julilili42/papers huggingface dataset:
Impaired DNA Damage Repair in Severe Congenital Neutropenia Patients
2016-12-02

Abstract Severe congenital neutropenia (CN) is a pre-malignant bone marrow failure syndrome with maturation arrest of granulopoiesis at the level of promyelocytes in the bone marrow. We hypothesized that increased genetic instability in hematopoietic stem and progenitor cells (HSPC) of CN patients caused by inherited mutations in ELANE (neutrophil elastase) or HAX1(mitochondrial HCLS1-associated protein X-1) may lead to high risk of malignant transformation. Treatment of CN patients with granulocyte-colony stimulating factor (G-CSF) overcomes maturation arrest by forcing unfit HSPC to proliferate and differentiate despite the presence of inherited mutations and thus increasing the risk of leukemogenic transformation. We first investigated differences in DNA damage susceptibility of CD34+ and CD33+ bone marrow cells from CN-ELANE (n = 3) and CN-HAX1 (n = 3) patients, as compared to healthy donors using short-term treatment (5 minutes) with bleomycin to induce DNA double-strand breaks. To detect DNA lesions we used the LORD-Q method, a high-sensitivity long-run real-time PCR-based technique for DNA damage quantification (Lehle S. et al., Nucleic Acids Research, 2014). We found no differences in DNA damage induction between both groups of CN patients and healthy donors. Therefore, we hypothesized that not DNA damage but DNA repair mechanisms may be affected in these patients. Indeed, Gene Set Enrichment Analysis (GSEA) of microarray data revealed a marked inhibition of gene expression in pathways associated with DNA double-strand break (DSB) repair, mismatch repair as well as cell cycle regulation in HSPC from CN patients as compared to cells from healthy individuals. Validation by qRT-PCR confirmed severe downregulation of genes related to DSB repair (BRCA1 and RAD51), mismatch repair (MSH2 and PCNA) as well ascell cycle regulation (CHEK2 and CDKN2C) in CD33+ of both CN groups as compared to healthy individuals. Interestingly, CN-ELANE and CN-HAX1 groups behaved similarly with some exceptions showing decreased expression of CDC25B, RAD50 and ATR expression in the CN-HAX1 group only and of MRE11A in the CN-ELANEgroup only. Taken together, disrupted DNA repair and impaired expression of cell cycle regulating genes resulting from inherited mutations in ELANE and HAX1 indicate that HSPC of CN patients are more susceptible to malignant transformation. Disclosures No relevant conflicts of interest to declare.
Knock-Down of the NFAT2 Long and Intermediate Isoforms Leads to CLL Acceleration
2016-12-02

Abstract NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700aa short isoform, 800aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation. We investigated primary samples from CLL patients (n=30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell lines was subsequently assessed in xeno-transplant experiments into NSG mice. While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a five fold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n=6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n=6) and MEC-1 cells with a complete NFAT2 knock-down (n=6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells. In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration Disclosures No relevant conflicts of interest to declare.
Attenuation of Apoptosis Stimulating Protein of p53-1 (ASPP1) Associates with Therapy Failure in Acute Myeloid Leukemia (AML)
2016-12-02

Abstract ASPP1 belongs to a family of p53-binding proteins and enhances apoptosis by stimulation of p53-transactivation of selected proapoptotic target genes. It is preferentially expressed in hematopoietic stem cells (HSC) and together with p53 preserves the genomic integrity of the HSC pool. Consequently, attenuated expression of ASPP1 which is linked to methylation of the promoter region has been associated with malignant transformation and development of acute lymphoblastic leukemia and lymphomas. We now provide evidence that ASPP1 is highly altered in AML suggesting a role in leukemogenesis as well as therapy response. ASPP1 mRNA and protein expression levels of freshly isolated native patient samples (68) and healthy bone marrow donors (29) were determined by qRT-PCR and western immunoblotting. Statistical analyseswere performed. To explore implications of attenuated ASPP1 levels with regard to apoptosis induction and proliferation, ASPP1-expressing leukemia cell lines (MOLM14, Jurkat), native patient blasts or native bone marrow donor samples were stably silenced using a retroviral shRNA approach. Vice versa, ASPP1 was stably overexpressed in AML cell lines expressing per se low ASPP1 levels. Expression was thereby confirmed by qRT-PCR and western blotting. XTT viability and annexin V-based apoptosis assays were performed using standard chemotherapeutics in comparison to empty vector controls. Decitabine was used as an epigenetic sensitizer via hypomethylation of the promoter region. ASPP1 mRNA expression was found to be frequently and highly statistically significantly (p=0.001) attenuated in AML. Low ASPP1 mRNA levels thereby translated into attenuated protein expression. Retroviral ASPP1-interference lead to perturbed proliferation capacities (up to 3-fold increase) and attenuated apoptosis upon standard chemotherapeutics in leukemia cell lines as well as native leukemia blasts. As expected, overexpression of ASPP1 resulted in significantly attenuated proliferation and higher induction of apoptosis in all tested cell lines and patient blasts. Intriguingly, epigenetic therapy using the hypomethylating agent decitabine resulted in upregulation of ASPP1 expression in leukemia cells with originally low basal ASPP1 levels as confirmed by qRT-PCR and western blotting. Consequently, decitabine pretreatment sensitized these patient samples towards chemotherapy with a favorable proapoptotic overall efficacy compared to chemotherapy alone. Our results demonstrate that dysfunctional regulation of ASPP1 expression likely contributes to the biology of leukemogenesis and to primary therapy resistance in a subgroup of patients with acute leukemia and seems to be linked to hypermethylation. Prospective clinical studies are warranted to evaluate the roleas a biomarker for risk stratification in leukemia patients and for monitoring therapy responses. Disclosures No relevant conflicts of interest to declare.
Oncogenic Splicing of the Apoptosis-Stimulating Protein of p53-2 (ASPP2) Is an Initiating Step in Leukemogenesis Facilitating Acquisition of Structural Genomic Mutations
2016-12-02

