The Nup98::Nsd1 fusion gene induces CD123 expression in 32D cells.

The NUP98::NSD1 fusion gene is associated with extremely poor prognosis in patients with acute myeloid leukemia (AML). NUP98::NSD1 induces self-renewal and blocks differentiation of hematopoietic stem cells, leading to development of leukemia. Despite its association with poor prognosis, targeted therapy for NUP98::NSD1-positive AML is lacking, as the details of NUP98::NSD1 function are unknown. Here, we generated 32D cells (a murine interleukin-3 (IL-3)-dependent myeloid progenitor cell line) expressing mouse Nup98::Nsd1 to explore the function of NUP98::NSD1 in AML, including comprehensive gene expression analysis. We identified two properties of Nup98::Nsd1 + 32D cells in vitro. First, Nup98::Nsd1 promoted blocking of AML cell differentiation, consistent with a previous report. Second, Nup98::Nsd1 increased dependence on IL-3 for cell proliferation, due to overexpression of the alpha subunit of the IL-3 receptor (IL3-RA, also known as CD123). Consistent with our in vitro data, IL3-RA was also upregulated in samples from patients with NUP98::NSD1-positive AML. These results highlight CD123 as a potential new therapeutic target in NUP98::NSD1-positive AML.

Two Closed Conformations of CRAF Require the 14-3-3 Binding Motifs and Cysteine-Rich Domain to be Intact in Live Cells.

The protein rapidly accelerated fibrosarcoma (RAF) is a kinase downstream of the membrane protein RAS in the cellular signal transduction system. In the structure of RAF, the N- and C-terminus domains are connected with a flexible linker. The open/close dynamics and dimerization of RAF are thought to regulate its activity, although the details of these conformations are unknown, especially in live cells. In this work, we used alternating laser excitation to measure cytosolic CRAF in live HeLa cells and obtained single-molecule Förster resonance energy transfer (smFRET) distributions of the structural states. We compared the results for wild-type (WT)-CRAF before and after epidermal growth factor (EGF) stimulation, with mutations of the 14-3-3 binding sites and cysteine-rich domain, and an N-terminus truncation. The smFRET distributions of full-length CRAFs were analyzed by global fitting with three beta distributions. Our results suggested that a 14-3-3 dimer bound to two sites on a single CRAF molecule and induced the formation of the autoinhibitory closed conformation. There were two closed conformations, which the majority of WT-CRAF adopted. These two conformations showed different responsiveness to EGF stimulation.

[HLA-mismatched bone marrow transplantation with post-transplant cyclophosphamide for pediatric patients with aplastic anemia].

Post-transplant cyclophosphamide (PTCy) has improved the efficacy of HLA-mismatched hematopoietic cell transplantation (HCT) by decreasing the risk of graft-versus-host disease (GVHD) and nonrelapse mortality. If an HLA-matched donor is not available, GVHD prophylaxis with PTCy can also be used for HLA-mismatched HCT in patients with pediatric aplastic anemia (AA). We report two cases of pediatric AA that were treated with HLA-mismatched HCT with reduced-intensity conditioning and PTCy. We administered 50 mg/kg/day Cy for GVHD prophylaxis on days 3 and 4, and tacrolimus and mycophenolate mofetil (or methotrexate) were initiated from day 5. In both the cases, the time to engraftment was favorable and GVHD and infection were controllable. PTCy probably allows us to expand donor candidates in pediatric AA when an HLA-matched donor is not available; however, further studies regarding optimal conditioning regimens and late complications are required.

The incidence of symptomatic osteonecrosis is similar between Japanese children and children in Western countries with acute lymphoblastic leukaemia treated with a Berlin-Frankfurt-Münster (BFM)95-based protocol.

In this study, we performed a retrospective analysis of a cohort of Japanese paediatric patients with B-cell precursor (BCP)-acute lymphoblastic leukaemia (ALL) treated with a Berlin-Frankfurt-Münster (BFM)95-based protocol, to clarify the incidence, clinical characteristics, and risk factors of osteonecrosis (ON) in comparison to the ALL-02 protocol. We identified a high frequency of ON with the BFM95-based protocol compared to the ALL-02 protocol. The incidence of symptomatic ON with the BFM95-based protocol is comparable to previous studies in Western countries. We believe that the type of treatment regimen has more impact on the incidence of symptomatic ON in paediatric ALL than ethnicity.

In-cell single-molecule FRET measurements reveal three conformational state changes in RAF protein.

