High D-index during mobilization predicts poor mobilization of CD34+ cells after anti-lymphoma salvage chemotherapy.

BACKGROUND: Performing stem cell collection after mobilization chemotherapy was a well-balanced strategy between anti-tumor effect and efficient collection of CD34+ cells, but deep and prolonged nadir exposed patients to risk of febrile neutropenia. Febrile neutropenia was known to be associated with lower yields of CD34+ cells, but quantitative data referring to association between yields of CD34+ cells and severity of neutropenia was lacking. We hypothesized that D-index, which was developed for quantitative evaluation of severity of neutropenia especially in the field of hematologic malignancies, could predict yields of CD34+ cells. METHODS: We performed a single center, retrospective analysis of patients with relapsed or refractory aggressive lymphoma who were mobilized with ESHAP or modified ESHAP. We evaluated the association between yields of CD34+ cells at first apheresis and D-index. RESULTS: Thirty-six patients were included, and we demonstrated that yields of CD34+ cells from patients with higher D-index were significantly lower than those from patients with lower D-index. Multivariate linear regression analysis and logistic regression analysis also demonstrated the significant predictive power of D-index. Further, D-index was significantly correlated to platelet count before starting mobilization chemotherapy. Platelet count was known to predict yields of CD34+ cells, and combination of platelet count and D-index could identify patients with lowest CD34+ yields. CONCLUSION: D-index could predict yields of CD34+ cells and it seemed that its predictive power was not less than that of platelet count. Prospective studies including more heterogeneous patients were needed to validate our study.

Reduction of cycles of bendamustine plus rituximab therapy in the cases with good response for indolent B-cell lymphomas.

Bendamusutine plus rituximab (BR) regimen is one of the standard regimens for indolent B-cell lymphomas, yet the possibility of reduction of cycles of BR therapy without compromising therapeutic effects is not still uncovered. We retrospectively surveyed 57 cases including 40 follicular lymphoma cases who underwent BR regimen in our institute. The overall response (OR) rate and complete response (CR) rate were 86.0% (95% confidential interval (CI), 74.2-93.7) and 54.4% (40.7-67.6), respectively. Five-year overall survival (OS) and 5-years progression-free survival (PFS) were 76.8% and 45.7%, respectively. We then grouped the patients by the number of administered cycles of BR regimen. PFS was significantly longer in 41 cases of the later cessation group (cycle 4-6) than in 16 cases of the earlier cessation group (cycle 1-3) (p = 0.012, 5-years PFS; 46.8% vs. 35.2%, respectively), and both of OR and CR rate of the former was better than the latter (OR rate; 95.1% vs. 62.5%, p < 0.01, CR rate; 61.4% vs. 31.3%, p = 0.04). Interestingly PFS of twenty-one (36.8%) cases receiving just 4 cycles was longer than that of 20 cases who received five or 6 cycles (p < 0.01, 5-years PFS; 71.8% vs. 23.2%, respectively). Focusing on the group of four cycles, the 12 case with CR revealed longer PFS than seven cases with partial response (PR), and median PFS was not reached in CR cases and 16.9 months in the PR cases (p < 0.01). These results suggest that four cycles at least should be administered if possible, and the outcome of the patients who discontinued BR after four cycles was not inferior to that of the cases who received five or six cycles. In conclusion, discontinuation after four cycles may be permissible in some cases with complete response to BR regimen.

Clarification of fast metal sphere movement in glass.

In this study, metal spheres were implanted into glass by continuous-wave (CW) laser illumination, which manipulated the metal sphere inside the glass. The spheres moved at approximately 100 mm/s, which is 100 times faster compared to conventional movement. The movement mechanism was clarified by in situ, cross-sectional, and microscopic observations. With a high laser power density, the metal spheres moved fast with plasma emission, and their trajectory contained fine iron particles. The temperatures of the metal sphere with slow (<0.1 mm/s) and fast (>1 mm/s) speeds were 1,900 and 2,900 K, respectively.

Live imaging of multicolor-labeled cells in Drosophila.

