Genetic and genomic analyses of musculoskeletal differences between BEH and BEL strains

Berlin high (BEH) and Berlin low (BEL) strains selected for divergent growth differ 3-fold in body weight. We aimed at examining muscle mass, which is a major contributor to body weight, by exploring anatomical characteristics of the soleus muscle, its fiber numbers and their cross sectional area (CSA), by analysing transcriptome of the gastrocnemius and by initiating quantitative trait locus (QTL) mapping. BEH muscles were 4-to-8 times larger compared to BEL strain. In sub-strain BEH+/+, mutant myostatin was replaced with a wild type allele, however, BEH+/+muscles still were 2-to-4 times larger compared to the BEL strain. BEH soleus contained 2-times more (P<0.0001) and 2-times larger in CSA (P<0.0001) fibers compared to BEL strain. In addition, soleus femoral attachment anomaly (SFAA) was observed in all BEL mice. One significant (chromosome 1) and four suggestive (chromosomes 3, 4, 6 and 9) muscle weight QTLs were mapped in 21-day old F2 intercross (n=296) between BEH and BEL strains. The frequency of SFAA incidence in the F2 and in the backcross to BEL strain (BCL) suggested the presence of more than one causative gene. Two suggestive SFAA QTLs were mapped in BCL, however, their peak markers were not associated with the phenotype in F2. RNA-Seq analysis revealed 2,148 differentially expressed (P<0.1) genes and 45,673 SNPs and >2,000 indels between BEH+/+ and BEL males. In conclusion, contrasting muscle traits, genomic and gene expression differences between BEH and BEL strains provide a promising model for the search of genes involved in muscle growth and musculoskeletal morphogenesis.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium

The basic helix-loop-helix protein achaete-scute homolog-1 (ASH1) is involved in lung neuroendocrine (NE) differentiation and tumor promotion in SV40 transgenic mice. Constitutive expression of human ASH-1 (hASH1) in mouse lung results in hyperplasia and remodeling that mimics bronchiolization of alveoli (BOA), a potentially premalignant lesion of human lung carcinomas. We now show that this is due to sustained cellular proliferation in terminal bronchioles and resistance to apoptosis. Throughout the airway epithelium the expression of anti-apoptotic Bcl-2 and c-Myb was increased and Akt/mTOR pathway activated. Moreover, the expression of matrix metalloproteases (MMPs) including MMP7 was specifically enhanced at the bronchiolo-alveolar duct junction and BOA suggesting that MMPs play a key role in this microenvironment during remodeling. We also detected MMP7 in 70% of human BOA lesions. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis. We also show that forced expression of hASH1 in immortalized human bronchial epithelial cells decreases apoptosis. We conclude that the impact of hASH1 is not limited to cells with NE phenotype. Rather, constitutive expression of hASH1 in lung epithelium promotes remodeling through multiple pathways that are commonly activated during lung carcinogenesis. The collective results suggest a novel model of BOA formation via hASH1-induced suppression of the apoptotic pathway. Our study yields a promising new preclinical tool for chemoprevention of peripheral lung carcinomas. © 2007 USCAP, Inc All rights reserved.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism. © 2012 Macmillan Publishers Limited

Proportioning variability in the dispensation of hand-mixed dental cements

Objectives: The purpose of this investigation was to determine for dispensed multiples (1 through 4) of powder (P) and liquid (L) in hand-mixed dental cement whether: (1) the mean (P/L) ratio (m/m) and (2) the maximum difference in (P/L) ratio is dependent on the number of multiples dispensed. The Null hypotheses were: (a) mean (P/L) ratio is independent of the number of multiples dispensed and (b) maximum difference in (P/L) ratio is independent of the number of multiples dispensed.
Methods: The materials investigated are listed in the Table. The masses of dispensed aliquots of powder and liquid were measured by a single operator (n=10, for multiples 1 through 4) on a 4-place analytical balance. All measurements were made independently and all possible (P/L) ratios calculated for each sample. The effect of multiple dispensations on (P/L) ratios and maximum (P/L) differences was by one-way ANOVA and linear regression, respectively, with the Tukey post-hoc correction for multiple comparisons.MULTIPLE DISPENSEDDISPENSED MU(x1)(x2)(x3)(x4)Zinc phosphateHeraeus12.271(0.691)a13.051(1.269)b13.215(0.824)b13.118(1.149)bFuji IXGC4.209(0.373)a4.085(0.275)b4.095(0.226)b4.095(0.217)bIRMDentsply7.933(0.767)a7.430(0.451)b7.977(0.729)a8.186(0.929)aKetac-Cem3M Espe9.6206(0.613)a9.714(0.523)a9.298(0.314)b9.321(0.292)bMean (SD) powder/liquid ratio (m/m). Superscript letters represent significances (α = 0.05) within each material
Results: Mean (SD) (P/L) ratios are presented in the Table. Null hypothesis (a) is rejected: either (x1) or (x2) dispensation yields a different (P/L) ratio to (x3) or (x4) (p < 0.05). Null hypothesis (b) is rejected: a negative correlation is observed in max (P/L) ratio difference with dispensed multiple for Ketac Cem (p = 0.029).
Conclusion: For hand-mixed dental cements: (1) more consistent (P/L) ratios may be observed with multiple dispensations of powder & liquid; (2) maximum differences in (P/L) ratio may be negatively correlated with dispensation multiple in some materials.

