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Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors
© Verbrugge et al.; licensee BioMed Central Ltd. 2013
Received: 4 January 2013
Accepted: 5 January 2013
Published: 10 January 2013
In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence.
Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC).
Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC.
These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.
KeywordsProteasome inhibitors Anti-CD20/rituximab therapy B cells Autoimmune disorders Resistance
Tumor Necrosis Factor- α
Since the initial approval of rituximab, a monoclonal antibody against CD20, in 1997, it has become the cornerstone in the treatment of B cell lymphoproliferative diseases . Additionally, B cells play a pivotal role in the pathogenesis of inflammatory autoimmune disorders such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), for which reason over the past decade rituximab has been added as B cell depleting therapy of auto-immune diseases [2–6]. However, it is well recognized that a considerable number of patients are either primary resistant to rituximab, or experience a limited period of response [5, 7]. The efficacy of rituximab is associated with proficient CD20 expression on the surface of B cells, and indeed, acquisition of rituximab resistance appears to be correlated with down-regulation of CD20 [8, 9]. Therefore, there is an urgent need for new therapeutic modalities. Beyond immunotherapy approaches, small molecule targeting of B-cells has been achieved with proteasome inhibitors (PIs) such as bortezomib (BTZ) . Bortezomib-induced apoptosis and cell death has been reported for a large panel of human B cell lines, including those resistant to rituximab therapy [11–13]. Accordingly, a clinical effect of BTZ in B cell driven diseases has been found. BTZ is highly effective in inducing plasma cell death, thus, is now being implemented both in first line and relapsed and refractory multiple myeloma (MM) . BTZ has also shown clinical efficacy in Mantle Cell Lymphoma  and Waldenström’s Macroglobulinemia , the latter also in combination with rituximab , indicating its effectivity in inducing cell death in earlier stages of B-cell development. In the autoimmune disease setting, clinical studies with PIs are far less advanced, although in pre-clinical studies, PI therapy demonstrated promising results as anti-arthritic treatment strategy, also by B cell depletion [18–22].
One hypothesis for proficient B cell targeting by PIs is that their substantial turnover and secretion of proteins renders these cells highly susceptible to interference in protein homeostasis, in particular following inhibition of the ubiquitin proteasome system (UPS) . The UPS is the master regulator of cellular homeostasis by mediating ubiquitination and degradation of proteins that control apoptosis induction, and also mediate pro-inflammatory cytokine production via NFκB activation [24–26]. Most mammalian cells express constitutive proteasomes which harbor the β5 subunit (PSMB5), the β1 subunit (PSMB6) and the β2 subunit (PSMB7) and cleave proteins (or long peptides) after hydrophobic, acidic, and basic amino acid residues, respectively [27, 28]. After stimulation with IFN-γ and/or TNF-α, these constitutive proteasome subunits are replaced by immunoproteasome subunits: β5i (LMP7), β1i (LMP2), and β2i (MECL1) . These proteasome entities are part of the immunoproteasome, which is mainly expressed in cells of hematological origin and can generate a distinct peptide repertoire for MHC-class I presentation . As a first generation PI, Bortezomib targets both constitutive and/or immunoproteasome subunits via a reversible binding to the proteasome, however, nowadays several next-generation PIs have been rationally designed and tested, harboring the capacity to specifically target the constitutive and/or immunoproteasome subunits through irreversible binding [23, 28, 29, 31–35].
Given the chronic nature of autoimmune-related diseases, with patients facing long-term treatment, it is anticipated that PI treatment may also be accompanied by loss of treatment efficacy/onset of drug resistance over time (Kumar et al., 2012), as has been described in extensively-treated MM patients [36, 37]. We, as well as others, have described the development of resistance to BTZ in several models of hematological cancer cells, including T-cells, myeloid cells, and plasma cells [38–41]. For both benign and neoplastic B cells, however, the long term effects of BTZ exposure are largely unexplored and deserve further investigation.
In this study, we investigated the long-term effects of B-cell exposure to BTZ and onset of resistance to BTZ using EBV-transformed JY lymphoblast cells as a benign autoimmune disorder in vitro B-cell model. Beyond characterizing the molecular basis of resistance to BTZ in JY cells, we report two modalities to overcome BTZ resistance in B cells; (a) by next-generation epoxyketone-based irreversible PIs, and (b) by enhanced rituximab-mediated complement-dependent cytotoxicity (CDC), exploiting the upregulated cell surface expression of CD20 in BTZ-resistant JY cells.
Acquisition of bortezomib resistance and cross-resistance to other proteasome inhibitors
The human JY B cell line was used as a model to examine the cellular effects of long-term exposure of B cells to BTZ, including the acquisition of BTZ resistance. Cell cultures were exposed to a low concentration of BTZ (IC10: 1.5 nM) which was gradually (stepwise) increased to 100 nM of BTZ over a period of 6 months. JY cells stably growing in BTZ concentrations of 35nM (JY/35) and 100nM (JY/100) were used for further characterization.