Abstract ASPP2 is an independent haploinsufficient tumor suppressor which initiates induction of apoptosis after cellular damage in a TP53-dependent manner. We recently reported the identification of two C-terminally truncated splice variants of ASPP2 with high frequency in acute leukemias. Tantalizingly, these variants lack the functionally relevant TP53 binding sites, suggesting a dominant-negative, oncogenic function. The most prevalent isoform lacking exon 17 (ASPP2k) is detected in up to 60% of acute leukemias. A cell sort for the CD34+ stem cell fraction confirmed ASPP2k-expression in the leukemia-initiating clone. Immunoblotting revealed translation of ASPP2k into a genuine protein isoform. Generation of isoform-specific antibodies allows to discriminate ASPP2k from the wildtype isoforms. Interestingly, ASPP2k is the sole isoform expressed in some native leukemia samples. Other samples reveal a heterozygous expression pattern suggesting a dominant-negative function of the C-terminally truncated ASPP2k isoform. Forced expression of ASPP2k using a HisMax plasmid vector in murine IL3-dependent pro-B Ba/F3 cells induced a more aggressive phenotype with mitotic failure and perturbed cellular proliferation compared to the empty vector controls. Of note, IL3 was successfully weaned in the ASPP2k cells - indicating that autoactivating genomic alterations must have occurred upholding cellular integrity and viability. Fractionated gamma irradiation accelerated this process. In contrast, the empty vector strains did not survive IL3 weaning. Chromosomal analyses of IL3-independent Ba/F3 ASPP2kcells revealed structural alterations including monosomies and additional marker chromosomes. Furthermore, we demonstrate that ASPP2k is stress-inducible. Changing culture conditions (e.g. temperature) of cell lines or ex vivo native AML samples resulted in a dramatic increase of the ASPP2k isoform. Consecutive treatment of cells with daunorubicin lead to relative resistance of pre-stressed cells compared to their counterparts cultured under standard conditions. Vice versa, specific knock-down of ASPP2k resulted in a significant increase in induction of apoptosis upon chemotherapy compared to empty vector controls. Our data demonstrate that ASPP2k plays a distinctive role as an anti-apoptotic regulator of the TP53 checkpoint rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates - and facilitates acquisition of genomic mutations, a first initiating step in leukemogenesis. Disclosures No relevant conflicts of interest to declare.
Loss of NFAT2 Leads to an Acceleration of Clonal Evolution in CLL
2016-12-02

