Line Deletion Research Articles

Structure, Activity and Function of the MLL2 (KMT2B) Protein Lysine Methyltransferase.

The Mixed Lineage Leukemia 2 (MLL2) protein, also known as KMT2B, belongs to the family of mammalian histone H3 lysine 4 (H3K4) methyltransferases. It is a large protein of 2715 amino acids, widely expressed in adult human tissues and a paralog of the MLL1 protein. MLL2 contains a characteristic C-terminal SET domain responsible for methyltransferase activity and forms a protein complex with WRAD (WDR5, RbBP5, ASH2L and DPY30), host cell factors 1/2 (HCF 1/2) and Menin. The MLL2 complex is responsible for H3K4 trimethylation (H3K4me3) on specific gene promoters and nearby cis-regulatory sites, regulating bivalent developmental genes as well as stem cell and germinal cell differentiation gene sets. Moreover, MLL2 plays a critical role in development and germ line deletions of Mll2 have been associated with early growth retardation, neural tube defects and apoptosis that leads to embryonic death. It has also been involved in the control of voluntary movement and the pathogenesis of early stage childhood dystonia. Additionally, tumor-promoting functions of MLL2 have been detected in several cancer types, including colorectal, hepatocellular, follicular cancer and gliomas. In this review, we discuss the main structural and functional aspects of the MLL2 methyltransferase with particular emphasis on transcriptional mechanisms, gene regulation and association with diseases.

Breast cancer bone metastases are attenuated in a Tgif1-deficient bone microenvironment

BackgroundOsteoclast activation is a hallmark of breast cancer-induced bone disease while little is known about the role of osteoblasts in this process. Recently, we identified the homeodomain protein TG-interacting factor-1 (Tgif1) as a crucial regulator of osteoblast function. In this study, we demonstrate that lack of Tgif1 also restricts the progression of breast cancer bone metastases.MethodsTranswell migration assays were used to investigate the osteoblast-breast cancer cell interaction in vitro. Molecular analyses included RNA sequencing, immunoblotting, and qRT-PCR. To determine the role of Tgif1 in metastatic bone disease, 4T1 breast cancer cells were injected intracardially into mice with a germ line deletion of Tgif1 (Tgif1−/−) or control littermates (Tgif1+/+). Progression of bone metastases and alterations in the bone microenvironment were assessed using bioluminescence imaging, immunofluorescence staining, confocal microscopy, and histomorphometry.ResultsMedium conditioned by osteoblasts stimulated breast cancer cell migration, indicating a potential role of osteoblasts during bone metastasis progression. Tgif1 expression was strongly increased in osteoblasts upon stimulation by breast cancer cells, demonstrating the implication of Tgif1 in the osteoblast-breast cancer cell interaction. Indeed, conditioned medium from osteoblasts of Tgif1−/− mice failed to induce breast cancer cell migration compared to control, suggesting that Tgif1 in osteoblasts augments cancer cell motility. Semaphorin 3E (Sema3E), which is abundantly secreted by Tgif1−/− osteoblasts, dose-dependently reduced breast cancer cell migration while silencing of Sema3E expression in Tgif1−/− osteoblasts partially restored the impaired migration. In vivo, we observed a decreased number of breast cancer bone metastases in Tgif1−/− mice compared to control littermates. Consistently, the presence of single breast cancer cells or micro-metastases in the tibiae was reduced in Tgif1−/− mice. Breast cancer cells localized in close proximity to Endomucin-positive vascular cells as well as to osteoblasts. Although Tgif1 deficiency did not affect the bone marrow vasculature, the number and activity of osteoblasts were reduced compared to control. This suggests that the protective effect on bone metastases might be mediated by osteoblasts rather than by the bone marrow vasculature.ConclusionWe propose that the lack of Tgif1 in osteoblasts increases Sema3E expression and attenuates breast cancer cell migration as well as metastases formation.