Molecular alterations in BTZ-resistant JY cells
Surface molecules involved in antigen-presentation
JY cells are efficient antigen-presenting cells  and we therefore examined whether or not BTZ selection would induce alterations in markers involved in antigen presentation. In fact, no significant changes were found in the cell surface expression of HLA-ABC (MHC-class I), HLA-DR (MHC-class II), and the co-stimulatory molecules CD80, CD86, and CD40 in JY/35 and JY/100 cells compared to JY/WT cells (Additional file 1: Figure S2). Further confirmation that BTZ selection had no impact on antigen presentation was provided by the observation that JY/WT and BTZ-resistant cells showed no difference in their capacity to induce allogeneic T-cell proliferation (Additional file 1: Figure S3). Of note, short term (24 hr) incubations with maximal non-toxic concentrations of BTZ also did not affect their ability to induce proliferation of allo-T-cells (Additional file 1: Figure S3).
Cytokine release capacity by BTZ-resistant JY cells
CD19 and CD20 expression in BTZ-resistant JY cells
Regulation of CD20 expression in BTZ-resistant JY cells
Increased sensitivity to anti-CD20/rituximab-mediated CDC in bortezomib-resistant JY cells
The current study describes two ways to overcome acquired resistance to BTZ in JY human B cells: (a) anti-CD20/rituximab-mediated CDC, and (b) next generation epoxyketone-based irreversible PIs.
B cell depleting therapies utilizing the anti-CD20/rituximab antibody approach, as well as the small molecule approach with the proteasome inhibitor BTZ, have demonstrated clinical efficacy in the treatment of lymphoproliferative disorders [1, 10, 23, 48]. In the treatment of autoimmune diseases, rituximab also now has an accepted place [3–5, 43], but BTZ or other PIs still await clinical evaluation. Ideally, based on their non-overlapping mechanisms of action, rituximab and bortezomib could potentially replace each other when development of resistance to one or the other occurred. In this context, it has been reported that rituximab-resistant B cells retain sensitivity for BTZ, but the reverse has not been demonstrated. In view of the need for chronic treatments in autoimmune diseases, there are concerns about the emergence of resistance , thus highlighting the need to unravel underlying mechanisms of resistance, in order to design strategies to overcome acquired resistance. To address these issues, we provoked acquired resistance to BTZ in a human B cell line model and investigated its impact on rituximab sensitivity and next-generation PIs designed to overcome BTZ-resistance.
Here we mimicked the onset of acquired BTZ resistance in human B cells using human JY lymphoblastic cells as benign autoimmune disorder in vitro model, by a classical approach of cell exposure to stepwise increasing concentrations of BTZ (5 through 100 nM), representing clinically-achievable plasma levels [10, 23]. Characterization of the BTZ-resistant JY cells (10–12 fold compared to WT cells) revealed the following main features: 1) acquisition of a point mutation in the PSMB5 gene (encoding for the constitutive β5 proteasome subunit) along with overexpression of β5 subunit protein, 2) retention of sensitivity to epoxyketone-based PIs including carfilzomib, 3) an altered cytokine environment indicated by a marked reduction in the secretion of the inflammatory cytokine TNF-α, and 4) immunophenotypically, a significantly reduced expression of the B cell surface marker CD19 and a concomitant increase in CD20 levels.
BTZ-resistance associated with a single point mutation in PSMB5 has been demonstrated by us and others for leukemia cell lines of lymphoid and myeloid origins, as well as solid tumor cell lines [38–41, 49]. These mutations introduced amino acid alterations that clustered in critical positions (Thr21, Met45, Ala49 and Cys52) in the BTZ binding pocket within the β5 subunit . Based on data from the present study, it is revealing that beyond hematological malignant cells, PSMB5 mutations can also be identified in BTZ-resistant JY lymphoblastic cells. It is noteworthy that the mutation in PSMB5 in JY/BTZ cells is heterozygous, indicating that cells still retained an intact WT allele. This may underlie a relatively lower level of BTZ resistance observed in JY/BTZ cells as compared to the substantially higher resistance levels (> 100-fold) noted in leukemic cells harboring homozygous mutations in PSMB5[38, 41]. Notwithstanding this fact, expression levels of mutant β5 subunits were upregulated in JY/BTZ cells, likely as a compensatory mechanism to sustain basal proteasome activity. The Met45Ile substitution identified in BTZ-resistant JY cells consistently resided in the highly-conserved substrate/inhibitor-binding domain of the β5 subunit. Met45 is well recognized to be essential for proteasome-substrate interactions, as well as for binding of the leucine boronic acid moiety (P1 site) of BTZ [29, 32, 50]. In this context, it may be anticipated that replacement of the non-polar, spatially flexible Met45 for another slightly more non-polar, bulkier, and more rigid amino acid as Ile, would affect optimal BTZ binding, thereby resulting in the conferring of drug resistance. From this perspective, it is intriguing to note that the Ala49 and Met45 mutations in the β5 subunit were also identified in marine bacteria as a self-protection mechanism against the natural PI, salinosporamide A, that they produce .
