Mechanism of Leukaemogenesis Downstream of CSF3R-, RUNX1-Mutations and Trisomy 21

The underlying study is based on the research work published in Blood by Skokowa et al. in 2014. The authors postulated a unique mechanism of leukemogenesis in a group of patients suffering from congenital neutropenia (CN), a disease characterized by a low absolute neutrophil count and a high susceptibility of malignant progression to MDS or AML, which occurs in approximately 20 % of CN patients [Skokowa et al., 2014, 2017]. They found that, in approximately 70 % of CN/AML patients, in addition to the inherited CN-associated mutations (e.g., ELANE, HAX1, GPT1 and WAS), the AML phenotype was observed when hematopoietic cell clones were positive for sporadic RUNX1 mutations which were acquired after sporadic CSF3R mutations. The authors postulated cooperating leukemogenic effects of RUNX1 and CSF3R mutations. In this Doctoral Thesis, we re-analyzed the CN/AML patients’ group investigated in 2014. Among those patients who underwent malignant transformation, we found hints of non-random distribution for sporadic missense (n = 7) and nonsense (n = 6) RUNX1 mutations (Fisher’s t-test: p = 0.0515) at AML stage. Furthermore, samples positive for missense RUNX1 mutations were also positive for trisomy 21. This was not observed in samples positive for nonsense RUNX1 mutations (table 3.1). Since RUNX1 is located on chromosome 21, it was of special interest to test whether trisomy 21 resulted in an increase of the mutant or the wild-type RUNX1 allelic fraction. Thus, we performed Sanger sequencing and digital PCR on samples of three selected CN/AML individuals all positive for missense RUNX1 mutations and trisomy 21 (UniProtKB:Q01196, p.R139G, p.D171N, p.R174L) (figures 3.1 to 3.6). We were able to confirm an increase of mutant RUNX1 allelic fraction over wild type RUNX1 allelic fraction in a 2:1 ratio in all three patients. Hence, we showed that the occurrence of trisomy 21 was accompanied by an increase of the mutant RUNX1 allele. Since in our patient cohort nonsense RUNX1 mutations were not associated with trisomy 21, we concluded that this was due to different mechanisms of leukemogenic progression between both groups (figure 4.1). Furthermore, we established a chromatin immunoprecipitation assay using a RUNX1 antibody which allows the identification of binding patterns of different mutated RUNX1 proteins to DNA or to other proteins, interaction partners of RUNX1 protein in the future. This might contribute to the better understanding of the patho-mechanisms underlying the effects of different RUNX1 mutations in leukemogenesis. The second objective reported in this thesis, was to investigate the role of microRNAs in CN pathogenesis. MicroRNAs are small, approximately 22 nts long noncoding RNAs, which exert diverse biologic functions including the post- transcriptional control of mRNAs [Lee, 1993]. First, we established a workflow for the isolation and expression quantification of microRNAs in CN patients. We were able to isolate and quantify microRNA-125b and let-7b and aimed to investigate microRNA-3151 (figures 3.11 to 3.14). We observed that miR-125b expression levels were down-regulated upon myeloid differentiation from CD34+ hematopoietic stem and progenitor cells to CD33+ promyelocytic cells. Those findings were in line with previous reports [O’Connell et al., 2011; Rajasekhar et al., 2018; Shaham et al., 2012]. Interestingly, we could not detect significant differences in miR- 125b expression levels between healthy donors and CN patients, neither in CD34+ nor in CD33+ cell populations. This was also true for miR-125b expression lev- els in CN samples, when grouped according to their inherited mutations (ELANE, HAX1, etc.). Of note, miR-3151 expression was not detected in any of the samples; either because it is not expressed by the cells investigated or due to technical issues of the methods used. In this study, we successfully identified and quantified microRNAs, known to be relevant for hematopoiesis, for the first time in our patient cohort. However, due to the small sample size and the small number of microRNAs examined, further research in this field is required in order to finally draw significant conclusions about the role of microRNA in CN pathogenesis. In summary, this study expands the understanding of leukemogenic progression in CN and provides valuable workflows for further investigation of the role of RUNX1 proteins as well as microRNA profiles in CN

