Pitfalls in TCR gene clonality testing: teaching cases

Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis

Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress

Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again

Fast motif recognition via application of statistical thresholds

Background: Improving the accuracy and efficiency of motif recognition is an important computational challenge that has application to detecting transcription factor binding sites in genomic data. Closely related to motif recognition is the Consensus String decision problem that asks, given a parameter d and a set of ℓ-length strings S = {s1,...,sn}, whether there exists a consensus string that has Hamming distance at most d from any string in S. A set of strings S is pairwise bounded if the Hamming distance between any pair of strings in S is at most 2d. It is trivial to determine whether a set is pairwise bounded, and a set cannot have a consensus string unless it is pairwise bounded. We use Consensus String to determine whether or not a pairwise bounded set has a consensus. Unfortunately, Consensus String is NP-complete. The lack of an efficient method to solve the Consensus String problem has caused it to become a computational bottleneck in MCL-WMR, a motif recognition program capable of solving difficult motif recognition problem instances. Results: We focus on the development of a method for solving Consensus String quickly with a small probability of error. We apply this heuristic to develop a new motif recognition program, sMCL-WMR, which has impressive accuracy and efficiency. We demonstrate the performance of sMCL-WMR in detecting weak motifs in large data sets and in real genomic data sets, and compare the performance to other leading motif recognitio

Clinical heterogeneity can hamper the diagnosis of patients with ZAP70 deficiency

One of the severe combined immunodeficiencies (SCIDs), which is caused by a genetic defect in the signal transduction pathways involved in T-cell activation, is the ZAP70 deficiency. Mutations in ZAP70 lead to both abnormal thymic development and defective T-cell receptor (TCR) signaling of peripheral T-cells. In contrast to the lymphopenia in most SCID patients, ZAP70-deficient patients have lymphocytosis, despite the selective absence of CD8+ T-cells. The clinical presentation is usually before 2 years of age with typical findings of SCID. Here, we present three new ZAP70-deficient patients who vary in their clinical presentation. One of the ZAP70-deficient patients presented as a classical SCID, the second patient presented as a healthy looking wheezy infant, whereas the third patient came to clinical attention for the eczematous skin lesions simulating atopic dermatitis with eosinophilia and elevated immunoglobulin E (IgE), similar to the Omenn syndrome. This study illustrates that awareness of the clinical heterogeneity of ZAP70 deficiency is of utmost importance for making a fast and accurate diagnosis, which will contribute to the improvement of the adequate treatment of this severe immunodeficiency

An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice.

Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new N-ethyl-N-nitrosurea (ENU)-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, Ocm, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric P-value analysis showed that hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally

Towards Heat-stable Oxytocin Formulations: Analysis of Degradation Kinetics and Identification of Degradation Products

Purpose. To investigate degradation kinetics of oxytocin as a function of temperature and pH, and identify the degradation products. Materials and Methods. Accelerated degradation of oxytocin formulated at pH 2.0, 4.5, 7.0 and 9.0 was performed at 40, 55, 70 and 80°C. Degradation rate constants were determined from RP-HPLC data. Formulations were characterized by HP-SEC, UV absorption and fluorescence spectroscopy. Degradation products were identified by ESI-MS/MS. Results. The loss of intact oxytocin in RP-HPLC was pH- and temperature-dependent and followed (pseudo) first order kinetics. Degradation was fastest at pH 9.0, followed by pH 7.0, pH 2.0 and pH 4.5. The Arrhenius equation proved suitable to describe the kinetics, with the highest activation energy (116.3 kJ/mol) being found for pH 4.5 formulations. At pH 2.0 deamidation of Gln 4, Asn 5, and Gly 9-NH2, as well as combinations thereof were found. At pH 4.5, 7.0 and 9.0, the formation of tri- and tetrasulfidecontaining oxytocin as well as different types of disulfide and dityrosine-linked dimers were found to occur. Beta-elimination and larger aggregates were also observed. At pH 9.0, mono-deamidation of Gln 4, Asn 5, and Gly 9-NH2 additionally occurred. Conclusions. Multiple degradation products of oxytocin have been identified unequivocally, including various deamidated species, intramolecular oligosulfides and covalent aggregates. The strongly pH dependent degradation can be described by the Arrhenius equation. KEY WORDS: aggregation; Arrhenius kinetics; degradation; mass spectrometry; oxytocin