Modelling H3+ in planetary atmospheres: effects of vertical gradients on observed quantities.

Since its detection in the aurorae of Jupiter approximately 30 years ago, the H3+ ion has served as an invaluable probe of giant planet upper atmospheres. However, the vast majority of monitoring of planetary H3+ radiation has followed from observations that rely on deriving parameters from column-integrated paths through the emitting layer. Here, we investigate the effects of density and temperature gradients along such paths on the measured H3+ spectrum and its resulting interpretation. In a non-isothermal atmosphere, H3+ column densities retrieved from such observations are found to represent a lower limit, reduced by 20% or more from the true atmospheric value. Global simulations of Uranus' ionosphere reveal that measured H3+ temperature variations are often attributable to well-understood solar zenith angle effects rather than indications of real atmospheric variability. Finally, based on these insights, a preliminary method of deriving vertical temperature structure is demonstrated at Jupiter using model reproductions of electron density and H3+ measurements. The sheer diversity and uncertainty of conditions in planetary atmospheres prohibits this work from providing blanket quantitative correction factors; nonetheless, we illustrate a few simple ways in which the already formidable utility of H3+ observations in understanding planetary atmospheres can be enhanced. This article is part of a discussion meeting issue 'Advances in hydrogen molecular ions: H3+, H5+ and beyond'.

A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing.

Recent improvements in high-throughput sequencing technologies underscore the pervasiveness of circular RNA (circRNA) expression in animal cells. CircRNAs are distinct from their linear counterparts because they lack the 5' caps and 3' tails that typically help determine the cellular fate of a transcript. However, due to the lack of free ends, circRNAs are impervious to exonucleases and thus can evade normal RNA turnover mechanisms. Most circRNAs are derived from protein-coding pre-mRNAs, via a mechanism called "back-splicing." Existing methods of circRNA expression thus typically involve genes that have been engineered to contain sequence elements that promote back-splicing. We recently uncovered an anciently conserved mechanism of RNA circularization in metazoans that involves splicing of tRNA introns. This splicing mechanism is completely independent from that of pre-mRNAs. In this chapter, we detail an orthogonal method that involves splicing of intron-containing tRNAs in order to produce circRNAs in vivo. We utilize fluorescence-based RNA reporters to characterize the expression, localization, and stability of these so-called tRNA intronic circular RNAs. Because tRNA biogenesis is essential for all cellular life, this method provides a means to express ultrastable, high-copy, circRNA effectors in a wide variety of metazoan cell types.

The Higher Classification of the Ant Subfamily Ponerinae (Hymenoptera: Formicidae), with a Review of Ponerine Ecology and Behavior.

The tribal and generic classification of the diverse ant subfamily Ponerinae (Hymenoptera: Formicidae) is revised to reflect recent molecular phylogenetic information and a reappraisal of ponerine morphological diversity. The monogeneric tribe Thaumatomyrmecini (Thaumatomyrmex) is newly synonymized under Ponerini (syn. nov.), and the diverse genus Pachycondyla is fragmented into 19 genera, largely along the lines of its junior synonyms: Bothroponera, Brachyponera (gen. rev.), Ectomomyrmex (gen. rev.), Euponera (gen. rev.), Hagensia (gen. rev.), Megaponera (gen. rev.), Mesoponera (gen. rev.), Neoponera (gen. rev.), Ophthalmopone (gen. rev.), Pachycondyla, Paltothyreus (gen. rev.), Pseudoneoponera (gen. rev.), Pseudoponera (gen. rev.), and 6 new genera: Austroponera (gen. nov.), Buniapone (gen. nov.), Fisheropone (gen. nov.), Mayaponera (gen. nov.), Parvaponera (gen. nov.) and Rasopone (gen. nov.). Some junior synonyms of Pachycondyla are transferred to junior synonym status under other genera: Wadeura as a junior synonym of Cryptopone (syn. nov.), and both Termitopone and Syntermitopone as junior synonyms of Neoponera (syn. nov.). A new genus, Iroponera (gen. nov.), based on the new species Iroponera odax (sp. nov.), is described from Australia. Molecular and morphological justifications for these taxonomic changes are given alongside discussions of phylogenetic relationships. Keys to the world genera of Ponerinae are provided, and morphological diagnoses and species lists are given for each genus. Finally, the available information on ponerine ecology and behavior is reviewed and synthesized.

Tumor suppressor TNFAIP3 (A20) is frequently deleted in Sézary syndrome.