We describe LOLLIbow, a Brainbow-based live imaging system with applications in developmental biology and neurobiology. The development of an animal, including the environmentally sensitive adaptation of its brain, is thought to proceed through continual orchestration among diverse cell types as they divide, migrate, transform and interact with one another within the body. To facilitate direct visualization of such dynamic morphogenesis by individual cells in vivo, we have modified the original Brainbow for Drosophila in which live imaging is practical during much of its development. Our system offers permanent fluorescent labels that reveal fine morphological details of individual cells without requiring dissection or fixation of the samples. It also features a non-invasive means to control the timing of stochastic tricolor cell labeling with a light pulse. We demonstrate applicability of the new system in a variety of settings that could benefit from direct imaging of the developing multicellular organism with single-cell resolution.

Synthesis of Substituted 3(2H)-Furanones Using Alkylative Intramolecular Cyclization of Sulfonium Salts.

The facile alkylative intramolecular cyclization of 3-alkoxycarbonyl-2-oxopropyldiphenylsulfonium salts is described. This simple method can be readily applied to the synthesis of a novel family of 4-alkylated 3(2H)-furanones in moderate to high yields under mild conditions via a one-pot process.

Strain Promoted Click Chemistry of 2- or 8-Azidopurine and 5-Azidopyrimidine Nucleosides and 8-Azidoadenosine Triphosphate with Cyclooctynes. Application to Living Cell Fluorescent Imaging.

Strain-promoted click chemistry of nucleosides and nucleotides with an azido group directly attached to the purine and pyrimidine rings with various cyclooctynes in aqueous solution at ambient temperature resulted in efficient formation (3 min to 3 h) of fluorescent, light-up, triazole products. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne, or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The 5-azidouridine and 5-azido-2'-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 min. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne in aqueous acetonitrile (3 h). The novel triazole adducts at the 2- or 8-position of adenine or 5-position of uracil rings induce fluorescence properties which were used for direct imaging in MCF-7 cancer cells without the need for traditional fluorogenic reporters. FLIM of the triazole click adducts demonstrated their potential utility for dynamic measuring and tracking of signaling events inside single living cancer cells.

Part-based motor neuron recognition in the Drosophila ventral nerve cord.

We exploit the morphological stereotypy and relative simplicity of the Drosophila nervous system to model the diverse neuronal morphologies of individual motor neurons and understand underlying principles of synaptic connectivity in a motor circuit. In our analysis, we use images depicting single neurons labeled with green fluorescent protein (GFP) and serially imaged with laser scanning confocal microscopy. We model morphology with a novel formulation of Conditional Random Fields, a hierarchical latent-state CRF, to capture the highly varying compartment-based structure of the neurons (soma-axon-dendrites). In the training phase, we follow two approaches: (i) hierarchical learning, where compartment labels are given, and (ii) latent-state learning, where compartment labels are not given in the samples. We demonstrate the accuracy of our approach using wild-type motor neurons in the larval ventral nerve cord. However, our method can also be used for the identification of motor neuron mutations, as well as the automated annotation of the motor circuitry in wild type and mutant animals. Our method is directly applicable to the recognition of compartment-defined structures.

Physical traits of male Japanese bush warblers (Cettia diphone) in summer and winter: hyperactive aspects of the vocal system and leg muscles in summer males.

The Japanese bush warbler has a very distinctive song, shows marked sexual size dimorphism, and has a polygynous mating system. However, the physical traits of males and seasonal variation in such traits have remained unknown. Twenty-five anatomical measurements representing physical traits of males in the breeding (summer, n = 5) and non-breeding (winter, n = 5) seasons were examined morphologically and compared statistically. Differences were evident between summer and winter (P < 0.05, t test) in the following seven items: body mass (19.8 ± 0.7 g vs. 15.6 ± 1.2 g [mean ± SD]), mass of male reproductive organs (184.0 ± 25.7 mg vs. 6.0 ± 1.4 mg), hind limb (3789.2 ± 346.2 mg vs. 3003.4 ± 226.8 mg), leg muscles (883.0 ± 63.5 mg vs. 581.4 ± 33.2 mg in either side), skin around the neck/throat (1280 ± 34.9 mg vs. 287.2 ± 84.7 mg), and syrinx (35.8 ± 2.39 mg vs. 25.0 ± 3.24 mg), and circumference of the neck/throat (52.1 ± 2.3 mm vs. 38.3 ± 2.6 mm). In contrast to winter males, summer males had thickened flabby skin prominently in the neck/throat area and an inflatable esophagus, perhaps a morphological basis for the throat sac as a vocal resonator. Also, the remarkable development of the flexor muscles of the legs of summer males suggests that perching and movement using the legs increases during the breeding season. These distinct characteristics of summer males may be related to the polygynous mating system of this species.