Evaluating single-subject study methods for personal transcriptomic interpretations to advance precision medicine

BackgroundGene expression profiling has benefited medicine by providing clinically relevant insights at the molecular candidate and systems levels. However, to adopt a more ‘precision’ approach that integrates individual variability including ‘omics data into risk assessments, diagnoses, and therapeutic decision making, whole transcriptome expression needs to be interpreted meaningfully for single subjects. We propose an “all-against-one” framework that uses biological replicates in isogenic conditions for testing differentially expressed genes (DEGs) in a single subject (ss) in the absence of an appropriate external reference standard or replicates. To evaluate our proposed “all-against-one” framework, we construct reference standards (RSs) with five conventional replicate-anchored analyses (NOISeq, DEGseq, edgeR, DESeq, DESeq2) and the remainder were treated separately as single-subject sample pairs for ss analyses (without replicates).ResultsEight ss methods (NOISeq, DEGseq, edgeR, mixture model, DESeq, DESeq2, iDEG, and ensemble) for identifying genes with differential expression were compared in Yeast (parental line versus snf2 deletion mutant; n = 42/condition) and a MCF7 breast-cancer cell line (baseline versus stimulated with estradiol; n = 7/condition). Receiver-operator characteristic (ROC) and precision-recall plots were determined for eight ss methods against each of the five RSs in both datasets. Consistent with prior analyses of these data, ~ 50% and ~ 15% DEGs were obtained in Yeast and MCF7 datasets respectively, regardless of the RSs method. NOISeq, edgeR, and DESeq were the most concordant for creating a RS. Single-subject versions of NOISeq, DEGseq, and an ensemble learner achieved the best median ROC-area-under-the-curve to compare two transcriptomes without replicates regardless of the RS method and dataset (> 90% in Yeast, > 0.75 in MCF7). Further, distinct specific single-subject methods perform better according to different proportions of DEGs.ConclusionsThe “all-against-one” framework provides a honest evaluation framework for single-subject DEG studies since these methods are evaluated, by design, against reference standards produced by unrelated DEG methods. The ss-ensemble method was the only one to reliably produce higher accuracies in all conditions tested in this conservative evaluation framework. However, single-subject methods for identifying DEGs from paired samples need improvement, as no method performed with precision> 90% and obtained moderate levels of recall.http://www.lussiergroup.org/publications/EnsembleBiomarker

Germ Line Deletion Reveals a Nonessential Role of Atypical Mitogen-Activated Protein Kinase 6/Extracellular Signal-Regulated Kinase 3.

Mitogen-activated protein kinase 6/extracellular signal-regulated kinase 3 (MAPK6/ERK3) is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary dysfunction and by defects in function, activation, and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon 3 flanked by loxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing the first evidence for the existence of the ERK3/MK5 signaling complex in vivo In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile and do not display pulmonary hypoplasia, acute respiratory failure, abnormal T-cell development, reduction of thymocyte numbers, or altered T-cell selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality and lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin resistance cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal, suggesting redundant functions of both protein kinases.

Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation

CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins.Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE.Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp).To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice.In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation. DisclosuresNo relevant conflicts of interest to declare.

Transferrin receptor 2 is a potential novel therapeutic target for β-thalassemia: evidence from a murine model

Abstractβ-thalassemias are genetic disorders characterized by anemia, ineffective erythropoiesis, and iron overload. Current treatment of severe cases is based on blood transfusion and iron chelation or allogeneic bone marrow (BM) transplantation. Novel approaches are explored for nontransfusion-dependent patients (thalassemia intermedia) who develop anemia and iron overload. Here, we investigated the erythropoietin (EPO) receptor partner, transferrin receptor 2 (TFR2), as a novel potential therapeutic target. We generated a murine model of thalassemia intermedia specifically lacking BM Tfr2: because their erythroid cells are more susceptible to EPO stimulation, mice show improved erythropoiesis and red blood cell morphology as well as partial correction of anemia and iron overload. The beneficial effects become attenuated over time, possibly due to insufficient iron availability to sustain the enhanced erythropoiesis. Germ line deletion of Tfr2, including haploinsufficiency, had a similar effect in the thalassemic model. Because targeting TFR2 enhances EPO-mediated effects exclusively in cells expressing both receptors, this approach may have advantages over erythropoiesis-stimulating agents in the treatment of other anemias.