BACKGROUND: The structures of proteins are intimately related to their functions. Significant efforts have been dedicated to the structural investigation of proteins, mainly those of purified proteins in in vitro environments. Proteins function in living cells and thus protein structures must be regulated by interactions with various molecules, some of which participate in reaction networks, depending on the states, conditions, or actions of the cell. Therefore, it is very important to understand the structural behavior of proteins in living cells. METHODS: Single-molecule Förster resonance energy transfer (smFRET) measurements were conducted using the alternative laser excitation (ALEX) technique. smFRET distributions of cytosolic Rapidly Accelerated Fibrosarcoma (RAF) proteins in living HeLa cells were obtained with exclusion of the negative effects of photobleached fluorophores and incompletely labeled proteins on smFRET. RESULTS: smFRET histograms of wildtype (wt) RAF in live cells exhibited two major peaks, whereas that of the S621A mutant, which has been thought to have an expanded structure, was almost single-peaked. A population shift involving the peaks for wt RAF was detected upon epidermal growth factor stimulation. Spontaneous transitions between the conformational states corresponding to the two peaks were also detected using the FRET-two-channel kernel-based density distribution estimator method in comparison to static double-stranded DNA samples. CONCLUSIONS: Cytosolic CRAF has at least three conformational states; in addition to the closed and open forms, the fully-open form was distinctly specified. Based on the results, we propose a speculative structural model for CRAF. GENERAL SIGNIFICANCE: Structural distribution and changes to proteins in live cells as a result of intracellular interactions were successfully identified. smFRET using ALEX is applicable to any other cytosolic proteins.

Single-Molecule Förster Resonance Energy Transfer Measurement Reveals the Dynamic Partially Ordered Structure of the Epidermal Growth Factor Receptor C-Tail Domain.

Intrinsically disordered proteins (IDPs) or regions (IDRs) are thought to exhibit unique functionalities without forming ordered structures. However, these molecular mechanisms are not easily elucidated, partly because of the difficultly of measuring structural information. In this study, we applied the alternative laser excitation (ALEX) method and circular dichroism (CD) spectroscopy to investigate the structure of the C-terminal tail (CTT) domain of the human epidermal growth factor receptor (EGFR). The single-molecule distributions of Förster resonance energy transfer (FRET) obtained by ALEX under solution conditions modified by the addition of potassium chloride (KCl), urea, or guanidinium chloride (GdmCl) allowed us to separately examine the influences of charge interactions and secondary structure formation. The CD spectrum analyses indicated the types of included secondary structure. The results suggested that the structure of the CTT is influenced by secondary structure formation, which is a principally antiparallel ß-sheet, rather than by charge interactions and that phosphorylation of the major Grb2-binding sites partially denatures that secondary structure. Our findings suggest that the EGFR CTT might regulate ligand binding kinetics by local ß-sheet formation or by the disruption associated with phosphorylation states.

[Pure red cell aplasia following the rapid reduction and discontinuation of cyclosporine for mixed chimerism after allogeneic bone marrow transplantation].

A 19-year-old male with therapy-related myelodysplastic syndrome underwent allogeneic bone marrow transplantation with reduced-intensity conditioning from his HLA-identical sibling whose ABO blood type exhibited major incompatibility with the patient. After post-transplantation 1 month, chimerism analysis of the bone marrow revealed mixed chimerism with 30% of recipient cells, and after post-transplantation 3 months, complete remission was maintained; however, recipient granulocytes were elevated up to 50% per the chimerism analysis. Next, pancytopenia developed following the rapid discontinuation of the immunosuppressive agent. Although neutrophils and platelets spontaneously recovered, anemia progressed. Based on severe erythroid hypoplasia in the bone marrow and the elevation of anti-ABO isohemagglutinin against donor-derived red blood cells, the patient was diagnosed with pure red cell aplasia (PRCA) following hematopoietic cell transplantation. Because complete chimerism was attained at the PRCA onset even for B cells, we decided to conservatively manage PRCA with only red blood cell transfusion. Notably, after 2 months of the PRCA onset, anemia improved. This case suggests that the therapeutic strategy for PRCA following hematopoietic cell transplantation should be determined by considering the status of each patient, including chimerism.

Lipid-Protein Interplay in Dimerization of Juxtamembrane Domains of Epidermal Growth Factor Receptor.

