Localisation, activity and targeting of Bcr-Abl in chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) is a myeloproliferative disease of stem cell origin. It is characterised by the Philadelphia chromosome and Bcr-Abl oncoprotein which is a constitutively activated tyrosine kinase that is causative in CML. Treatment of CML was revolutionized by tyrosine kinase inhibitors (TKIs) in the last decade. TKIs target Bcr-Abl and block its tyrosine kinase activity. Despite the success of TKI in eliminating differentiated CML cells, primitive quiescent CML stem cells still resist or persist with TKI treatment. In this study, several strategies have been investigated towards elimination of TKI-insensitive primitive CML stem cells. At first, the effect of nuclear entrapment of Bcr-Abl protein in CML cells was studied. Although Bcr-Abl protein is located in the cytoplasm of CML cells, TKIs, such as imatinib mesylate (IM), can induce the nuclear translocation of Bcr-Abl. Nuclear Bcr-Abl tyrosine kinase is capable of inducing apoptosis after drug washout, and leptomycin B (LMB) can trap translocated Bcr-Abl in the nucleus. Primary CML CD34+ cells were treated with IM and LMB either continuously or sequentially. It was found that neither regimen significantly increased the anti-proliferative effect of IM in primary CML CD34+ cells. It was also observed that the majority of Bcr-Abl was still retained in the cytoplasm of CML cells treated with IM and LMB, suggesting other mechanisms apart from its tyrosine kinase activity retained Bcr-Abl protein in the cytoplasm of CML cells. Next the subcellular distribution of Bcr-Abl was investigated in TKI-insensitive CML progenitors which survived 12-Day treatment of a potent TKI dasatinib (150nM). It was demonstrated that about 50% of Bcr-Abl was still retained in the cytoplasm of TKI-insensitive primary CML progenitors, although there was a significant increase in nuclear Bcr-Abl level in these survived cells compared to the no drug control (NDC). It was hypothesised that the cytoplasmic retention of Bcr-Abl was caused by its cytoplasmic binding partners, and it was found that a proportion of Bcr-Abl protein was associated with 14-3-3 proteins in the surviving cells, indicating the cytoplasmic retention of Bcr-Abl might be due to its binding with 14-3-3 proteins. In addition, due to the rapid nature of kinase inhibition and nuclear transportation, studies are required to measure the earliest time-point of inhibition of Bcr-Abl tyrosine kinase by IM. The common method to do this is to employ p-CrkL which has been widely used as a surrogate marker of Bcr-Abl tyrosine kinase activity, but the accuracy of p-CrkL as an indicator of Bcr-Abl tyrosine kinase status at early time-points during in vitro TKI treatment has not been examined. It was demonstrated that p-CrkL was not a reliable indicator of Bcr-Abl kinase activity within 24 hours of IM treatment in vitro and indicated that the early responses to IM and dasatinib were different. It was also observed that there was a rapid and active dephosphorylation of Bcr-Abl within 1 hour of TKI treatment, driven at least in part by protein tyrosine phosphatase activity. Furthermore, a farnesyltransferase inhibitor (FTI) BMS-214662 preferentially induces apoptosis in CML stem and progenitor cells compared to their normal counterparts, but another similar FTI BMS-225975 does not. However, the mechanism of action of BMS-214662 in inducing apoptosis of CML cells is not clear. When BMS-214662 was used as a negative control in the p-CrkL study, it was unexpectedly observed that the drug accumulated Bcr-Abl protein in CML cells. Thus, it was hypothesised that BMS-214662 may function as a proteasome inhibitor, which could result in accumulation of Bcr-Abl protein and further elevation of intracellular ROS level, leading to apoptosis of CML cells. Although BMS-214662 treatment induced accumulation of total ubiquitinated proteins and specifically Bcr-Abl protein in K562 cells, BMS-225975 had very similar effects, and this might result from the common mechanism of BMS-214662 and BMS-225975, which is their FTI activity. On the other hand, BMS-214662 induced significantly higher levels of intracellular ROS in both proliferating and non-proliferating K562 cells with respect to BMS-225975, indicating the production of ROS may be involved in the non-FTI mechanism of action of BMS-214662. However it was concluded that BMS-214662 was not a proteasome inhibitor like bortezomib. Finally, the use of synthetic low density lipoprotein (sLDL) as a vehicle for drug delivery to overcome the insufficient intracellular drug concentration in CML stem cells was investigated. Uptake and internalization of unloaded sLDL particles by CML cell line K562 and for the first time by CML stem cells was observed, demonstrating the targeting potential of sLDL particles in CML when they are loaded with drugs. Overall, this study provides further understanding of CML treatment and identifies some alternative strategies to target CML stem cells that may be used in combination with TKI to enhance the eradication of this stem cell-driven disease