The Fat Mass- and Obesity-Associated (FTO) Gene to Obesity: Lessons from Mouse Models.

Genetic variants at the fat mass- and obesity-associated (FTO) locus are strongly associated with obesity-related traits by regulating neighboring genes. Nevertheless, it is possible that FTO protein is directly involved in mechanisms regulating body composition and adiposity. Here, the in vivo biological functions of FTO in the risk for obesity were studied by reviewing murine models. The effects of the locus-specific manipulations of the murine Fto gene on metabolic-related phenotypes in genetically modified mouse models were reviewed and summarized into the following three categories: growth and body composition, eating behaviors, and metabolic homeostasis. The mouse models showed different phenotypes depending on target tissues and methods for gene manipulation. Mice harboring deletions or point mutations at the Fto locus had high metabolic rates, while FTO-overexpressing mice showed dyslipidemia. Both deletion and overexpression of the Fto gene led to abnormal eating behaviors. Intriguingly, several phenotypes were differently expressed depending on developmental timing of the genetic manipulations. For instance, a germ line deletion decreased total body fat mass, while the deletion in adult mice increased it. The results highlight that FTO is critical not only for body composition but also normal development, and its function might differ depending on the stage of development.

Screening of melon genotypes identifies gummy stem blight resistance associated with Gsb1 resistant loci

Md Zahid Hassan, Arif Hasan Khan Robin, Md Abdur Rahim, Sathishkumar Natarajan, Hoy-Taek Kim, Jong-In Park, and Ill-Sup Nou. J Plant Biotechnol 2018;45:217-27. https://doi.org/10.5010/JPB.2018.45.3.217

A novel large germ line deletion in adenomatous polyposis coli (APC) gene associated with familial adenomatous polyposis.

BackgroundFamilial adenomatous polyposis (FAP) is a familial colorectal cancer predisposition syndrome characterized by the development of numerous colorectal polyps, which is inherited in an autosomal dominant manner. FAP is caused by germ line mutations in adenomatous polyposis coli (APC) gene. Here, we described the identification of a causative APC gene deletion associated with FAP in an Iranian family.MethodsDiagnosis of FAP was based on clinical findings, family history, and medical records (colonoscopy and histopathological data) after the patients were referred to Reza Radiotherapy and Oncology Center, Iran, for colonoscopy. Blood samples were collected, and genomic DNA was extracted. APC mutation screening was conducted by target next‐generation sequencing and quantitative real‐time PCR.ResultsA novel heterozygous large deletion mutation, c.(135+1_136–1)_(*2113+1_*2114–1) spanning exon 3 to 16 [EX3_16 DEL] of APC gene (GenBank Accession# MG712911), was detected in a proband and all her affected relatives in five generations, which was absent in unaffected family members and normal controls.ConclusionsThis novel deletion is the first report, describing the largest deletion of APC gene. Our novel finding contributes to a more comprehensive database of germ line mutations of APC gene that could be used in medical practice for the molecular diagnosis, risk assessment susceptibility of the disease for the FAP patients.