Radiative hydrodynamic modelling and observations of the X-class solar flare on 2011 March 9

Aims. We investigated the response of the solar atmosphere to non-thermal electron beam heating using the radiative transfer and hydrodynamics modelling code RADYN. The temporal evolution of the parameters that describe the non-thermal electron energy distribution were derived from hard X-ray observations of a particular flare, and we compared the modelled and observed parameters.

Methods. The evolution of the non-thermal electron beam parameters during the X1.5 solar flare on 2011 March 9 were obtained from analysis of RHESSI X-ray spectra. The RADYN flare model was allowed to evolve for 110 s, after which the electron beam heating was ended, and was then allowed to continue evolving for a further 300 s. The modelled flare parameters were compared to the observed parameters determined from extreme-ultraviolet spectroscopy.

Results. The model produced a hotter and denser flare loop than that observed and also cooled more rapidly, suggesting that additional energy input in the decay phase of the flare is required. In the explosive evaporation phase a region of high-density cool material propagated upward through the corona. This material underwent a rapid increase in temperature as it was unable to radiate away all of the energy deposited across it by the non-thermal electron beam and via thermal conduction. A narrow and high-density (ne ≤ 1015 cm-3) region at the base of the flare transition region was the source of optical line emission in the model atmosphere. The collision-stopping depth of electrons was calculated throughout the evolution of the flare, and it was found that the compression of the lower atmosphere may permit electrons to penetrate farther into a flaring atmosphere compared to a quiet Sun atmosphere.

Prostaglandin E2 as a regulator of germ cells during ovarian development

CONTEXT: The formation of primordial follicles occurs during fetal life yet is critical to the determination of adult female fertility. Prior to this stage, germ cells proliferate, enter meiosis, and associate with somatic cells. Growth and survival factors implicated in these processes include activin A (INHBA), the neurotrophins BDNF and NT4 (NTF5), and MCL1. The prostaglandins have pleiotrophic roles in reproduction, notably in ovulation and implantation, but there are no data regarding roles for prostaglandins in human fetal ovarian development.

OBJECTIVE: The aim of the study was to investigate a possible role for prostaglandin (PG) E(2) in human fetal ovary development.

DESIGN: In vitro analysis of ovarian development between 8 and 20 wk gestation was performed.

MAIN OUTCOME MEASURE(S): The expression patterns of PG synthesis enzymes and the PGE(2) receptors EP2 and EP4 in the ovary were assessed, and downstream effects of PGE(2) on gene expression were analyzed.

RESULTS: Ovarian germ cells express the PG synthetic enzymes COX2 and PTGES as well as the EP2 and EP4 receptors, whereas COX1 is expressed by ovarian somatic cells. Treatment in vitro with PGE(2) increased the expression of BDNF mRNA 1.7 +/- 0.16-fold (P = 0.004); INHBA mRNA, 2.1 +/- 0.51-fold (P = 0.04); and MCL1 mRNA, 1.15 +/- 0.06-fold (P = 0.04), but not that of OCT4, DAZL, VASA, NTF5, or SMAD3.

CONCLUSIONS: These data indicate novel roles for PGE(2) in the regulation of germ cell development in the human ovary and show that these effects may be mediated by the regulation of factors including BDNF, activin A, and MCL1.