Uptake of synthetic low density lipoprotein by leukemic stem cells — a potential stem cell targeted drug delivery strategy

Chronic Myeloid Leukemia (CML) stem/progenitor cells, which over-express Bcr-Abl, respond to imatinib by a reversible block in proliferation without significant apoptosis. As a result, patients are unlikely to be cured owing to the persistence of leukemic quiescent stem cells (QSC) capable of initiating relapse. Previously, we have reported that intracellular levels of imatinib in primary primitive CML cells (CD34<sup>+</sup>38<sup>lo/−</sup>), are significantly lower than in CML progenitor cells (total CD34<sup>+</sup>) and leukemic cell lines. The aim of this study was to determine if potentially sub-therapeutic intracellular drug concentrations in persistent leukemic QSC may be overcome by targeted drug delivery using synthetic Low Density Lipoprotein (sLDL) particles. As a first step towards this goal, however, the extent of uptake of sLDL by leukemic cell lines and CML patient stem/progenitor cells was investigated. Results with non-drug loaded particles have shown an increased and preferential uptake of sLDL by Bcr-Abl positive cell lines in comparison to Bcr-Abl negative. Furthermore, CML CD34<sup>+</sup> and primitive CD34<sup>+</sup>38<sup>lo/−</sup> cells accumulated significantly higher levels of sLDL when compared with non-CML CD34<sup>+</sup> cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication

Low BACH2 Expression Predicts Adverse Outcome in Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a highly variable clinical outcome. There are well‐established CLL prognostic biomarkers that have transformed treatment and improved the understanding of CLL biology. Here, we have studied the clinical significance of two crucial B cell regulators, BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) and BCL6 (B‐cell CLL/lymphoma 6), in a cohort of 102 CLL patients and determined the protein interaction networks that they participate in using MEC‐1 CLL cells. We observed that CLL patients expressing low levels of BCL6 and BACH2 RNA had significantly shorter overall survival (OS) than high BCL6‐ and BACH2‐expressing cases. Notably, their low expression specifically decreased the OS of immunoglobulin heavy chain variable region‐mutated (IGHV‐M) CLL patients, as well as those with 11q and 13q deletions. Similar to the RNA data, a low BACH2 protein expression was associated with a significantly shorter OS than a high expression. There was no direct interaction observed between BACH2 and BCL6 in MEC‐1 CLL cells, but they shared protein networks that included fifty different proteins. Interestingly, a prognostic index (PI) model that we generated, using integrative risk score values of BACH2 RNA expression, age, and 17p deletion status, predicted patient outcomes in our cohort. Taken together, these data have shown for the first time a possible prognostic role for BACH2 in CLL and have revealed protein interaction networks shared by BCL6 and BACH2, indicating a significant role for BACH2 and BCL6 in key cellular processes, including ubiquitination mediated B‐cell receptor functions, nucleic acid metabolism, protein degradation, and homeostasis in CLL biology

A Nonlinear Model and Parameter Identification Method for Rubber Isolators under Shock Excitation in Underwater Vehicles