A major finding in the current study is that JY/BTZ cells harboring the Met45Ile mutation in the β5 subunit retained full sensitivity to carfilzomib, an epoxyketone-based PI. Previous studies from our laboratory also indicated that carfilzomib retained appreciable activity against BTZ-resistant leukemia [41, 52] and solid tumor cell lines [23, 35, 49]; similar findings were observed with ONX 0912 , an orally- bioavailable analog of carfilzomib which provokes irreversible binding via the epoxyketone group to the active site Thr1 residue in the β5 subunit. This unique property, rather than reversible binding by boronate-based BTZ, may still facilitate inhibition of the catalytic activity in mutated β5, thereby resulting in retention of drug sensitivity. Since levels of immunoproteasome subunits were largely unaltered and no mutations were observed in the β5i subunit in JY/BTZ cells, it may also explain the marginal levels of cross-resistance that were observed for the immunoproteasome-specific inhibitor ONX 0914. In this context, it is worthwhile mentioning that recent crystal structure studies identified Met45 in β5i as a major determinant in the efficient docking of ONX 0914 to the catalytic binding site . The Met45Ile mutation in the constitutive β5 subunit has apparently lesser consequences for ONX 0914 activity . Together, constitutive and immunoproteasome targeted epoxyketone-based PIs like carfilzomib, currently evaluated in phase I-III clinical trials , as well as ONX 0914 may readily overcome BTZ-resistance.
Besides antiproliferative effects, BTZ can also elicit potential anti-inflammatory effects by suppression of the release of pro-inflammatory cytokines through inhibition of NF-κB activation [18–20, 25, 26]. In this respect, TNF-α has received the most attention as one of the critical cytokines in the pathophysiology of arthritis, and is thus a major druggable target for biological agents and small molecules [20–22, 48]. Consistent with data from the Komano group , we noted that JY/WT cells could produce appreciable levels of TNF-α, which could be partially inhibited by BTZ. Of additional interest was the notion that alongside these short term effects, acquisition of BTZ resistance in JY cells provoked a dramatic reduction in the basal levels of TNF-α production, being most pronounced in JY/100 cells. It remains to be established whether or not this effect is due to diminished response to activation stimuli, or aberrations in TNF-α secretion per se. In either case, prolonged BTZ exposure may elicit potential anti-inflammatory effects even when anti-proliferative effects are compromised.
Acquisition of BTZ resistance in JY cells was associated with opposite impacts on cell surface expression of two common B-cell surface markers: down regulation of CD19 and upregulation of CD20. CD19 is a transmembrane protein and co-receptor molecule that modulates signals through the B cell antigen receptor (BCR) to control B cell-differentiation from pro/pre-B cells to resting pre-B cells in bone marrow. Consistently, CD19−/− mice have exhibited defects at stages of early B cell development and reduced levels of circulating B-cells, as reviewed in Del Nagro et al. . It is yet unclear whether reduced CD19 levels in JY/BTZ cells would result in a B cell differentiation arrest; this should be subject for further studies using panels of B cell differentiation-restricted cell surface markers.
CD20 serves as a Ca2+-permeable cation channel protein expressed on B cells from immature to mature stages of differentiation, as recently reviewed by Thaunat et al. . BTZ has been reported to induce disregulation of intracellular Ca2+, triggering caspase activation and initiating apoptosis . It is unknown whether this off-target effect of BTZ alters CD20 function and expression during resistance development. Rather, recent studies have pointed to the role of the ubiquitin proteasome system in the regulation of CD20 expression, as rituximab-resistant lymphoma cells have displayed upregulated expression of proteasome subunits , and treating Raji human Burkitt lymphoma cells with BTZ for 24–48 hours has down-regulated CD20 expression . In accordance with these studies, we now showed that impaired (poly) ubiquitination of proteins upon 24–48 hour BTZ treatment in resistant JY/BTZ cells attenuated degradation of CD20, resulting in a net 3-fold increase in CD20 expression in JY/100 cells and markedly enhanced rituximab-mediated CDC in JY/100 cells as compared to JY/WT cells. Based on the differential impact on CD20 expression in BTZ-sensitive and BTZ-resistant cells, one should take caution when considering combining rituximab and BTZ for B cell targeting; efficacy may only be realized in BTZ-resistant cells. Moreover, the current findings for JY lymphoblastic cells should also await further corroboration for B-cell neoplasms (e.g. mantle cell lymphoma) for which BTZ is a therapeutic option. Finally, it would be of additional future interest to explore whether aberrations in protein ubiquitination provoked by prolonged exposure to either BTZ or other types of PIs, may be instrumental to upregulated expression of other B cell surface markers that could elicit antibody-mediated CDC.
In conclusion, this study presented two novel strategies to overcome acquired resistance to BTZ in human B lymphoblastic cells: one is a small molecule approach with irreversible epoxyketone-based PIs, whereas the second relates to an immunological approach by anti-CD20/rituximab-mediated CDC, exploiting the upregulation of CD20 in BTZ-resistant cells. This deserves further exploration in order to improve the clinical outcome in patients with bortezomib-resistant diseases.