Ultrashort self-assembling peptidomimetic nanomaterials target resistant pathogenic infections

The impending and increasing threat of antimicrobial resistance has led to a greater focus into developing alternative therapies as substitutes for traditional antibiotics for the treatment of multi-drug resistant infections.1 Our group has developed a library of short, cost-effective, diphenylalanine-based peptides (X1-FF-X2) which selective eradicate (viability reduced >90% in 24 hours) the most resistant biofilm forms of a range of Gram-positive and negative pathogens including: methicillin resistant and sensitive Staphyloccoccus aureus and Staphyloccoccus epidermidis; Pseudomonas aeruginosa, Proteus mirabilis and Escherichia coli. They demonstrate a reduced cell cytotoxic profile (NCTC929 murine fibroblast) and limited haemolysis.2 Our molecules have the ability respond to subtle changes in pH, associated with bacterial infection, self-assembling to form β-sheet secondary structures and supramolecular hydrogels at low concentrations (~0.5%w/v). Conjugation of variety of aromatic-based drugs at the X1 position, including non-steroidal anti-inflammatories (NSAIDs), confer further pharmacological properties to the peptide motif enhancing their therapeutic potential. In vivo studies using waxworms (Galleria mellonella) provide promising preliminary results demonstrating the low toxicity and high antimicrobial activity of these low molecular weight gelators in animal models. This work shows biofunctional peptide-based nanomaterials hold great promise for future translation to patients as antimicrobial drug delivery and biomaterial platforms.3 [1] G. Laverty, S.P. Gorman and B.F. Gilmore. Int.J.Mol.Sci. 2011, 12, 6566-6596. [2] G. Laverty, A.P. McCloskey, B.F. Gilmore, D.S. Jones, J Zhou, B Xu. Biomacromolecules. 2014, 15, 9, 3429-3439. [3] A.P. McCloskey, B.F. Gilmore and G.Laverty. Pathogens. 2014, 3, 791-821.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce anti-proliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G2/M arrest. G2/M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xL and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G2/M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-xL. Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOX-induced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

The novel pyrrolo-1,5-benzoxazepine, PBOX-21, potentiates the apoptotic efficacy of STI571 (imatinib mesylate) in human chronic myeloid leukaemia cells

The Bcr-Abl kinase inhibitor, STI571, is the first line treatment for chronic myeloid leukaemia (CML), but the recent emergence of STI571 resistance has led to the examination of combination therapies. In this report, we describe how a novel non-toxic G1-arresting compound, pyrrolo-1,5-benzoxazepine (PBOX)-21, potentiates the apoptotic ability of STI571 in Bcr-Abl-positive CML cells. Co-treatment of CML cells with PBOX-21 and STI571 induced more apoptosis than either drug alone in parental (K562S and LAMA84) and STI571-resistant cells lines (K562R). This potentiation of apoptosis was specific to Bcr-Abl-positive leukaemia cells with no effect observed on Bcr-Abl-negative HL-60 acute myeloid leukaemia cells. Apoptosis induced by PBOX-21/STI571 resulted in activation of caspase-8, cleavage of PARP and Bcl-2, upregulation of the pro-apoptotic protein Bim and a downregulation of Bcr-Abl. Repression of proteins involved in Bcr-Abl transformation, the anti-apoptotic proteins Mcl-1 and Bcl-(XL) was also observed. The combined lack of an early change in mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9 suggests that this pathway is not involved in the initiation of apoptosis by PBOX-21/STI571. Apoptosis was significantly reduced following pre-treatment with either the general caspase inhibitor Boc-FMK or the chymotrypsin-like serine protease inhibitor TPCK, but was completely abrogated following pre-treatment with a combination of these inhibitors. This demonstrates the important role for each of these protease families in this apoptotic pathway. In conclusion, our data highlights the potential of PBOX-21 in combination with STI571 as an effective therapy against CML.