Despite recent therapeutic improvements, the prognosis for patients suffering from Sézary syndrome (SS), a disseminated form of cutaneous T-cell lymphomas, is still poor. We identified bi- and monoallelic deletions of the tumor necrosis factor-α-induced protein 3 gene (TNFAIP3; A20) in a high proportion of SS patients as well as biallelic A20 deletion in the SS-derived cell line SeAx. Furthermore, we demonstrate that inhibition of A20 activates the NF-κB pathway thereby increasing the proliferation of normal T lymphocytes. On the other hand, the reconstitution of A20 expression slowed down the cell cycle in SeAx cells. Recently A20 inactivation has been reported in various B-cell lymphomas. In this study, we show that A20 is also a putative tumor suppressor in the T-cell malignancy-SS.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

Inhibition of BCL11B expression leads to apoptosis of malignant but not normal mature T cells.

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.

Disruption of the BCL11B gene through inv(14)(q11.2q32.31) results in the expression of BCL11B-TRDC fusion transcripts and is associated with the absence of wild-type BCL11B transcripts in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is associated with chromosomal aberrations characterized by juxtaposition of proto-oncogenes to T-cell receptor gene loci (TCR), resulting in the deregulated transcription of these proto-oncogenes. Here, we describe the molecular characterization of a novel chromosomal aberration, inv(14)(q11.2q32.31), in a T-ALL sample, involving the recently described BCL11B gene and the TCRD locus. The inversion joined the 5' part of BCL11B, including exons 1-3, to the TRDD3 gene segment of the TCRD locus, whereas the reciprocal breakpoint fused the TRDV1 gene segment to the fourth exon of BCL11B. The TRDV1-BCL11B joining region was 1344 bp long and contained fragments derived from 20q11.22, 3p21.33 and from 11p12, indicating the complex character of this aberration. A strong expression of in-frame transcripts with truncated BCL11B and TCRD constant region (TRDC) were observed, but in contrast to normal T cells and other T-ALL samples, no wild-type BCL11B transcripts were detected in the T-ALL sample. Screening of 37 other T-ALLs revealed one additional case with expression of the BCL11B-TRDC fusion transcript. As BCL11B appears to play a key role in T-cell differentiation, BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in man.

[Lung metastases of colorectal tumors: clinical background and development of care supply]. / Lungenmetastasen kolorektaler Tumoren: klinischer Hintergrund und Entwicklung des Versorgungsbedarfs.

The incidence of most cancers increases with age, including colorectal-, lung- and breast carcinomas. Each year, approximately 50,000 new cases of colorectal carcinoma (CRC) are diagnosed in Germany with a peak incidence around the age of 65. At diagnosis, 50% of CRC-cases show already metastases. Cure of metastatic disease with chemotherapy, radiology or surgery alone or in combination can be rarely achieved in this situation. However, palliative therapy regimens can significantly prolong life in most cases. Besides systemic therapy, minimal invasive techniques for tumor reduction are an interesting option in the palliative situation, especially in elderly patients. Yet the clinical impact of these new techniques has to be determined in future studies.

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4 percent for the brilliant cresyl blue method, 14.1-30.8 percent for the selective hemolysis method and 8.5-19.7 percent for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2 percent), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Comparative evaluation of the human whole blood and human peripheral blood monocyte tests for pyrogens.

Two different in vitro tests for pyrogens, using human peripheral blood monocytes (PBMNC) and diluted whole blood (WBC), respectively, were applied to different classes of parenteral medicinal products. Many of these products did not have a specified endotoxin limit concentration that was established as the maximum valid dilution to comply with the test. The results of the in vitro tests for pyrogens were compared with the results from the Limulus amoebocyte lysate (LAL) and rabbit pyrogen tests. The Second International Standard for endotoxin was used to calibrate all of the assays and the International Standard for IL-6 was used to calibrate the IL-6 ELISA which provided the readout for the in vitro tests for pyrogens. Preparatory tests were conducted to ensure that the "criteria for validity and precision of the standard curve" were satisfied and that the drugs being tested did not interfere in the tests. The PBMNC/IL-6 test had a detection limit of 0.06 EU/ml and spike recoveries were 62-165%. The whole blood/IL-6 test also had a detection limit of 0.06 EU/ml and spike recoveries were 58-132%. The application to the detection of non-endotoxin pyrogens needs to be evaluated in more detail, but the two in vitro tests for pyrogens showed good agreement overall, both with each other and with the LAL test and the rabbit pyrogen test for the detection of endotoxins.