Cell culture and development of BTZ-resistant cell lines
The human EBV-transformed B-lymphoblastic cell line JY (ATCC) was cultured in IMDM-medium (PAA, The Cell Culture Company, PAA Laboratories GmbH, Germany) supplemented with 10% FCS, 1% β-mercaptoethanol, 1% penicillin, 1% streptomycin and 2 mM L-glutamine. BTZ-resistant JY cell lines were established by stepwise increasing concentrations of BTZ over a period of 6 months, starting at a BTZ concentration of 1.5 nM (IC10 concentration; establishing 10% cell growth inhibition compared to the untreated control) up to a concentration of 100 nM. Cells stably growing in the presence of 35 nM (JY/35) and 100 nM BTZ (JY/100) were isolated after 3 months and 6 months of stepwise selection, respectively. Unless otherwise indicated, experiments were performed with cells cultured in drug-free medium for at least 4 days.
Reagents / chemicals / antibodies
Bortezomib (BTZ)/Velcade™ was provided by Millennium Pharmaceuticals (Cambridge, USA). Carfilzomib and ONX 0914 were generously provided by Onyx Pharmaceuticals, Inc. (South San Francisco, USA). MG132 (Z-Leu-Leu-Leucinal) was purchased from Calbiochem/ Merck (Nottingham, UK). Phorbol-12-myristate 13-acetate (PMA) and ionomycin were from Sigma (St. Louis, MO, USA).
Rituximab, a chimeric immunoglobulin G1, was purchased from Roche (Roche Netherlands B.V., Woerden, The Netherlands). Other antibodies were from the following sources: ubiquitin: (P4D1, Santa Cruz Biotechnology, sc-8017) and β-actin (C4, Santa Cruz, sc-47778) mouse monoclonal antibodies; goat-anti-mouse secondary antibody conjugated to IRDye® 800CW (Odyssey; L1-COR Biosciences, Nebraska, USA).
Isolation of mRNA, cDNA synthesis, and quantitative RT-PCR
Following RNA isolation with the RNeasy Mini kit (Qiagen), cDNA was synthesized using 0.75U/ul M-MLV reverse transcriptase (Invitrogen) in a RT buffer (Invitrogen), containing 5 mM DTT (Invitrogen), 2 mM dNTP (Roche), pdN6 96ug/ml (Roche), and 2U/ulRNAsin (HT Biotechnology Ltd., Cambridge, UK). mRNA expression levels of proteasome subunits PSMB5 (β5), PSMB6 (β1), PSMB7 (β2), PSMB8 (ß5i), PSMB9 (ß1i), PSMB10 (ß2i), and the endogenous housekeeping gene β-glucuronidase (GUS) as a reference, were quantified using real-time PCR analysis (Taqman) using an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) as described previously . Briefly, for PSMB5, a Taqman gene expression assay was used according to the manufacturers’ instructions (Hs00605652_m1, Applied Biosystems). All other primers and probes were designed using Primer Express software (Applied Biosystems) and are indicated in Additional file 1: Table S1. Probes were labeled with 5’-FAM and 3’-BHQ1 as a reporter. Real-time PCR was performed in a total reaction volume of 25 μl containing TaqMan buffer A (Applied Biosystems), 4 mM MgCl2, 0.25 mM of each dNTP (Amersham Pharmacia Biotech), and 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems). Samples were heated for 10 min at 95°C to activate the AmpliTaq Gold DNA polymerase and amplified during 40 cycles of 15 s at 95°C and 60 s at 60°C. Relative mRNA expression levels of the target genes in each sample were calculated using the comparative cycle time (Ct) method. The Ct of the target gene was normalized to the GUS Ct value by subtracting the GUS Ct value from the target Ct value. The mRNA expression level for each target PCR relative to GUS was calculated using the following equation: mRNA expression = 2(Ct target-Ct GUS) × 100%.
Proteasome constitutive-immuno subunit ELISA assay (ProCISE)
The ProCISE assay for quantification of individual constitutive (β5, β1, β2) and immunoproteasome (β5i, β1i, β2i) subunits was performed as previously described [33, 34]. Expression of each c20S and i20S subunit in JY/WT, JY/35, and JY/100 cells was examined after cells were cultured in the absence of BTZ for one week. Pellets of 10 x 106 -15 x 106 cells were washed three times with PBS, frozen on dry ice, thawed, then incubated in 100 μL of lysis buffer (20 mM Tris–HCl, pH 8.0, 5 mM EDTA) for 15 minutes on ice. Lysed pellets were micro-centrifuged (15 min, 14,000 rpm, 4°C), the supernatant collected, and protein content determined by BCA assay (Pierce), utilizing a BSA standard. Cell lysates were diluted in lysis buffer to 1 mg/mL, and then incubated with a proteasome active site probe (PABP; 5 μM) for 2 hours at 25°C. Samples were denatured with 8 M guanidine hydrochloride (Fisher Scientific) and subunits bound to PABP were captured with streptavidin-conjugated sepharose beads (GE Healthcare) in 96-well 0.65 μm porous filter plates (Millipore). Individual subunits were probed with subunit-specific primary antibodies, followed by secondary antibodies conjugated to horseradish peroxidase (HRP). The SuperSignal ELISA Pico chemiluminescent substrate kit (Pierce) was utilized to generate luminescent signal associated with HRP binding. Luminescence after 5 minutes of signal development was monitored on a plate reader (Tecan Safire). Subunit content was expressed as nanograms of subunit per microgram of total protein, as calculated from a standard curve of human purified constitutive or immunoproteasome, assayed simultaneously with the cell lysate samples.