Tibiofemoral and patellofemoral joint 3D-kinematics in patients with posterior cruciate ligament deficiency compared to healthy volunteers

Background: The posterior cruciate ligament (PCL) plays an important role in maintaining physiological kinematics and function of the knee joint. To date mainly in-vitro models or combined magnetic resonance and fluoroscopic systems have been used for quantifying the importance of the PCL. We hypothesized, that both tibiofemoral and patellofemoral kinematic patterns are changed in PCL-deficient knees, which is increased by isometric muscle flexion. Therefore the aim of this study was to simultaneously investigate tibiofemoral and patellofemoral 3D kinematics in patients suffering from PCL deficiency during different knee flexion angles and under neuromuscular activation. Methods: We enrolled 12 patients with isolated PCL-insufficiency as well as 20 healthy volunteers. Sagittal MR-images of the knee joint were acquired in different positions of the knee joint (0[degree sign], 30[degree sign], 90[degree sign] flexion, with and without flexing isometric muscle activity) on a 0.2 Tesla open MR-scanner. After segmentation of the patella, femur and tibia local coordinate systems were established to define the spatial position of these structures in relation to each other. Results: At full extension and 30[degree sign] flexion no significant difference was observed in PCL-deficient knee joints neither for tibiofemoral nor for patellofemoral kinematics. At 90[degree sign] flexion the femur of PCL-deficient patients was positioned significantly more anteriorly in relation to the tibia and both, the patellar tilt and the patellar shift to the lateral side, significantly increased compared to healthy knee joints. While no significant effect of isometric flexing muscle activity was observed in healthy individuals, in PCL-deficient knee joints an increased paradoxical anterior translation of the femur was observed at 90[degree sign] flexion compared to the status of muscle relaxation. Conclusions: Significant changes in tibiofemoral and patellofemoral joint kinematics occur in patients with isolated PCL-insufficiency above 30 degrees of flexion compared to healthy volunteers. Since this could be one reasonable mechanism in the development of OA our results might help to understand the long-term development of tibiofemoral and/or patellofemoral osteoarthritis in PCL-insufficient knee joints

Selective aerobic oxidation of 5‐(hydroxymethyl)furfural over heterogeneous silver‐gold nanoparticle catalysts

Bimetallic silver‐gold alloy nanoparticles on zirconia with varying Ag/Au ratios were designed by a rational approach and tested as catalysts for the selective oxidation of the promising biomass platform molecule 5‐(hydroxymethyl)furfural (HMF). For this purpose, colloidal Ag$_{x}$Au$_{10-x}$ particles with molar compositions x=1/3/5/7/9 were prepared by laser ablation in liquids, a surfactant‐free method for the preparation of highly pure nanoparticles, before adsorption on zirconia. In‐depth characterization of the supported catalysts evidenced alloyed nanoparticles with distinct trends of the surface and bulk composition depending on the overall Ag/Au molar ratio as determined by X‐ray photoelectron spectroscopy (XPS) and X‐ray absorption spectroscopy (XAS), respectively. To uncover the synergistic effect of the Ag/Au ratio, the catalysts were further studied in terms of the catalytic activity and selectivity in HMF oxidation. Either the aldehyde moiety or both functional groups of HMF were selectively oxidized depending on the Ag/Au composition resulting in 5‐hydroxymethyl‐2‐furan‐carboxylic acid (HFCA) or 2,5‐furandicarboxylic acid (FDCA), respectively. Optimization of the reaction conditions allowed the quantitative production of HFCA over most catalysts, also after re‐use. Only gold rich catalysts Ag$_{1}$Au$_{9}$/ZrO$_{2}$ and particularly Ag$_{3}$Au$_{7}$/ZrO$_{2}$ were highly active in FDCA synthesis. While Ag$_{3}$Au$_{7}$/ZrO$_{2}$ deactivated upon re‐use due to sintering, no structural changes were observed for the other catalysts and all catalysts were stable against metal leaching. The present work thus provides fundamental insights into the synergistic effect of Ag and Au in alloyed nanoparticles as active and stable catalysts for the oxidation of HMF

LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro

<p>Abstract</p> <p>Background</p> <p>Relapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML.</p> <p>Results</p> <p>About 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75.</p> <p>Conclusion</p> <p>Our results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.</p

[Disability] justice dictated by the surfeit of love:Simone Weil in Nigeria

How is Nigeria’s failure to fulfil its obligations as a signatory of the United Nations Convention on the Rights of Persons with Disabilities to be appreciated or even resolved? Answers to this are sought through a seminal criticism of human rights, namely, Simone Weil’s 1942 essay Human Personality. Weil questioned the ability of human rights concepts to cause the powerful to develop the emotional dispositions of empathy for those who suffer. Weil’s insights provide a convincing explanation that the indifference of Nigerian authorities towards the Convention may be accounted for by the weakness of human rights discourse to foster human capacity for empathy and care for those who suffer. Weil’s criticisms will serve as a point of departure for a particular way to circumvent this inadequacy of human rights discourse to achieve disability justice in Nigeria through other means. I argue that Weil, through her concept of attention, grappled with and offers a consciousness of suffering and vulnerability that is not only uncommon to existing juridical human rights approaches, but is achievable through the active participation in the very forms of suffering and vulnerability in which amelioration is sought. To provide empirical content to this argument, I turn to a short-lived initiative of the Nigerian disability movement, which if ethico-politically refined and widely applied, can supply an action-theoretical grounding for and be combined with Weil’s work to elevate agitations for disability justice above human rights to the realm of human obligations

PepX: a structural database of non-redundant protein–peptide complexes

Although protein–peptide interactions are estimated to constitute up to 40% of all protein interactions, relatively little information is available for the structural details of these interactions. Peptide-mediated interactions are a prime target for drug design because they are predominantly present in signaling and regulatory networks. A reliable data set of nonredundant protein–peptide complexes is indispensable as a basis for modeling and design, but current data sets for protein–peptide interactions are often biased towards specific types of interactions or are limited to interactions with small ligands. In PepX (http://pepx.switchlab.org), we have designed an unbiased and exhaustive data set of all protein–peptide complexes available in the Protein Data Bank with peptide lengths up to 35 residues. In addition, these complexes have been clustered based on their binding interfaces rather than sequence homology, providing a set of structurally diverse protein–peptide interactions. The final data set contains 505 unique protein–peptide interface clusters from 1431 complexes. Thorough annotation of each complex with both biological and structural information facilitates searching for and browsing through individual complexes and clusters. Moreover, we provide an additional source of data for peptide design by annotating peptides with naturally occurring backbone variations using fragment clusters from the BriX database

Morphology and luminosity segregation of galaxies in nearby loose groups

We study morphology and luminosity segregation of galaxies in loose groups. We analyze the two catalogs of groups which have been identified in the Nearby Optical Galaxy (NOG) sample, by means of hierarchical and percolation ``friends-of-friends'' methods (HG and PG catalogs, respectively). In the first part of our analysis we consider 387 and 436 groups of HG and PG, respectively, and compare morphology- (luminosity-) weighted to unweighted group properties: velocity dispersion, mean pairwise distance, and mean groupcentric distance of member galaxies. The second part of our analysis is based on two ensemble systems, one for each catalog, built by suitably combining together galaxies of all groups (1584 and 1882 galaxies for HG and PG groups, respectively). We find that earlier-type (brighter) galaxies are more clustered and lie closer to the group centers, both in position and in velocity, than later-type (fainter) galaxies. Spatial segregations are stronger than kinematical segregations. These effects are generally detected at the larger than 3-sigma level. Luminosity segregation is shown to be independent of morphology segregation. Our main conclusions are strengthened by the detection of segregation in both hierarchical and percolation catalogs. Our results agree with a continuum of segregation properties of galaxies in systems, from low-mass groups to massive clusters.Comment: 12 pages, 8 eps figures, submitted for publication in A

Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5′-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P–epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35°C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL

Affine term structure models : a time-changed approach with perfect fit to market curves

We address the so-called calibration problem which consists of fitting in a tractable way a given model to a specified term structure like, e.g., yield or default probability curves. Time-homogeneous jump-diffusions like Vasicek or Cox-Ingersoll-Ross (possibly coupled with compounded Poisson jumps, JCIR), are tractable processes but have limited flexibility; they fail to replicate actual market curves. The deterministic shift extension of the latter (Hull-White or JCIR++) is a simple but yet efficient solution that is widely used by both academics and practitioners. However, the shift approach is often not appropriate when positivity is required, which is a common constraint when dealing with credit spreads or default intensities. In this paper, we tackle this problem by adopting a time change approach. On the top of providing an elegant solution to the calibration problem under positivity constraint, our model features additional interesting properties in terms of implied volatilities. It is compared to the shift extension on various credit risk applications such as credit default swap, credit default swaption and credit valuation adjustment under wrong-way risk. The time change approach is able to generate much larger volatility and covariance effects under the positivity constraint. Our model offers an appealing alternative to the shift in such cases.Comment: 44 pages, figures and table

Effectiveness of a home-based re-injury prevention program on motor control, return to sport and recurrence rates after anterior cruciate ligament reconstruction: study protocol for a multicenter, single-blind, randomized controlled trial (PReP)

Background: Although anterior cruciate ligament (ACL) tear-prevention programs may be effective in the (secondary) prevention of a subsequent ACL injury, little is known, yet, on their effectiveness and feasibility. This study assesses the effects and implementation capacity of a secondary preventive motor-control training (the Stop-X program) after ACL reconstruction. Methods and design: A multicenter, single-blind, randomized controlled, prospective, superiority, two-arm design is adopted. Subsequent patients (18–35 years) with primary arthroscopic unilateral ACL reconstruction with autologous hamstring graft are enrolled. Postoperative guideline rehabilitation plus Classic follow-up treatment and guideline rehabilitation plus the Stop-X intervention will be compared. The onset of the Stop-X program as part of the postoperative follow-up treatment is individualized and function based. The participants must be released for the training components. The endpoint is the unrestricted return to sport (RTS) decision. Before (where applicable) reconstruction and after the clearance for the intervention (aimed at 4–8 months post surgery) until the unrestricted RTS decision (but at least until 12 months post surgery), all outcomes will be assessed once a month. Each participant is consequently measured at least five times to a maximum of 12 times. Twelve, 18 and 24 months after the surgery, follow-up-measurements and recurrence monitoring will follow. The primary outcome assessement (normalized knee-separation distance at the Drop Jump Screening Test (DJST)) is followed by the functional secondary outcomes assessements. The latter consist of quality assessments during simple (combined) balance side, balance front and single-leg hops for distance. All hop/jump tests are self-administered and filmed from the frontal view (3-m distance). All videos are transferred using safe big content transfer and subsequently (and blinded) expertly video-rated. Secondary outcomes are questionnaires on patient-reported knee function, kinesiophobia, RTS after ACL injury and training/therapy volume (frequency – intensity – type and time). All questionnaires are completed online using the participants’ pseudonym only. Group allocation is executed randomly. The training intervention (Stop-X arm) consists of self-administered home-based exercises. The exercises are step-wise graduated and follow wound healing and functional restoration criteria. The training frequency for both arms is scheduled to be three times per week, each time for a 30 min duration. The program follows current (secondary) prevention guidelines. Repeated measurements gain-score analyses using analyses of (co-)variance are performed for all outcomes. Trial registration: German Clinical Trials Register, identification number DRKS00015313. Registered on 1 October 2018