Biological evaluation of recombinant human erythropoietin in pharmaceutical products.

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.

Quantitation of target mRNAs using the reverse-transcription polymerase chain reaction found a widespread field of application in diverse biomedical diagnostic assays. However, the problem of varying sample quality has to be solved by correcting target molecule amounts through detection of an endogenous control template. The choice of an appropriate reference gene is still object of debate as pseudogene co-amplification and expression level variations may limit the usefulness of some currently used reference reactions. We compared quantitative expression levels of the commonly used endogenous reference genes beta-actin (beta-actin), beta-2-microglobulin (beta2-MG) and porphobilinogen deaminase (PBDG) using the TaqMan chemistry. With these assays we investigated the respective expression patterns in K562 cells and leucocytes of normal individuals as well as of malignoma patients. In K562 cells 1544+246 beta-actin, 65+30 beta2-MG and 22+/-8 PBDG copies/cell were detected. In normal leucocytes 491+/-97 beta-actin, 40+/-17 beta2-MG and <1 PBDG copies/cell were quantified. Leucocytes of various malignancies exhibited 84+/-51 beta-actin, 106+/-8 beta2-MG and <1 PBDG copies/cell. We conclude that beta2-MG is the most suitable reference gene tested as its variation between different sample origins and within distinct cell types was acceptable low.

Selection of B-cell chronic lymphocytic leukemia cell variants by therapy with anti-CD20 monoclonal antibody rituximab.

OBJECTIVE: Anti-CD20 chimeric monoclonal antibody rituximab (Mabthera; IDEC-C2B8) is currently tested in several clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). In the present study, we investigated whether rituximab therapy may select for CD20(-) subclones. MATERIALS AND METHODS: Leukemic B-CLL cells were isolated from patients with B-CLL and sensitivity to rituximab-induced cell death was examined. Levels of CD20 protein and mRNA were determined using flow cytometry and real-time PCR, respectively. Clonality analyses of leukemic cells throughout rituximab therapy were performed by GeneScan analysis of patient clone specific rearrangements of the complementarity determining region III of the heavy chain immunoglobulin. RESULTS: Cytotoxicity of rituximab in vitro did not depend on the protein levels of CD20. During therapy with rituximab CD20(+) B-CLL cells were depleted and CD20(-) leukemic cells emerged. After treatment, the initial CD20(+) B-CLL cell clone reexpanded. CD20(-) B-CLL cells retained their capacity to synthesize the CD20 molecule. CONCLUSIONS: These data support the concept that in B-CLL rituximab treatment may not lead to the emergence of CD20(-) leukemic variants. Our findings support clinical studies investigating the benefit of prolonged period of rituximab therapy in B-CLL disease.

Novel T-cell receptor delta gene rearrangement involving a recombining element located 2.6 kb 3' from the Vdelta2 gene segment.

In this study, we describe a novel T-cell receptor delta (TCRdelta) gene rearrangement observed in acute myeloid leukemia with coexpression of T-lymphoid antigens (Ly+AML) and in peripheral blood leukocytes (PBL) from one out of ten healthy donors. The rearrangement was identified by Southern blot analysis using a joining region (Jdelta1) specific probe and amplified by polymerase chain reaction (PCR) with a variable region (Vdelta2) and Jdelta1 specific primers. The nucleotide sequence analysis of an atypical 3000 bp PCR product allowed localization of the breakpoint within the TCRdelta gene locus, 2.6 kb 3' from the Vdelta2 gene segment. A regular Ddelta2-Ddelta3-Jdelta1 joining was found at the 3' end of the breakpoint, indicating that the rearrangement was mediated by the VDJ recombinase, but no TCRdelta gene segment was detected at the 5' end. Analysis of the germline sequence 3' from the breakpoint revealed an isolated recombination signal sequence (RSS) capable of initiating a rearrangement. The RSS motif described by us is the second TCRdelta recombining element (deltaRec2). The deltaRec2(Ddelta)Jdelta1 recombination is a rather rare event and can be found in acute leukemia and in PBL from healthy individuals. Most likely, the nonfunctional deltaRec2(Ddelta)Jdelta1 rearrangement is a transient step during the VDJ recombination. It may potentially lead to deletion of the deltaRec2(Ddelta)Jdelta1 complex and either to direct joining of a Vdelta region to one of the downstream Jdelta regions or to a rearrangement of the TCRalpha gene.