Cytokine release assay
JY cells were seeded in 96-wells plates at a cell density of 5 x 104/200μl and stimulated with 10 ng/ml PMA (Sigma) and 750 ng/ml ionomycin (Sigma). After 24 hours, culture supernatants were collected and analyzed using the cytometric bead array (CBA) human inflammatory cytokines kit (BD Biosciences, San Jose, CA) according to the manufacturer’s protocol. Quantitative measurements of TNF-α were performed using a FACSCalibur flow cytometer (Becton and Dickinson, San Jose, CA) with the CellQuest program.
FACS analysis – immunophenotyping
JY cells were immunophenotyped using FITC-, PE- and/or APC-conjugated monoclonal antibodies (Mabs) directed against: CD80 (1:10), CD86 (1:10), CD40 (1:10) HLA-ABC (1:25), HLA-DR (1:25), CD19 (1:10), and CD20 (1:10) (BD Biosciences, San Jose, CA) and CD54 (1:10) (Leinco Technologies Inc., St. Louis, USA). In short, 3 x 104 – 8 x 104 cells were washed with PBS supplemented with 0.1% BSA and 0.02% NaN3, and incubated with specific or corresponding isotype-matched control Mabs for 30 minutes at 4°C. Cells were washed and analyzed with a FACSCalibur flow cytometer (Becton and Dickinson, San Jose, CA) using the Cell Quest program. Results were expressed as percentages of positive cells and the mean fluorescence index was calculated based on mean fluorescence intensities.
Western blot analysis
Accumulation of ubiquitinated proteins upon treatment with BTZ was determined by Western blot analysis as described previously . In short, 3 × 106 JY/WT, JY/35 and JY/100 cells were incubated in 10 ml growth medium in the absence or presence of 10nM, 35nM and 100nM BTZ, respectively. After 24 hours of incubation, cells were harvested and washed 3 times with ice-cold PBS at pH 7.4. Total cell lysates were prepared by resuspending in 0.1 ml lysis buffer (Cell Signaling Technology) containing: 20% PIC (Protease Inhibitor Cocktail) and 10% NaVO4. The suspension was sonicated (MSE sonicator, amplitude 7, for 3 × 5 seconds with 20-second time intervals at 4°C) and centrifuged in an Eppendorf micro-centrifuge (5 min, 12,000 rpm, 4°C). Protein content of the supernatant was determined by the Bio-Rad Protein Assay using a bovine serum albumin (BSA) standard. Protein aliquots (30 μg) of total cell lysates were fractionated on a 4-20% TGX pre-cast gel (BioRad) containing SDS, and transferred onto a PVDF membrane. The membranes were pre-incubated for 1 hour in blocking buffer (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) to prevent non-specific antibody binding. After blocking, the membranes were incubated for 1 hour at room temperature with an ubiquitin-specific antibody (1:200). After 3 washing steps with Tris-buffered saline with 0.5% Tween, the membranes were incubated for 1 hour with goat-anti-mouse secondary antibody conjugated to IRDye ® 800CW (1:10000, Odyssey; L1-CORBiosciences, Nebraska, USA). Detection of antibody binding was obtained using the LI-COR Odyssey scanner (Biosciences) according to the manufacturers’ instructions, and digital image acquisition and quantification was performed using the Odyssey infrared imaging system software (version 3.0.16, LI-COR Biosciences, Nebraska, USA). Expression of ubiquitinated proteins was detected using a mouse monoclonal antibody (1:200) as described above. β-actin (1:1000 diluted antibody) was used as the control for equal loading.
Complement-dependent cytotoxicity (R-CDC) assay
JY cells (5 x 104cells) were plated in triplicate in 96-well plates in IMDM medium with or without rituximab (concentrations ranging from 0.1 μg-50 μg/ml) and 5% baby rabbit complement serum (Cedarlane, Ontario, Canada). After 1 hour of incubation at 37°C, the numbers of non-viable cells were determined after propidium iodide (PI) staining using flow cytometry. The percentage of lysed (PI+) cells was calculated based on a total of 1.2 x 104 measured events. Cell lysis in control conditions (without baby rabbit serum) was subtracted to obtain specific complement-dependent lysis.
For comparisons between groups, ANOVA and Student’s t-test were used. Differences were considered to be significant at p < 0.05.
The authors would like to thank Dr. Saskia A. Cillessen (Department of Pathology), VU University Medical Center, Amsterdam, The Netherlands, for critical reading of the manuscript.