A Hybrid Domain Color Image Watermarking Based on DWT–SVD

In recent years, digital image watermarking has been emerged as a promising solution for copyright protection and authentication problem of digital images. Among several existing watermarking schemes, very few have been designed for the authenticity and copyright protection problem of color images. To protect the copyright and validate the authenticity of color images, in this paper a hybrid robust image watermarking scheme based on discrete wavelet transform (DWT) and singular value decomposition is proposed and simulated. Initially, RGB color image is switched to YCbCr color space from which only Y achromatic component is considered for watermark data insertion. In the next step, Y component of the YCbCr color model is decomposed into non-overlapping blocks and subsequently DWT are applied to each block. In this work, non-overlapping decomposed gray watermark image blocks are embedded into singular values of DWT-transformed cover image blocks. Proposed technique’s simulation result established the robustness by extracting adequate watermark data from the restructured cover image after applying common geometric transformation attacks (like rotation, flip operation, cropping, scaling, shearing and deletion of lines or column operation), common enhancement technique attacks (like lowpass filtering, histogram equalization, sharpening, gamma correction, noise addition), JPEG compression attacks and combinational attacks (like combination of geometric transformation attacks and common enhancement technique attacks, combination of geometric transformation attacks and JPEG compression attacks, combination of common enhancement technique attacks and JPEG compression attacks, and combination of geometric transformation attacks and geometric transformation attacks).

A parental requirement for dual-specificity phosphatase 6 in zebrafish

BackgroundSignaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in embryogenesis remain to be fully understood.ResultsUsing CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the 1-cell stage for several hours before dying by 10 h post fertilization. We demonstrate that dusp6 is expressed in gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults.ConclusionsThe fact that offspring of homozygous dusp6 mutants stall prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis and/or parentally-directed early development. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for proper embryogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other regulators of the ERK pathway compensate for loss of dusp6 at later stages.

Potent suppression of both spontaneous and carcinogen-induced colitis-associated colorectal cancer in mice by dietary celastrol supplementation.

Celastrol is an anti-inflammatory natural triterpenoid, isolated from the herb Tripterygium wilfordii or thunder god vine. Here, we define mechanisms mediating anti-inflammatory activity of celastrol and demonstrate efficacy of a dietary celastrol supplement for chemoprevention of inflammation-driven carcinogenesis in mice. Dietary celastrol (31.25 ppm in rodent diet from 8 weeks to 25 weeks of age) is well tolerated and protects against LPS-induced acute inflammation in C57BL/6 mice, potently suppressing LPS-induction of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, Interleukin (IL)-6 and IL-1β. To test whether dietary celastrol suppresses inflammation-driven colorectal cancer (CRC), we employed a unique model of spontaneous, inflammation-driven CRC in mice harboring a germ line deletion of the p27Kip1 gene and a T cell-specific deletion of Smad4 gene (Smad4co/co;Lck-crep27Kip1-/-or DKO), which develop severe intestinal inflammation and carcinogenesis as early as 3 months of age. Exposure of DKO mice to daily dietary celastrol (12.5 ppm in diet) from 6 weeks of age significantly suppressed development of colitis-associated CRC (CAC). Celastrol chemoprevention of CAC in this new model of intestinal neoplasia was associated with significant suppression of iNOS at 4 months of age, and iNOS, COX-2 and NFκB at 6 months of age, with significant reduction in inflammatory cytokines, IL-6 and IL-1β. Chemoprevetion of CAC by dietary celastrol was further confirmed in the model of azoxymethane (AOM) plus dextran sodium sulfate (DSS)-induced carcinogenesis in C57BL/6 mice. These data suggest the potential for celastrol as a safe and effective dietary supplement in the chemoprevention of CAC in humans.