Moving force of metal particle migration induced by laser irradiation in borosilicate glass.

We optically manipulated a metal particle in borosilicate glass. The glass in the neighborhood of the laser-heated metal particle softened; hence, the metal particle was able to migrate in the glass. In this letter, the driving force of the metal particle toward the light source in the glass provided by laser illumination was investigated. The variation in the surface tension of the glass at the interface between the glass and the metal particle induced by the temperature gradient was calculated via a numerical temperature calculation. It was found that the temperature at the laser-illuminated surface of a stainless-steel particle with a radius of 40 µm was ~320 K higher than that on the nonilluminated side. The force applied to the metal particle from the surrounding glass was calculated to be ~100 µN, which was approximately equal to the viscous resistance force. In addition, the experimental and numerically calculated speeds of the moving particle, which was measured while varying the laser power, are discussed.

Daratumumab plus lenalidomide and dexamethasone for relapsed POEMS syndrome with bone plasmacytoma harboring 17p deletion.

The standard therapies for polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes (POEMS) syndrome are radiation therapy, high-dose chemotherapy followed by autologous stem cell transplantation, and lenalidomide combined with dexamethasone. Daratumumab was reported to be effective for treatment-naive and relapsed POEMS syndrome, but treatment options for relapsed POEMS syndrome with poor prognostic factors or cytogenetic abnormalities have not been established due to a lack of studies in these patients. Here, we describe a case of relapsed POEMS syndrome with bone plasmacytoma harboring a newly detected 17p deletion after high-dose chemotherapy followed by autologous stem cell transplantation and radiation therapy in a male patient. He was successfully treated with daratumumab plus lenalidomide and dexamethasone (Dara-Rd). Dara-Rd could be effective in relapsed POEMS syndrome with 17p deletion, which is known as a poor cytogenetic abnormality in multiple myeloma. This report may broaden the application of Dara-Rd for POEMS syndrome.

EVI1 exerts distinct roles in AML via ERG and cyclin D1 promoting a chemoresistant and immune-suppressive environment.

Aberrant expression of ecotropic viral integration site-1 (EVI1+) is associated with very poor outcomes in acute myeloid leukemia (AML), mechanisms of which are only partially understood. Using the green fluorescent protein reporter system to monitor EVI1 promoter activity, we demonstrated that Evi1high KMT2A-MLLT1-transformed AML cells possess distinct features from Evi1low cells: the potential for aggressive disease independent of stem cell activity and resistance to cytotoxic chemotherapy, along with the consistent gene expression profiles. RNA sequencing and chromatin immunoprecipitation sequencing in EVI1-transformed AML cells and normal hematopoietic cells combined with functional screening by cell proliferation-related short hairpin RNAs revealed that the erythroblast transformation-specific transcription factor ERG (E26 transformation-specific [ETS]-related gene) and cyclin D1 were downstream targets and therapeutic vulnerabilities of EVI1+ AML. Silencing Erg in murine EVI1+ AML models severely impaired cell proliferation, chemoresistance, and leukemogenic capacity. Cyclin D1 is also requisite for efficient EVI1-AML development, associated with gene expression profiles related to chemokine production and interferon signature, and T- and natural killer-cell exhaustion phenotype, depending on the interferon gamma (IFN-γ)/STAT1 pathway but not on CDK4/CDK6. Inhibiting the IFN-γ/STAT1 pathway alleviated immune exhaustion and impaired EVI1-AML development. Overexpression of EVI1 and cyclin D1 was associated with IFN-γ signature and increased expression of chemokines, with increased exhaustion molecules in T cells also in human AML data sets. These data collectively suggest that ERG and cyclin D1 play pivotal roles in the biology of EVI1+ AML, where ERG contributes to aggressive disease nature and chemoresistance, and cyclin D1 leads to IFN-γ signature and exhausted T-cell phenotypes, which could potentially be targeted.

Computational identification of tissue-specific alternative splicing elements in mouse genes from RNA-Seq.