This study is supported by Cancer Center Amsterdam (CCA)/VU- Institute of Cancer and Immunology (V-ICI) [Grant 07/36]. Y.G. Assaraf is recipient of an Award Visiting Professor Fellowships from the Royal Netherlands Academy of Arts and Sciences (KNAW), the Netherlands Organization for Scientific Research (NWO), and the Dutch Arthritis Foundation.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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In June, the European Commission launched sector competition inquiries into financial services and energy. The first such inquiry since modernisation relates to sports services over 3G mobile telephony networks. The methodology and preliminary results of that inquiry were recently made public, and contain some relevant lessons for future inquiries
Regulators face difficult challenges in allocating indirect costs such as overheads and network costs. One method of doing so, equi-proportionate mark-up, has recently been adopted in the telecoms sector. Based on this precedent, Postcomm, the UK postal services regulator, has concluded that it is also appropriate for the postal sector. Is this the case?
As the demand for flights continues to grow, there is increasing concern about congestion at airports and the noise and emissions produced by aircraft. Airport charges can play a key role in ensuring that existing infrastructure is used efficiently, and in encouraging airlines to use quieter and less-polluting planes.
The 2004 water industry price control review, and the parallel review in the electricity distribution industry, tackled important issues that are relevant for the future of utility regulation in the UK in these sectors and more widely. Keith Palmer, NM Rothschild & Son and Cambridge Economic Policy Associates, and Hannah Nixon, Cambridge Economic Policy Associates, discuss the lessons that can be learnt.
Since the publication in 2004 of Oxera’s study on over-indebtedness in the UK, consumer borrowing has continued to rise, passing the £1 trillion threshold, and the level of household debt continues to make the headlines. Yet the latest data does not suggest that current levels of indebtedness are unsustainable. What are the recent trends in indebtedness and what progress have the government and the banking community made in this area in the past year?
Following the debate over the fierce price war between supermarkets in the Netherlands, the Dutch government has announced its decision not to introduce a rule that would prevent retailers from selling below the purchase price. Several other European countries do have such a rule in place. This article explores the economic effects of minimum price rules and discusses the existing empirical evidence, including a study by Oxera.
In light of the current debate surrounding the future of electricity generation in the UK, Oxera has developed an investment model to determine the conditions under which private investors could finance a new fleet of nuclear power stations
Julian Franks, Professor of Finance, London Business School, and Oxera Director, explains how recent research into bankruptcy codes reveals significant and sometimes unexpected differences between bankruptcy procedures in the UK and those of other countries.

domain: law


text:
ERIC Number: ED270576
Record Type: RIE
Publication Date: 1986-Jun
Reference Count: 0
Making the Connection: A Report for Literacy Volunteers Working with Out-of-School Youth. Revised Edition.
Conklin, Nancy Faires; Hurtig, Janise
This report for individuals and organizations engaged in literacy outreach activities explores a youth agency-based, social service professional-facilitated strategy for reaching out-of-school youth. Chapter I describes the growing problem of school dropout. Chapter II synthesizes governmental and scholarly studies of youth and schooling, reporting findings on the numbers and identity of early school leavers and their social and economic prospects. Various analyses of dropouts' attitudes toward schooling and reasons for leaving are reviewed. In Chapter III, descriptions of youth life--in alternative educational programs, in the labor force, as heads of households, and on the streets--are offered. Chapter IV reviews literature findings about dropouts' actual skill levels and explores actual functions of literacy in the youth street culture. Chapter V outlines the advocated intervention strategy, which draws on two critical perspectives: the functional approach to literacy training developed by adult educators and based on use of materials of immediate interest and use to the learner and youth service workers' approaches to working with out-of-school youth. Chapter VI is an annotated listing of reports, teaching materials, and other resources for youth literacy program developers. (YLB)
Publication Type: Information Analyses; Reference Materials - Bibliographies
Education Level: N/A
Sponsor: Department of Education, Washington, DC.
Authoring Institution: Northwest Regional Educational Lab., Portland, OR.
Note: Portions of the document contain small print.

domain: library


text:
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domain: medical


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IF YOU ARE BETWEEN TWO SIZES CHOOSE THE LARGER SIZE.

domain: design


TRAILER TOWING WIRING HARNESS
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text:
Mestni trg 4
Phone: +386 1 2411 818
Library details: Government Office for Legislation Library is a Government Agency library.
This library is affiliated with Slovenia -- Office for Legislation.
(Use this link to refer back to this listing.)
Administration: The director of the library is Ksenija Mihovar Globokar .
Record History: This listing was created on Oct 7, 2008 and was last modified on Dec 8, 2016.


text:
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Everyone wants to have a beautiful life. But really what is a beautiful life?
The definition of beauty according to Merriam Webster dictionary is “the quality or aggregate of qualities in a person or thing that gives pleasure to the senses or pleasurably exalts the mind or spirit.” Finding the beauty in life has always been a struggle for me. I used to constantly look for perfection as my standard of beauty. Perfection, my friends, is not always beautiful and can cause you immense anxiety. It also isn’t realistic to expect perfection in most aspects of one’s life and seeking it can make you miss the beauty in the messy, imperfect parts of life.