Abstract Chronic lymphocytic leukemia (CLL) may be defined as a clonal expansion of mature B cells with stereotypic B cell receptors (BCR). Somatic hypermutation of the BCR heavy chains (IGVH) defines subgroups with different prognosis. In up to 10% of patients Richter's transformation to a high grade lymphoma with a mostly dismal prognosis is observed. The TCL1 transgenic mouse is a well accepted model of human CLL. Upon B cell-specific knock out of NFAT2, TCL1 mice develop a disease resembling Richter's syndrome with a significantly more aggressive disease phenotype. While TCL1 B cells exhibit tonic anergic BCR signaling characteristic of human CLL, loss of NFAT2 leads to readily activated BCRs indicating different BCR usage with altered downstream signaling. Here, we analyzed BCR usage in C57BL/6 wildtype, TCL1, and TCL1-NFAT2-/-mice. Splenocyte cDNA of the respective animals at an age of 7 months was amplified by multiplex PCR covering known heavy chain VDJ alleles. The PCR products were subsequently cloned into standard bacterial plasmids and subjected to conventional DNA sequencing. In addition, PCR products were analyzed by next-generation-sequencing (NGS). For data analysis, the IMGT/HightV-QUEST online tool was applied. A very diverse and polyclonal BCR usage was found in wild type mice with more than 4000 different clones identified. Although TCL1 mice at 7 months of age exhibit all features of CLL, their BCR usage as analyzed by unambiguously identified VDJ recombination was still polyclonal. Only with respect to VH usage, TCL1 mice were found to use a more limited set of V alleles compared to wild type mice. Loss of NFAT2 by conditional knockout on the contrary, lead to an oligoclonal usage of VDJ recombination, to a further limitation of V alleles, and also to the usage of identical VDJ recombination in different mice with high frequency - indicating BCR selection either by antigen or self-signaling in these mice. Further analyses by sequencing of hypervariable regions showed preferential usage of mutated BCRs in TCL1 mice and of unmutated BCRs in NFAT2 ko mice reflecting benign and aggressive disease in humans. In summary, the loss of NFAT2 signaling in CLL prefers the selection of unmutated BCRs as well as the preferential usage of certain VDJ recombinations which results in the accelerated development of true oligoclonal disease. Disclosures No relevant conflicts of interest to declare.
True Positive Rate And False Positive Rate Used To Generate S2_Fig1A And S2_Table2
2016-12-02

In the attached file, first column corresponds to true positive (TP), second column corresponds to false positive (FP), third column corresponds to false negative (FN), fourth column corresponds to true negative (TN), fifth column corresponds to true positive rate (TPR) and sixth column corresponds to false positive rate (FPR), while each row corresponds to the session number. The data was used to generate patient's F, "Receiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier" and "Contingency table" during training session, S2_Fig1A and S2_Table2, respectively.
True positive rate and false positive rate used to generate S2_Fig4B and S2_Table9
2016-12-02

&lt;p&gt;In the attached file, first column corresponds to true positive (TP), second column corresponds to false positive (FP), third column corresponds to false negative (FN), fourth column corresponds to true negative (TN), fifth column corresponds to true positive rate (TPR) and sixth column corresponds to false positive rate (FPR), while each row corresponds to the session number. The data was used to generate patient's W, "Receiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier" and "Contingency table" during training session, S2_Fig4B and S2_Table9, respectively.&lt;/p&gt;
True Positive Rate And False Positive Rate Used To Generate S2_Fig4A And S2_Table8
2016-12-02

In the attached file, first column corresponds to true positive (TP), second column corresponds to false positive (FP), third column corresponds to false negative (FN), fourth column corresponds to true negative (TN), fifth column corresponds to true positive rate (TPR) and sixth column corresponds to false positive rate (FPR), while each row corresponds to the session number. The data was used to generate patient's W, "Receiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier" and "Contingency table" during training session, S2_Fig4A and S2_Table8, respectively.
True Positive Rate And False Positive Rate Used To Generate S2_Fig1B And S2_Table3
2016-12-02

In the attached file, first column corresponds to true positive (TP), second column corresponds to false positive (FP), third column corresponds to false negative (FN), fourth column corresponds to true negative (TN), fifth column corresponds to true positive rate (TPR) and sixth column corresponds to false positive rate (FPR), while each row corresponds to the session number. The data was used to generate patient's F, "Receiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier" and "Contingency table" during feedback session, S2_Fig1B and S2_Table3, respectively.
True Positive Rate And False Positive Rate Used To Generate S2_Fig2A And S2_Table4
2016-12-02

In the attached file, first column corresponds to true positive (TP), second column corresponds to false positive (FP), third column corresponds to false negative (FN), fourth column corresponds to true negative (TN), fifth column corresponds to true positive rate (TPR) and sixth column corresponds to false positive rate (FPR), while each row corresponds to the session number. The data was used to generate patient's G, "Receiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier" and "Contingency table" during training session, S2_Fig2A and S2_Table4, respectively.
True Positive Rate And False Positive Rate Used To Generate S2_Fig2B And S2_Table5
2016-12-02

In the attached file, first column corresponds to true positive (TP), second column corresponds to false positive (FP), third column corresponds to false negative (FN), fourth column corresponds to true negative (TN), fifth column corresponds to true positive rate (TPR) and sixth column corresponds to false positive rate (FPR), while each row corresponds to the session number. The data was used to generate patient's G, "Receiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier" and "Contingency table" during feedback session, S2_Fig2B and S2_Table5, respectively.
True Positive Rate And False Positive Rate Used To Generate S2_Fig3A And S2_Table6
2016-12-02