An SVD Based Location Specific Robust Color Image Watermarking Scheme Using RDWT and Arnold Scrambling

In recent times, effectiveness of the robust image watermarking scheme almost successfully solves the authenticity and copyright protection problem of images. Among several existing robust watermarking schemes, very few have been designed for color images. In this paper, the authors have proposed a redundant discrete wavelet transform (RDWT) and singular value decomposition based hybrid robust color image watermarking method where scrambled watermark is used for embedding purpose to provide extra security to authentic logo. At first, RGB color image is converted to YCbCr color image and then the Y component of YCbCr color space is used to insert Arnold scrambled grayscale watermark. In the next step, Y component of the YCbCr color model is decomposed into non overlapping blocks and subsequently RDWT are applied to each block. For better imperceptibility and effectiveness, in this proposed algorithm some singular values of RDWT transformed Y component blocks are modified by non overlapping decomposed and scrambled watermark blocks. Robustness of the proposed method is experimented against common geometric transformation attacks (like rotation, flip operation, cropping, scaling, shearing and deletion of lines or column operation etc.), common enhancement technique attacks (like lowpass filtering, histogram equalization, sharpening, gamma correction, noise addition etc.), jpeg compression attacks, some combinational attacks and satisfactory results are achieved. For better evaluation, proposed scheme is verified against standard benchmark software “Checkmark”.

GATA2 haploinsufficiency accelerates EVI1-driven leukemogenesis

AbstractChromosomal rearrangements between 3q21 and 3q26 induce inappropriate EVI1 expression by recruiting a GATA2-distal hematopoietic enhancer (G2DHE) to the proximity of the EVI1 gene, leading to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The acquisition of G2DHE by the EVI1 gene reciprocally deprives this enhancer of 1 of the 2 GATA2 alleles, resulting in a loss-of-function genetic reduction in GATA2 abundance. Because GATA2 haploinsufficiency is strongly associated with MDS and AML, we asked whether EVI1 misexpression and GATA2 haploinsufficiency both contributed to the observed leukemogenesis by using a 3q21q26 mouse model that recapitulates the G2DHE-driven EVI1 misexpression, but in this case, it was coupled to a Gata2 heterozygous germ line deletion. Of note, the Gata2 heterozygous deletion promoted the EVI1-provoked leukemic transformation, resulting in early onset of leukemia. The 3q21q26 mice suffered from leukemia in which B220+ cells and/or Gr1+ leukemic cells occupied their bone marrows. We found that the B220+Gr1–c-Kit+ population contained leukemia-initiating cells and supplied Gr1+ leukemia cells in the 3q21q26 leukemia. When Gata2 expression levels in the B220+Gr1–c-Kit+ cells were decreased as a result of Gata2 heterozygous deletion or spontaneous phenomenon, myeloid differentiation of the B220+Gr1–c-Kit+ cells was suppressed, and the cells acquired induced proliferation as well as B-lymphoid–primed characteristics. Competitive transplantation analysis revealed that Gata2 heterozygous deletion confers selective advantage to EVI1-expressing leukemia cell expansion in recipient mice. These results demonstrate that both the inappropriate stimulation of EVI1 and the loss of 1 allele equivalent of Gata2 expression contribute to the acceleration of leukemogenesis.

Unphosphorylated STAT3 regulates the antiproliferative, antiviral, and gene-inducing actions of type I interferons

Type I interferon (IFNα/β) induces antiviral and antiproliferative responses in cells through the induction of IFN-stimulated genes (ISGs). Although the roles of IFN-activated STAT1 and STAT2 in the IFN response are well described, the function of STAT3 is poorly characterized. We investigated the role of STAT3 in the biological response to IFNα/β in mouse embryonic fibroblasts (MEFs) with a germ line deletion of STAT3. These STAT3 knockout (STAT3-KO) MEFs were reconstituted with STAT3 or the F705-STAT3 mutant (unphosphorylated STAT3) where the canonical Y705 tyrosine phosphorylation site was mutated. We show that both STAT3 and unphosphorylated STAT3 expression enhance the sensitivity of MEFs to the antiviral, antiproliferative and gene-inducing actions of IFN. By chromatin immunoprecipitation assays, unphosphorylated STAT3 appears to bind, albeit weakly, to select gene promoters to enhance their expression. These results suggest that unphosphorylated STAT3 plays an important role in the IFN response pathway.

An indirect watermark hiding in discrete cosine transform-singular value decomposition domain for copyright protection.