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I have made efforts to allow myself to live in the moment and experience the full range of feelings. They may be messy, but they are most definitely beautiful. What makes you feel most alive? That is what makes life beautiful.Share

domain: literature


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A Family Is A Family Is A FamilyBook - 2016
One child is worried that her family is just too different to explain, but listens as her classmates talk about what makes their families special. One is raised by a grandmother, and another has two dads. One is full of stepsiblings, and another has a new baby.
As one by one, her classmates describe who they live with and who loves them -- family of every shape, size and every kind of relation -- the child realizes that as long as her family is full of caring people, her family is special.
A warm and whimsical look at many types of families written by award-winning author Sara O'Leary, A Family is a Family springs to life with quirky and sweet illustrations by Qin Leng.
From Library Staff
A child living in foster care is reluctant to talk about her family with peers until she realizes that families are special in all kinds of ways. A contemporary portrayal of families that reflects community diversity.
From the critics
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domain: sociology


text:
Understanding the Great Commission (Church Basics)
Limited Stock Title: This is a ‘Last Chance’ title. Once we run out of stock this title will no longer be available on our site. It is therefore non-returnable. In the event we are not able to fulfill this part of your order it will be canceled automatically. The remainder of your order will ship according to our regular schedule.
God means to fulfill the Great Commission through local churches. How did the apostles and the churches of the New Testament obey the Great Commission? By gathering Christians together in churches. The church is God s plan for evangelism, discipleship, and the Great Commission. This connection between the Great Commission and the church dramatically impacts how both leaders and members should think about their work of making disciples. We do it together.


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domain: entertainment


US 1418717 A
Description (OCR text may contain errors)
COAL BAG. APPLICATION FILED JAN. 6.192].
1 418 717. PatentedJune 6, 1922.
10 U Hu W 2 3 11v VEN TOR.
flap y Jase 0126022. BY
HARRY JOSEPHSON, OF BRIDGEPORT, CONNECTICUT, ASSIGNOR TO THE JOSEPHSON BAG COMPANY, OF BRIDGEPORT, CONNECTICUT.
Specification of Letters Patent.
Patented June 6, 1922.
Application filed January 6, 1921. Serial No. 435,419.
T 0 all whom 2'23 may concern:
Be it known that I, HARRY JosnPHsoN, acitizen of the United States, and resident of Bridgeport, in the county of Fairfield and State of Connecticut, have invented certain new and useful Improvements in Coal Bags, of which the following is a specification.
An object of this invention is to produce a noveland inexpensive coal bag of practical and durable construction that will be capable of withstanding, for a long period of time and without liability of becoining impaired, the ordinary strains of hard usage.
A further object is to provide a coal bag the body of which may be, in the; main, of usual construction, and the bottom of which consists of a plurality of slats, spaced from each other to allow proper drainage of the contents of the bag, and associated with the lower portion of said body insuch manner that it is impossible for the slats to become removed from the body under the most severe conditions of use.
Another object is'to provide a coal bag the spaced apart slats constituting the bottom of which are each composed of one or more metallic plates of proper construction (1) to support the contents of the bag at considerable distance from the bearing surface of said bottom in order that there can be'no interference with proper drainage of said'contents, and (2) to provide bearing surfaces of reduced area for said bottom in order that the coal bag can be dragged with but a minimum of friction.
A still further object is to provide in a coal bag of the present type, a bottom which is capable of being rolled.
With the above and other objects in view, the invention comprises the construction, arrangement and combination of parts now to be fully described and hereinafter specifi cally claimed.
In the accompanying drawing forming a part of this specification,
Fig. 1 is a perspective view of the improved coal bag, showing several of the slats of the bottom thereof removed.
Fig. 2 is avertical transverse sectional view thereof. i
Fig. 3 is an elevational view on an enlarged scale, showing a ribbed plate and a flat plate that may be associated with each other and with the body of the bag to form a slat for the bottom thereof.
Fig. 4 is a top plan view of the ribbed plate of Fig. 3, which may, by itself, constitute a slat.
Fig. 5 is an enlarged sectional view on line 5-5 in Fig. 4, and
Fig. 6 is a fragmentary perspective view, on a scale corresponding with Fig. 5, showing in detail an end portion of a ribbed plate.
The body of the bag, denoted by numeral 7, may be of canvas or other suitable mate .rial, and is preferably rectangular in horizontal cross-section, it having side walls 8 and end walls 9. The upper edges of the side and end walls are turned outwardly and stitched to the body, as denoted by numeral 10 in Fig. l, to add strength to the upper portion thereof. a
In preparing the body to receive its bottom, the lower edges of the side and end walls may be first turned inwardly for some considerable distance and then laid back upon the main body and stitched down-as indicated by numeral 11 in Fig. 2. The resulting folded over portions of said side and end. walls denoted by 12 and 13, respectively, provide a continuous flange forming the border of a restricted opening in'the bottom of said body, and it is to this border that the bottom of the bag is adapted to be secured. Adjacent ends of said folded over portions of the side and end walls may, if preferred,
end portions 13, and is arranged in the bottom of the body at about the longitudinal center thereof, and 16 denotes the usual handles for the body.