Prospective randomized trial to assess the value of preemptive oral therapy for CMV infection following liver transplantation.

BACKGROUND: With the development of sensitive tests to detect cytomegalovirus (CMV) viremia, preemptive approaches become a reasonable alternative to general CMV prophylaxis. We performed a randomized trial comparing pp65-antigenemia guided preemptive therapy using oral ganciclovir with symptom-triggered intravenous ganciclovir treatment. METHODS: Eighty-eight of 372 liver transplant recipients developed antigenemia early after orthotopic liver transplantation. Twenty-eight symptomatic patients with antigenemia were excluded from randomization and treated with intravenous ganciclovir. Sixty pp65-antigen-positive asymptomatic patients were randomized to receive either oral ganciclovir 3x1 g/day for 14 days (group 1) or no preemptive treatment (group 2). Patients that developed CMV disease were treated with intravenous ganciclovir 2x5 mg/kg body weight for 14 days. The high-risk (Donor+/Recipient-) patients were equally distributed in the two study groups. RESULTS: Three of 30 (10%) patients on oral ganciclovir developed mild to moderate CMV disease compared with 6/30 (20%) patients in the control group. In the Donor+/Recipient- patients, the incidence of CMV disease was 1/6 and 3/7. All disease episodes resolved after intravenous treatment. The 1- and 3-year patient and organ survival was the same in the study groups and in the patients with or without CMV infection. No deaths related to CMV occurred. CONCLUSIONS: The positive predictive value of pp65-antigenemia for the development of CMV disease was very low, and, in 28/88 patients (32%), antigenemia did not precede symptoms. Therefore, pp65-antigenemia is of limited value in deciding on the timing and need for ganciclovir therapy after liver transplantation.

Fluorescent 5'-exonuclease assay for the absolute quantification of Wilms' tumour gene (WT1) mRNA: implications for monitoring human leukaemias.

The Wilms' tumour gene (WT1) has been suggested as a powerful parameter for molecular monitoring of minimal residual disease (MRD) in leukaemias. However, molecular monitoring via WT1 RNA levels is far from being routinely performed, which is possibly owing to the complex and inaccurate quantitative reverse transcription polymerase chain reaction (RT-PCR) procedures. Using a newly-developed quantitative real time RT-PCR, we measured WT1 transcripts in peripheral blood leucocytes of patients with acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid leukaemia (CML). While healthy blood donors did not show measurable amounts of WT1 transcripts, WT1 RNA levels were detectable in all types of leukaemia. Furthermore, intraindividual WT1 transcript kinetics were exclusively dependent on disease progression, treatment and subsequent disease outcome. Using this approach, we could distinguish between treatment response and failure within the first days of therapeutic intervention. Moreover, gradually rising WT1 levels over a period of weeks and months paralleled long-term disease progression and appeared to be a prognostic indicator for subsequent clinical relapse. A linear correlation between quantities of WT1 and bcr/abl fusion transcripts could be seen in CML. We conclude that quantitative assessment of WT1 transcripts using real-time PCR is an appropriate method for molecular monitoring of AML, ALL and CML, and can be used independently for both short- and long-term monitoring of leukaemia patients.

What can we learn from leukemia as for the process of lineage commitment in hematopoiesis?

Most contemporary models of hematopoiesis assume lineage fidelity of early progenitor cells. Along with this concept normal hematopoietic cells and the majority of leukemias express exclusively myeloid or lymphoid specific antigens. On the other hand, growing evidence exists challenging the lineage fidelity model. Chronic myeloid leukemia (CML) in the blast crisis may switch to acute lymphoblastic leukemia (ALL) and as a result of the chemotherapy ALL may converse to acute myeloid leukemia (AML). Furthermore, a substantial portion of leukemia cases, named acute mixed-lineage leukemia (AMLL), show simultaneous expression of both myeloid and lymphoid antigens. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements, correlating with myeloid-lymphoid immunophenotype in AMLL, support the hypothesis of lineage infidelity of early progenitor cells, rather than the aberrant antigen expression. Based on a detailed characterization of AMLL we present a modified model of a "common myeloid/lymphoid progenitor cell". This hypothetical very early hematopoietic progenitor cell shows a transient expression of myeloid and B- or T-lymphoid antigen and may also have rearranged its Ig and/or TCR genes. Subsequently, myeloid or lymphoid markers are downregulated and the hematopoietic cell enters either myeloid, T-lymphoid or B-lymphoid differentiation pathway.