Transmembrane (TM) helix and juxtamembrane (JM) domains (TM-JM) bridge the extracellular and intracellular domains of single-pass membrane proteins, including epidermal growth factor receptor (EGFR). TM-JM dimerization plays a crucial role in regulation of EGFR kinase activity at the cytoplasmic side. Although the interaction of JM with membrane lipids is thought to be important to turn on EGF signaling, and phosphorylation of Thr654 on JM leads to desensitization, the underlying kinetic mechanisms remain unclear. In particular, how Thr654 phosphorylation regulates EGFR activity is largely unknown. Here, combining single-pair FRET imaging and nanodisc techniques, we showed that phosphatidylinositol 4,5-bis phosphate (PIP2) facilitated JM dimerization effectively. We also found that Thr654 phosphorylation dissociated JM dimers in the membranes containing acidic lipids, suggesting that Thr654 phosphorylation electrostatically prevented the interaction with basic residues in JM and acidic lipids. Based on the single-molecule experiment, we clarified the kinetic pathways of the monomer (inactive state)-to-dimer (active state) transition of JM domains and alteration in the pathways depending on the membrane lipid species and Thr654 phosphorylation.

Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals.

To develop a testing strategy incorporating the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non-sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h-CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS-based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h-CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water-soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance.

Live cell single-molecule detection in systems biology.

In this review, we describe technology and use of single-fluorophore imaging and detection in living cells with regard to application in systems biology and medicine. Because all biological reactions occur under aqueous conditions, the realization of single-fluorophore imaging using an optical microscope has led to the direct observation of biological molecules at work. Today, we can observe single molecules in individual living cells and even higher multicellular organisms. Using single-molecule imaging, we can determine the absolute values of kinetic and dynamic parameters of molecular reactions as a whole and during fluctuations and distribution. In addition, identification of the coordinate of single molecules has enabled super-localization techniques to virtually improve spatial resolution of optical microscopy. Single-molecule detection that depends on point detection instead of imaging is also useful in detecting concentrations, diffusive movements, and molecular interactions in living cells, especially in the cytoplasm. The precise and absolute values of positional, kinetic, and dynamic parameters that are determined by single-molecule imaging and detection in living cells constitute valuable data on unitary biological reactions, because they are obtained without destroying the integrity of complex cellular systems. Moreover, most parameters that are determined by single-molecule measurements can be substituted directly into equations that describe kinetic and dynamic models in systems biology and medicine.

LSFG findings of proliferative diabetic retinopathy after intravitreal injection of bevacizumab.

The authors investigate the changes of chorioretinal blood flow using laser speckle flowgraphy (LSFG) in efficacy of treatment. Intravitreal bevacizumab was injected in a patient with proliferative diabetic retinopathy. LSFG measures the relative velocity index of erythrocytes (mean blur rate) in a previously confirmed area, with neovascularization elsewhere (NVE), neovascularization of the disc (NVD), and without neovascularization. The authors compared mean blur rate before and after bevacizumab injection in each area. In LSFG images, regression of blood flow was observed at the area of neovascularization sequentially as the change of color pattern. Finally, decrease of the mean blur rate of an average 32.7% was observed in the NVE area. Similarly, a reduction of 31.9% of mean blur rate was observed in the NVD area. However, in the area of without neovascularization, reduction of mean blur rate was not observed. This suggested the useful possibility of measuring chorioretinal blood flow changes by drug intervention using LSFG analysis.

LSFG findings of proliferative diabetic retinopathy after intravitreal injection of bevacizumab.

The authors investigate the changes of chorioretinal blood flow using laser speckle flowgraphy (LSFG) in efficacy of treatment. Intravitreal bevacizumab was injected in a patient with proliferative diabetic retinopathy. LSFG measures the relative velocity index of erythrocytes (mean blur rate) in a previously confirmed area, with neovascularization elsewhere (NVE), neovascularization of the disc (NVD), and without neovascularization. The authors compared mean blur rate before and after bevacizumab injection in each area. In LSFG images, regression of blood flow was observed at the area of neovascularization sequentially as the change of color pattern. Finally, decrease of the mean blur rate of an average 32.7% was observed in the NVE area. Similarly, a reduction of 31.9% of mean blur rate was observed in the NVD area. However, in the area of without neovascularization, reduction of mean blur rate was not observed. This suggested the useful possibility of measuring chorioretinal blood flow changes by drug intervention using LSFG analysis.

Effects of GroESL coexpression on the folding of nicotinoprotein formaldehyde dismutase from Pseudomonas putida F61.

The overexpression of fdm, which encodes the formaldehyde dismutase from Pseudomonas putida F61, resulted in the formation of inclusion bodies made up of aggregated enzyme, leaving little activity in the soluble fraction of the transformant cells. On the other hand, coexpression of groESL along with fdm facilitated in vivo solubilization of the enzyme protein in its active form. When coexpressed with groESL, formaldehyde dismutase purified from E. coli had the same crystalline form (i.e., a regular octahedron) as the native enzyme, and like the native enzyme, it bound 1 mol of NAD(H) and 2 mol of zinc in each subunit.