Digital image watermarking has emerged as a promising solution for copyright protection. In this paper, a discrete cosine transform (DCT) and singular value decomposition (SVD) based hybrid robust image watermarking method using Arnold scrambling is proposed and simulated to protect the copyright of natural images. In this proposed scheme, before embedding, watermark is scrambled with Arnold scrambling. Then, the greyscale cover image and encrypted watermark logo are decomposed into non-overlapping blocks and subsequently some selected image blocks are transformed into the DCT domain for inserting the watermark blocks permanently. For better imperceptibility and effectiveness, in this proposed algorithm, watermark image blocks are embedded into singular values of selected blocks by multiplying with a feasible scaling factor. Simulation result demonstrates that robustness is achieved by recovering satisfactory watermark data from the reconstructed cover image after applying common geometric transformation attacks (such as rotation, flip operation, cropping, scaling, shearing and deletion of lines or columns operation), common enhancement technique attacks (such as low-pass filtering, histogram equalization, sharpening, gamma correction, noise addition) and jpeg compression attacks.

IBRUTINIB FOR RELAPSED / REFRACTORY CLL: AN UPDATE OF THE UK AND IRELAND ANALYSIS OF OUTCOMES IN 315 PATIENTS

Introduction: The UK CLL Forum recently published the largest real-world data set of patients treated with ibrutinib for relapsed/refractory CLL (Haematologica, 2016). We have now updated our database with an additional 14 months median follow-up. Methods: Data were collected retrospectively from 62 centres, and discontinuation free survival (DFS) and overall survival (OS) were analysed and stratified by baseline and treatment-related characteristics. Results: Of the original 315 patients, 103 have now died, 37 are alive but have stopped ibrutinib, and 175 continue to take the drug. With a median 30 months follow-up, 54.4% (95% CI: 48.5–60.0) of patients are still taking ibrutinib and 66.1% (60.4–71.3) are still alive. Despite the additional follow-up, with univariable analysis, we could still not show any deleterious effect of 17p deletion or number of prior lines of therapy on discontinuation-free survival or overall survival (OS) (OS for 17p deleted vs 17p wildtype: 68.6% vs 67.8%; OS for number of prior lines 1 vs 2 vs 3+: 66.0% vs 64.9% vs 66.6%). However, increased age and poorer pre-treatment performance status remain associated with ibrutinib discontinuation and earlier death. With our original publication, we were unable to demonstrate any survival detriment for dose-reduced patients, although patients who had treatment breaks >14 days, had inferior outcomes. Of the 48 patients who had dose-reduced ibrutinib without treatment breaks in the first year, 17 have now stopped the drug, 10 have died, 25 continue on dose-reduced ibrutinib and 6 are back to standard dose. To analyse on-going potential effects of first year dose reductions and treatment breaks, we classified all patients who were alive at 1 year into one of 4 different groups depending on first year treatment, namely, A: standard dosing with no breaks >14 days; B: dose reductions but no breaks >14 days; C1: temporary treatment breaks >14 days (but re-taking ibrutinib by 1 year); C2: permanent discontinuation of ibrutinib within the first year. Figure 1 plots DFS and OS with 18 months median follow-up for these patients beyond one year. There is no statistical difference in DFS and OS between groups A and B (DFS; 76.7% (68.3–83.1) vs 71.1% (53.3–83.1), p = 0.23: OS; 88.1% (81.6–92.5) vs 83.7% (66.8–92.5), p = 0.75). However, patients who had temporary breaks >14 days (C1) or permanently stopped ibrutinib (C2) within the first year have impaired survival over the next 18 months compared with groups A and B (OS C1: 63% (41.4–78.5); OS C2: 40.6% (22.7–57.8).This represents a 3.1-fold and 7.7-fold respective increase risk of death for groups C1 and C2 compared with standard dosed patients. Keywords: chronic lymphocytic leukemia (CLL); ibrutinib.