STI-571 (imatinib mesylate) enhances the apoptotic efficacy of pyrrolo-1,5-benzoxazepine-6, a novel microtubule-targeting agent, in both STI-571-sensitive and -resistant Bcr-Abl-positive human chronic myeloid leukemia cells

Interactions between the Bcr-Abl kinase inhibitor STI-571 (imatinib mesylate) and a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-6, were investigated in STI-571-sensitive and -resistant human chronic myeloid leukemia (CML) cells. Cotreatment of PBOX-6 with STI-571 induced significantly more apoptosis in Bcr-Abl-positive CML cell lines (K562 and LAMA-84) than either drug alone (P < 0.01). Cell cycle analysis of propidium iodide-stained cells showed that STI-571 significantly reduced PBOX-6-induced G2M arrest and polyploid formation with a concomitant increase in apoptosis. Similar results were obtained in K562 CML cells using lead MTAs (paclitaxel and nocodazole) in combination with STI-571. Potentiation of PBOX-6-induced apoptosis by STI-571 was specific to Bcr-Abl-positive leukemia cells with no cytoxic effects observed on normal peripheral blood cells. The combined treatment of STI-571 and PBOX-6 was associated with the down-regulation of Bcr-Abl and repression of proteins involved in Bcr-Abl transformation, namely the antiapoptotic proteins Bcl-x(L) and Mcl-1. Importantly, PBOX-6/STI-571 combinations were also effective in STI-571-resistant cells. Together, these findings highlight the potential clinical benefits in simultaneously targeting the microtubules and the Bcr-Abl oncoprotein in STI-571-sensitive and -resistant CML cells.

Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas, respectively

Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB targets and their expression distinguished small and aggressive B-cell lymphomas, respectively. Interestingly, in the vast majority of B-cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

Departure of high temperature iron lines from the equilibrium state in flaring solar plasmas

The aim of this study is to clarify if the assumption of ionization equilibrium and a Maxwellian electron energy distribution is valid in flaring solar plasmas. We analyze the 2014 December 20 X1.8 flare, in which the \ion{Fe}{xxi} 187~\AA, \ion{Fe}{xxii} 253~\AA, \ion{Fe}{xxiii} 263~\AA\ and \ion{Fe}{xxiv} 255~\AA\ emission lines were simultaneously observed by the EUV Imaging Spectrometer onboard the Hinode satellite. Intensity ratios among these high temperature Fe lines are compared and departures from isothermal conditions and ionization equilibrium examined. Temperatures derived from intensity ratios involving these four lines show significant discrepancies at the flare footpoints in the impulsive phase, and at the looptop in the gradual phase. Among these, the temperature derived from the \ion{Fe}{xxii}/\ion{Fe}{xxiv} intensity ratio is the lowest, which cannot be explained if we assume a Maxwellian electron distribution and ionization equilibrium, even in the case of a multi-thermal structure. This result suggests that the assumption of ionization equilibrium and/or a Maxwellian electron energy distribution can be violated in evaporating solar plasma around 10MK.

Improved clonality assessment in germinal centre/post-germinal centre non-Hodgkin's lymphomas with high rates of somatic hypermutation.

BACKGROUND: PCR detects clonal rearrangements of the Ig gene in lymphoproliferative disorders. False negativity occurs in germinal centre/post-germinal centre lymphomas (GC/PGCLs) as they display a high rate of somatic hypermutation (SHM), which causes primer mismatching when detecting Ig rearrangements by PCR. AIMS: To investigate the degree of SHM in a group of GC/PGCLs and assess the rate of false negativity when using BIOMED-2 PCR when compared with previously published strategies. METHODS: DNA was isolated from snap-frozen tissue from 49 patients with GC/PGCL (23 diffuse large B cell lymphomas (DLBCLs), 26 follicular lymphomas (FLs)) and PCR-amplified for complete (VDJH), incomplete (DJH) and Ig kappa/lambda rearrangements using the BIOMED-2 protocols, and compared with previously published methods using consensus primers. Germinal centre phenotype was defined by immunohistochemistry based on CD10, Bcl-6 and MUM-1. RESULTS: Clonality detection by amplifying Ig rearrangements using BIOMED-2 family-specific primers was considerably higher than that found using consensus primers (74% DLBCL and 96% FL vs 69% DLBCL and 73% FL). Addition of BIOMED-2 DJH rearrangements increased detection of clonality by 22% in DLBCL. SHM was present in VDJH rearrangements from all patients with DLBCL (median (range) 5.7% (2.5-13.5)) and FL (median (range) 5.3% (2.3-11.9)) with a clonal rearrangement. CONCLUSIONS: Use of BIOMED-2 primers has significantly reduced the false negative rate associated with GC/PGCL when compared with consensus primers, and the inclusion of DJH rearrangements represents a potential complementary target for clonality assessment, as SHM is thought not to occur in these types of rearrangements.