In the attached file, first column corresponds to true positive (TP), second column corresponds to false positive (FP), third column corresponds to false negative (FN), fourth column corresponds to true negative (TN), fifth column corresponds to true positive rate (TPR) and sixth column corresponds to false positive rate (FPR), while each row corresponds to the session number. The data was used to generate patient's B, "Receiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier" and "Contingency table" during training session, S2_Fig3A and S2_Table6, respectively.
Endoresection with adjuvant ruthenium brachytherapy for selected uveal melanoma patients – the Tuebingen experience
2016-12-03

Abstract Purpose To evaluate the treatment of selected patients with uveal melanoma with endoresection and adjuvant ruthenium brachytherapy. Methods Thirty‐five patients with uveal melanoma not suitable for ruthenium plaque monotherapy were treated with endoresection and adjuvant ruthenium brachytherapy between January 2001 and October 2013. Recurrence‐free survival, globe retention, course of visual acuity ( VA ), occurrence of therapy‐related complications and metastasis‐free and overall survival were analysed retrospectively. Results Eight patients (22.9%) had a tumour recurrence after a median follow‐up of 49.5 months (range: 21–134 months). Enucleation was necessary in eight patients. Thirty‐two patients (91%) had a loss of VA with a median loss of nine lines (range: 0 to −39 lines); VA was stable in three patients and no patients had a gain in VA . Four patients (11.4%) developed radiation retinopathy. Metastases were detected in seven patients (20.0%) during follow‐up. The occurrence of metastasis was significantly associated with monosomy 3 (p &lt; 0.0001). Twenty‐four patients (68.6%) were alive at the end of follow‐up. Five patients (14.3%) died because of uveal melanoma (UM) metastasis. Conclusions Endoresection with adjuvant ruthenium brachytherapy is an option for selected patients with UM who cannot be treated with brachytherapy as monotherapy. About two‐thirds of eyes can be retained long term without recurrences. Visual acuity cannot be maintained in most cases, and may even decrease considerably. Radiation complications are comparatively rare and not a significant problem.
Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster
2016-12-05

The abrupt onslaught of the syphilis pandemic that started in the late fifteenth century established this devastating infectious disease as one of the most feared in human history<sup>1</sup>. Surprisingly, despite the availability of effective antibiotic treatment since the mid-twentieth century, this bacterial infection, which is caused by Treponema pallidum subsp. pallidum (TPA), has been re-emerging globally in the last few decades with an estimated 10.6 million cases in 2008 (ref. 2). Although resistance to penicillin has not yet been identified, an increasing number of strains fail to respond to the second-line antibiotic azithromycin<sup>3</sup>. Little is known about the genetic patterns in current infections or the evolutionary origins of the disease due to the low quantities of treponemal DNA in clinical samples and difficulties in cultivating the pathogen<sup>4</sup>. Here, we used DNA capture and whole-genome sequencing to successfully interrogate genome-wide variation from syphilis patient specimens, combined with laboratory samples of TPA and two other subspecies. Phylogenetic comparisons based on the sequenced genomes indicate that the TPA strains examined share a common ancestor after the fifteenth century, within the early modern era. Moreover, most contemporary strains are azithromycin-resistant and are members of a globally dominant cluster, named here as SS14-Ω. The cluster diversified from a common ancestor in the mid-twentieth century subsequent to the discovery of antibiotics. Its recent phylogenetic divergence and global presence point to the emergence of a pandemic strain cluster.
Emotional and motivational outcomes of lab work in the secondary intermediate track: The contribution of a science center outreach lab
2016-12-05

Abstract Students’ interest in science declines in secondary school. Therefore, motivating students to become competent and engaged in science topics that are relevant for their everyday lives is an important goal, so they can be better citizens and decision makers with socioscientific issues (e.g., climate change and waste disposal). The present study contributes to research on activity emotions (state) and motivational outcomes (situational interest and situational competence) in science education. The study compared three experimental groups in a guided inquiry lab work on the chemistry of starch and a control group. The three groups were (i) 13 science classes taught at school; (ii) 14 classes taught in a Science Center Outreach Lab (SCOL) visit; and (iii) 17 classes taught in a combined setting of (i) and (ii); the groups were taught on the same topic. The large sample of classes was randomly assigned to the experimental conditions and a control group and data were analyzed for the theoretical part and the practical part of the intervention. The nested data were analyzed using item response theory and multilevel analyses. The three lab‐work conditions were quite successful in comparison with the control group. There were some differences between the SCOL and school groups; the students experienced higher enjoyment in the theoretical part of the intervention in the school group and higher situational competence in the practical lab work at the combined setting. Especially low achievers profited from the intervention, supposedly because the topic was close to students’ everyday lives. Conscientiousness was a major predictor of emotional and motivational outcomes. In conclusion, experimental lab work at a SCOL can be seen as a supplement to a worthwhile STEM education. © 2016 Wiley Periodicals, Inc. J Res Sci Teach 54: 3–28, 2017