An empirical study of crash-inducing commits in Mozilla Firefox

Software crashes are dreaded by both software organisations and end-users. Many software organisations have automatic crash reporting tools embedded in their software systems to help quality-assurance teams track and fix crash-related bugs. Previous approaches, which focused on the triaging of crash-types and crash-related bugs, can help software organisations increase their debugging efficiency of crashes. However, these approaches can only be applied after the software systems have been crashing for a certain period of time. To help software organisations detect and fix crash-prone code earlier, we examine the characteristics of commits that lead to crashes, which we call crash-inducing commits, in Mozilla Firefox. We observe that crash-inducing commits are often submitted by developers with less experience and that developers perform more addition and deletion of lines of code in crash-inducing commits but also that they need less effort to fix the bugs caused by these commits. We also characterise commits that would lead to frequent crashes, which impact a large user base, which we call highly impactful crash-inducing commits. Compared to other crash-related bugs, we observe that bugs due to highly impactful crash-inducing commits were less reopened by developers and tend to be fixed by a single commit. We build predictive models to help software organisations detect and fix crash-prone bugs early, when their developers commit code. Our predictive models achieve a precision of 61.2% and a recall of 94.5% to predict crash-inducing commits and a precision of 60.9% and a recall of 91.1% to predict highly impactful crash-inducing commits. Software organisations could use our models and approach to track and fix crash-prone commits early, before they negatively impact users, thus increasing bug fixing efficiency and user-perceived quality.

A Patient with Interstitial 5q21 Deletion, Familial Adenomatous Polyposis, Dysmorphic Features, and Profound Neurologic Dysfunction

Familial adenomatous polyposis (FAP) is a hereditary autosomal dominant cancer syndrome, results from germ line mutation or deletion of the Adenomatous Polyposis Coli (APC) gene on chromosome 5q21. Patients with FAP suffer from multiple polyps mainly at the colorectal region as well as other parts of the gastrointestinal tract, which has propensity to transform into carcinoma. FAP has also been well described in association with various syndromic extra-gastrointestinal manifestations. Less commonly, patients with FAP present with varying degrees of cognitive dysfunction and developmental delay, though the reason for the association is unclear. Herein, we report the case of a male patient born with an interstitial deletion of chromosome 5q, 46,XY, del(5) (q14q23), presenting with familial adenomatous polyposis (FAP), profound developmental delay, cognitive dysfunction, and multiple congenital anomalies including talipes equinovarus, agenesis of the corpus callosum, and dysmorphic facial features.

14-3-3epsilon controls multiple developmental processes in the mouse heart.

14-3-3ε plays an important role in the maturation of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via p27kip1 . However, additional cardiac defects are possible given the ubiquitous expression pattern of this protein. Germ line deletion of 14-3-3ε led to malalignment of both the outflow tract (OFT) and atrioventricular (AV) cushions, with resulting tricuspid stenosis and atresia, mitral valve abnormalities, and perimembranous ventricular septal defects (VSDs). We confirmed myocardial non-compaction and detected a spongy septum with muscular VSDs and blebbing of the epicardium. These defects were associated with abnormal patterning of p27kip1 expression in the subendocardial and possibly the epicardial cell populations. In addition to abnormal pharyngeal arch artery patterning, we found deep endocardial recesses and paucity of intramyocardial coronary vasculature as a result of defective coronary plexus remodeling. The malalignment of both endocardial cushions provides a new explanation for tricuspid and mitral valve defects, while myocardial non-compaction provides the basis for the abnormal coronary vasculature patterning. These abnormalities might arise from p27kip1 dysregulation and a resulting defect in epithelial-to-mesenchymal transformation. These data suggest that 14-3-3ε, in addition to left ventricular non-compaction (LVNC), might be linked to different forms of congenital heart disease (CHD). Developmental Dynamics 245:1107-1123, 2016. © 2016 Wiley Periodicals, Inc.