Tissue-specific alternative splicing is a key mechanism for generating tissue-specific proteomic diversity in eukaryotes. Splicing regulatory elements (SREs) in pre-mature messenger RNA play a very important role in regulating alternative splicing. In this article, we use mouse RNA-Seq data to determine a positive data set where SREs are over-represented and a reliable negative data set where the same SREs are most likely under-represented for a specific tissue and then employ a powerful discriminative approach to identify SREs. We identified 456 putative splicing enhancers or silencers, of which 221 were predicted to be tissue-specific. Most of our tissue-specific SREs are likely different from constitutive SREs, since only 18% of our exonic splicing enhancers (ESEs) are contained in constitutive RESCUE-ESEs. A relatively small portion (20%) of our SREs is included in tissue-specific SREs in human identified in two recent studies. In the analysis of position distribution of SREs, we found that a dozen of SREs were biased to a specific region. We also identified two very interesting SREs that can function as an enhancer in one tissue but a silencer in another tissue from the same intronic region. These findings provide insight into the mechanism of tissue-specific alternative splicing and give a set of valuable putative SREs for further experimental investigations.

Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals.

Memory and learning in animals are mediated by neurotransmitters that are released from vesicles clustered at the synapse. As a synapse is used more frequently, its neurotransmission efficiency increases, partly because of increased vesicle clustering in the presynaptic neuron. Vesicle clustering has been believed to result primarily from biochemical signaling processes that require the connectivity of the presynaptic terminal with the cell body, the central nervous system, and the postsynaptic cell. Our in vivo experiments on the embryonic Drosophila nervous system show that vesicle clustering at the neuromuscular presynaptic terminal depends on mechanical tension within the axons. Vesicle clustering vanishes upon severing the axon from the cell body, but is restored when mechanical tension is applied to the severed end of the axon. Clustering increases when intact axons are stretched mechanically by pulling the postsynaptic muscle. Using micro mechanical force sensors, we find that embryonic axons that have formed neuromuscular junctions maintain a rest tension of approximately 1 nanonewton. If the rest tension is perturbed mechanically, axons restore the rest tension either by relaxing or by contracting over a period of approximately 15 min. Our results suggest that neuromuscular synapses employ mechanical tension as a signal to modulate vesicle accumulation and synaptic plasticity.

Conserved alternative splicing and expression patterns of arthropod N-cadherin.

Metazoan development requires complex mechanisms to generate cells with diverse function. Alternative splicing of pre-mRNA not only expands proteomic diversity but also provides a means to regulate tissue-specific molecular expression. The N-Cadherin gene in Drosophila contains three pairs of mutually-exclusive alternatively-spliced exons (MEs). However, no significant differences among the resulting protein isoforms have been successfully demonstrated in vivo. Furthermore, while the N-Cadherin gene products exhibit a complex spatiotemporal expression pattern within embryos, its underlying mechanisms and significance remain unknown. Here, we present results that suggest a critical role for alternative splicing in producing a crucial and reproducible complexity in the expression pattern of arthropod N-Cadherin. We demonstrate that the arthropod N-Cadherin gene has maintained the three sets of MEs for over 400 million years using in silico and in vivo approaches. Expression of isoforms derived from these MEs receives precise spatiotemporal control critical during development. Both Drosophila and Tribolium use ME-13a and ME-13b in "neural" and "mesodermal" splice variants, respectively. As proteins, either ME-13a- or ME-13b-containing isoform can cell-autonomously rescue the embryonic lethality caused by genetic loss of N-Cadherin. Ectopic muscle expression of either isoform beyond the time it normally ceases leads to paralysis and lethality. Together, our results offer an example of well-conserved alternative splicing increasing cellular diversity in metazoans.

Bing-Neel Syndrome Successfully Treated with Tirabrutinib.

Bing-Neel syndrome (BNS) is a rare central nervous system manifestation of lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM). We herein report a 62-year-old man with LPL/WM after multiple chemotherapies. He had weakness of lower extremities and elevated serum IgM levels. A bone marrow examination showed lymphoplasmacytic cells infiltration. Contrast-enhanced magnetic resonance imaging suggested enhancing lesions in the cauda equina roots. He was diagnosed with BNS and started on treatment with tirabrutinib 480 mg daily. Within three months, he showed clinical and radiologic improvement. Tirabrutinib may have utility as an effective treatment for BNS.

Spermiophagy in the male reproductive tract of some passerine birds.

