes in different species. We have focused on the spermine synthase gene in human Xp22. 1. We show that whereas the human gene spans a genomic region of 54 kb, the Fugu rubripes gene is encompassed in a 4.7-kb region. However, we could not find conserved synteny between this region of human Xp22 and the equivalent F. rubripes region. A cosmid clone containing the F. rubripes gene does not contain other X-linked genes. Instead we identified homologs of human genes that are autosomally localized: the ryanodine receptor type I (RYRI), which is implicated in malignant hyperthermia and central core disease, and the HE6 gene. Comparison of the F. rubripes, Tetraodon fluviatilis, mouse, human, and Danio rerio 5'UTRs of spermine synthase highlights conserved sequences potentially involved in regulation. Interestingly, pseudogenes of this gene that are present in the human and mouse genomes seem to be absent in the compact F. rubripes genome. Analysis of a D. rerio PAC clone containing spermine synthase shows an intermediate genomic size in this fish. Sequence analysis of this PAC clone did not reveal other known genes: neither the RYRI gene, nor the HE6 gene, nor other human Xp22 genes were identified.

Transcription mapping in a medulloblastoma breakpoint interval and Smith-Magenis syndrome candidate region: identification of 53 transcriptional units and new candidate genes.

The chromosomal band 17p11.2 is associated with a number of neurological disorders and malignant diseases. This region is also characterized by the presence of complex repeat elements that are probably responsible for the frequent occurrence of interstitial deletions, duplications, and isochromosome formation. In the course of the molecular analysis of this interval, an integrated map with YACs, PACs, and cosmids covering approximately 6 Mb was established. Focusing on the 1.4-Mb interval containing the Smith-Magenis syndrome critical region and the breakpoint region for medulloblastomas, we constructed a detailed transcript map between the marker PS2 and the proximal CMT1A repeat. FISH analysis of the PACs allowed determination of the position of the transcripts with respect to the SMS critical region and the presumptive chromosomal breakpoint in medulloblastomas. One PAC (G21100) provided evidence for the presence of a novel complex repeat unit, indicating that there are at least three independent repeat elements within 2 Mb. Five genes were mapped to clone G21100 and are likely to form part of this novel complex sequence repeat. In summary, 53 new transcripts were isolated by using cDNA selection and exon trapping. This included 8 known but previously unmapped genes and 45 novel transcripts. The expression profile of 21 transcripts was determined by RT-PCR. Based on their homologies to known genes or proteins, some of the novel genes are considered candidate genes either for malignant diseases or for the Smith-Magenis syndrome.

Crystal structure of narbonin at 1.8 A resolution.

The three-dimensional structure of narbonin, a seed protein from Vicia narbonensis L, has been determined at 1.8 A resolution. Phase information was obtained by multiple isomorphous replacement and optimized anomalous dispersion. The narbonin structure was initially traced with only 17% amino-acid sequence information and preliminarily refined to a crystallographic R-factor of 16.5%. It is now refined to 15.9% using full sequence information derived from cDNA and after the addition of more solvent molecules. The monomeric molecule of narbonin is an eight-stranded parallel beta-barrel surrounded by alpha-helices in a beta/alpha-topology similar to that first observed in triose phosphate isomerase. Differences exist in the N-terminal part of the polypeptide chain, where the first helix is replaced by a loop and the second beta-strand is followed by an additional antiparallel alpha-sheet placed parallel on top of alpha-helices alpha3 and alpha4. Two short additional secondary structures are present. The first, an alpha-helix, is situated between the seventh beta-strand and the following helix, and the second, which is a 3(10) helix, between the eighth strand and the C-terminal helix. The most striking observation is the lack of a known enzymatic function for narbonin, because all TIM-like structures known so far are enzymes.

A monoclonal antibody Fab fragment crystallized with and without a peptide epitope from HIV.

The Fab fragment of CB 4-1, a monoclonal murine antibody against HIV protein p24, has been produced. It forms a complex with a synthetic antigen, an epitope of p24 made up of 11 amino acids, with the binding constant Kd = 3.6 x 10(-9) M. Crystals of hexagonal and orthorhombic space group has been obtained by cocrystallization of the Fab with the epitope and crystallization without the epitope, respectively. In either case, the crystals are suitable for X-ray structural analysis. Crystals of the Fab fragment cocrystallized with the peptide have the space group P 6(3)22 with cell dimensions of a = b = 105 A, c = 297 A. Fab crystals without the epitope are in space group C 222 with cell dimensions a = 110.1 A, b = 110.2 A, c = 150.1 A.

Adenosine triphosphate: glutamine synthetase adenylyltransferase of Escherichia coli: two active molecular forms.

Two active forms of purified ATP:glutamine synthetase adenylyl-transferase from Escherichia coli are apparent on polyacrylamide gel electrophoresis at pH 8. The slower migrating component, which is identical to the P(I)-protein fraction of the glutamine synthetase deadenylylating enzyme system, has S(20.w) congruent with 5.1 S and a molecular weight of about 130,000. The more rapidly migrating adenylyltransferase component has S(20.w) congruent with 4.0 S and a molecular weight of about 70,000. During storage at 4 degrees C, the larger adenylyltransferase component (P(I)) converts to the smaller active unit with a concomitant loss of both P(I) deadenylylating activity and soluble protein. It is concluded that the low-molecular weight form of the adenylyltransferase is a subunit of the deadenylylating P(I)-protein.

Association of ATP: glutamine synthetase adenylyltransferase activity with the P1 component of the glutamine synthetase deadenylylation system.

Regulation of glutamine synthetase (EC 6.3.1.2) in Escherichia coli is mediated by adenylylation and deadenylylation of the enzyme. The present studies show that one protein is a common component of both the adenylylation and deadenylylation systems. Thus, the ATP:glutamine synthetase adenylyltransferase, which catalyzes adenylylation of glutamine synthetase, and one of the two proteins required for deadenylylation (the P(I) protein) are inseparable by a variety of fractionation procedures. The adenylyltransferase and P(I)-deadenylylating activities behave as a single protein upon filtration through Agarose A 0.5 gel, and during chromatography on DE32 cellulose and hydroxyapatite columns. They migrate as a single protein band during electrophoresis on polyacrylamide gel and have identical susceptibilities to heat inactivation. These data indicate that the adenylyltransferase and the P(I)-deadenylylation activity are associated with the same protein complex.