Rubber isolators are usually used to protect high-precision equipment of autonomous underwater vehicles (AUVs), avoiding damage from overlarge dynamic excitation. Considering the nonlinear properties of the rubber material, the nonlinear behavior of rubber isolators under shock exaltation is hard to be predict accurately without the available modal and accurate parameters. In view of this, the present study proposes a nonlinear model and parameter identification method of rubber isolators to present their transient responses under shock excitation. First, a nonlinear model of rubber isolators is introduced for simulating their amplitude and frequency-dependent deformation under shock excitation. A corresponding dynamic equation of the isolation system is proposed and analytically solved by the Newmark method and the Newton-arithmetic mean method. Secondly, a multilayer feed-forward neural network (MFFNN) is constructed with the current model to search the parameters, in which the differences between the estimated and tested responses are minimized. The sine-sweep and drop test are planned with MFFNN to build the parameter identification process of rubber isolators. Then, a T-shaped isolator composed of high-damping silicon rubber is selected as a sample, and its parameters were determined by the current identification process. The transient responses of the isolation system are reconstructed by the current mode with the identified parameter, which show good agreement with measured responses. The accuracy of the proposed model and parameter identification method is proved. Finally, the errors between the reconstructed responses and tested responses are analyzed, and the main mode of energy attenuation in the rubber isolator is discussed in order to provide an inside view of the current model

Additional file 3 of The relationship between menopausal syndrome and gut microbes

Additional file 3. Gut microbiota composition of phyla, genera, and species. There are 43 phyla, 281 genera, and 163 species were identified in the P (n = 77) and H (n = 24) groups

Additional file 4 of The relationship between menopausal syndrome and gut microbes

Additional file 4. KEGG level3 pathways of functional prediction. 50 KEGG level3 pathways enriched in the P group were found to be significantly higher than in H group

Large-scale assembly of isotropic nanofiber aerogels based on columnar-equiaxed crystal transition

Abstract Ice-templating technology holds great potential to construct industrial porous materials from nanometers to the macroscopic scale for tailoring thermal, electronic, or acoustic transport. Herein, we describe a general ice-templating technology through freezing the material on a rotating cryogenic drum surface, crushing it, and then re-casting the nanofiber slurry. Through decoupling the ice nucleation and growth processes, we achieved the columnar-equiaxed crystal transition in the freezing procedure. The highly random stacking and integrating of equiaxed ice crystals can organize nanofibers into thousands of repeating microscale units with a tortuous channel topology. Owing to the spatially well-defined isotropic structure, the obtained Al2O3·SiO2 nanofiber aerogels exhibit ultralow thermal conductivity, superelasticity, good damage tolerance, and fatigue resistance. These features, together with their natural stability up to 1200 °C, make them highly robust for thermal insulation under extreme thermomechanical environments. Cascading thermal runaway propagation in a high-capacity lithium-ion battery module consisting of LiNi0.8Co0.1Mn0.1O2 cathode, with ultrahigh thermal shock power of 215 kW, can be completely prevented by a thin nanofiber aerogel layer. These findings not only establish a general production route for nanomaterial assemblies that is conventionally challenging, but also demonstrate a high-energy-density battery module configuration with a high safety standard that is critical for practical applications

Additional file 2 of The relationship between menopausal syndrome and gut microbes

Additional file 2. The rarefaction curve of richness in different groups. (A-B) The curve in each group is nearly smooth with a sufficient amount of sequencing data and few new undetected genes

Single-cell DNA sequencing identifies risk-associated clonal complexity and evolutionary trajectories in childhood medulloblastoma development

We reconstructed the natural history and temporal evolution of the most common childhood brain malignancy, medulloblastoma, by single-cell whole-genome sequencing (sc-WGS) of tumours representing its major molecular sub-classes and clinical risk groups. Favourable-risk disease sub-types assessed (MB and infant desmoplastic/nodular MB ) typically comprised a single clone with no evidence of further evolution. In contrast, highest risk sub-classes (MYC-amplified MB and TP53-mutated MB ) were most clonally diverse and displayed gradual evolutionary trajectories. Clinically adopted biomarkers (e.g. chromosome 6/17 aberrations; CTNNB1/TP53 mutations) were typically early-clonal/initiating events, exploitable as targets for early-disease detection; in analyses of spatially distinct tumour regions, a single biopsy was sufficient to assess their status. Importantly, sc-WGS revealed novel events which arise later and/or sub-clonally and more commonly display spatial diversity; their clinical significance and role in disease evolution post-diagnosis now require establishment. These findings reveal diverse modes of tumour initiation and evolution in the major medulloblastoma sub-classes, with pathogenic relevance and clinical potential