In order to elucidate the locus and means of spermiophagy in passerine birds, we examined histologically the entire male reproductive tract of sexually mature birds of three passerine species with different forms of sperm competition, namely, the alpine accentor (Prunella collaris), the redflanked bush robin (Tarsiger cyanurus), and the Bengalese finch (Lonchura striata var. domestica). Spermiophagy occurred consistently and frequently in the epithelial layer of the seminal glomera and ejaculatory duct in each species, which were regularly identified by non-ciliated epithelial cells. The epithelial spermiophagy was occasional or infrequent in other portions of the seminal tract, and spermiophagy by macrophages was uncommon throughout the tract. Quantitative data in the seminal glomera and ejaculatory duct gave no clear answer concerning a possible relationship between the epithelial spermiophagy and different levels of sperm competition among these passerine species. In conclusion, the epithelial lining of the terminal region of the seminal tract is the main site for spermiophagy in the male reproductive tract of these passerine species, which activity serves to maintain the quality of semen by eliminating infertile spermatozoa as well as sperm remaining at the end of the breeding season.

The variable transmembrane domain of Drosophila N-cadherin regulates adhesive activity.

Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, we found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage.

Thyroid lesions in the great cormorant (Phalacrocorax carbo) of Niigata, central Japan: a possible association with dioxin accumulation.

Necropsy and histopathologic examination of three Great Cormorants (Phalacrocorax carbo) shot in Niigata, central Japan, revealed goitrous changes in the thyroids. Thyroids had a hypertrophic follicular epithelium, loss or deficiency of luminal colloid, occasional small follicles suggesting hyperplasia, and occasional collapsed follicles. Irregularly shaped follicles were frequent, and hyperemia, deposition of dark pigment, and sporadic lymphoid aggregates were also seen. Chemical analysis simultaneously conducted showed higher than normal levels of dioxins in the liver, muscle, and fat, i.e., polychlorinated dibenzo-dioxins, polychlorinated dibenzo-furans, and coplanar polychlorinated biphenyls. The present results, together with those of relevant previous studies, strongly suggest an association between these pollutants and thyroid lesions in the Great Cormorant.

Robo and Frazzled/DCC mediate dendritic guidance at the CNS midline.

Neuronal connectivity is established by the axo-dendritic polarity, correct guidance and targeting of neurons. Unlike for axons, the mechanisms responsible for directed outgrowth of dendrites are not well understood. Using single-cell labeling, we describe specific guidance defects in dendrites of identified neurons in frazzled, robo, netrin and commissureless mutant embryos of Drosophila melanogaster. We found that the cell-surface molecules Frazzled and Robo work as guidance molecules not only for axons but also for dendrites as they navigate within the CNS. Furthermore, we report that each neuron showed a cell-autonomous and independent use of guidance molecules.

Chronic myelogenous leukemia presenting with central nervous system infiltration, successfully treated with central nervous system-directed chemotherapy followed by allogeneic stem cell transplantation.

With the introduction of tyrosine kinase inhibitors (TKIs), prognosis of chronic myelogenous leukemia (CML) has improved dramatically. However, treatment for blast phase (BP) CML remains a challenge. CML infiltration of the central nervous system (CNS) is particularly rare and no effective treatment strategy has been established. The present case reports a 30-year-old man presenting with sensory deafness. Marked leukocytosis with p210 BCR-ABL1 mRNA positivity and Philadelphia chromosome detected by bone marrow biopsy confirmed the diagnosis of CML. Dura thickening in brain MRI and immature cells with Philadelphia chromosome in spinal fluid confirmed CNS invasion of CML and he was diagnosed with BP-CML. Two cycles of hyper-CVAD/MA (cyclophosphamide, vincristine, doxorubicin and dexamethasone/ high-dose methotrexate and cytarabine) therapy with dasatinib and concomitant intrathecal chemotherapy induced complete cytogenetic response and remission of CNS involvement. Bone marrow transplantation from an unrelated HLA-mismatched donor was performed and complete molecular response in bone marrow and complete remission in CNS disease was achieved. To our knowledge, this the first report of BP-CML with CNS infiltration at initial diagnosis, and shows that CNS-directed chemotherapy with dasatinib followed by allogeneic hematopoietic stem cell transplantation is useful in the treatment for BP-CML with CNS invasion in the TKI era.