The bottom of the bag consists of a plurality of spaced apart slats, constructed as now to be described to permit proper drainage of the contents of the bag, secured to the continuous flange of the body in such manner that the slats cannot become removed from the body even under the most severe strains of the hardest usage. The slats are all duplicates ofeach other and may each consist of a ribbed plate 17 and a flat plate 18 associated with each other and with the material of the body, or each slat may con sist of a ribbed plate 1? alone. The slats extend transversely of the body and are designed to support the contents of the bag at considerable distance from the bearing surfaces for the bottom thereof to thus olfcr no interference to the proper drainage of said contents, and each slat is further designed to provide a bearing surface or reduced area to allow the bag to bcdraggcdi with but a minimum of friction.
The flat and ribbed plat-cs mentioned are preferably of metal. The flat plates are about the width of the body and when these plates are utilized they rest in the bottom of the body and against the flange defining the restricted opening. Holes 19 in the flat plates serve for the connection of the ribbed plates as will later be explained. The ribbed plates are preferably a trifle shorter than the width of the body and each ribbed plate has a smooth, level portion 20 and a ribbed or shaped portion 21 that nreiterably extends downwardly from about the central part of the smooth level portion and is of slightly shorter length than said smooth portion. Drain holes 22 in the ribbed portions perform an obvious function.
The ribbed plates are adapted to be fastened against the under face of the continuous flange of the body of the bag, and for this purpose I preferably provide the smooth, level portions of these plates with pointed lugs 23 adapted to pierce the material of said flange. Evidently, other securing means adapted to pass throngh the material of the continuous flange may be substituted for lugs 23. When the ribbed plates are utilized by themselves to constitute the slats of the bottom of the bag. the pointed lugs or other securing means, after being forced through the material of the continuous flange, are bent down against the upper face of said flange. hen the ribbed and flat plates are associated to pro vide the slats, the lugs or other securing means are passed through the holes of the flat plates and bent down against the upper faces of said flat plates. And. it will be apparent, a plurality of washers can be sub stit-uted for the flat plates, a single washer for each separate lug or other securin member of the ribbed plates. As shown, lugs 23 may also pass through strip of material 15 to be fastened down against the upper face of said strip or against a flat plate or washer arranged there-above. The intermediate lugs or other securing means of the end slats preferably pass through the ma terial of turned in portions 13 of the end walls, as will be understood.
By making the ribbed plates shorter than thewidth of the body of the bag, I preclude the possibility of the plat-es becoming engaged and torn loose when the bag. is being dragged and by providing said plates with downwardly extending ribbed or shaped portions, I provide bearing surfaces for the bottom of the bag adapted to sup port the contents of the bag at considerable distance from said bearing surfaces, as is evident. As is also evident, these bearing surfaces may be of any preferred dimension to better allow dragging of the bags.
By providing spaced apart slats between which are flexible connections, said connections comprising portions of the lower end of the body of the bag, the bottom of the bag is rendered foldable, and, as will be evident, the bags can consequently be rolled when not in use.
Any number of slats can be applied to the bottom of the body and the slats can be spaced any desired distances apart.
While I have shown and described one specific embodiment of the invention, it is to be understood that such changes in details of construction and arrangement of parts may be made as fall within the scope of the appended claims.
hat I desire to claim is:
1. A coal bag comprising a flexible body open at its lower end and a rollable bottom consisting of spaced apart, disconnected slats having. draining spaces therebetween, each of said slats being secured to said flexible body.
2. A coal bag comprising a flexible body consisting of side walls and open at its lower end, a rollable. bottom consisting of spaced apart, disconnected slats haying draining spaces therebetween, and means for separately securing each slat to the flexible body, whereby proper drainage of the bag is provided for.
3. A coal bag comprising a flexible body open at its lower end, and a rollable bottom adapted to be secured to the lower portion of said body. said bottom consisting of spaced apart, disconnected slats eachcomprising a plate having a downwardly extending rib, and said ribs being of such co11- struction that the bearing surfaces for said bottom are of reduced area for the purpose specified.
4. A coal bag comprising a flexible body open at its lower end, and a rollable bottom adapted to be secured. to said body, said bottom consisting of spaced apart, disconnected slats each comprising a ribbed plate and a. flat plate secured to each other and to said body.
5. A coal bag comprising a body shaped at its lower end to produce a continuous flange providing a restricted opening, and a foldable bottom consisting of spaced apart disconnected slats each comprising a ribbed plate and a flat plate secured to each other and to said flange, said flange being gripped between said plates.
6. A coal bag comprising a body having side and end Walls of flexible material shaped at their lower ends to produce a continuous flange providing a restricted opening, a bottom consisting of spaced apart disconnected slats each comprising a flat plate adapted to rest against said flange and above said restricted opening and a ribbed plate adapted to rest against said flange and beneath said opening, and means for fastening said flat and ribbed plates to each other, said means adapted to pass through the material of said flange. 4
7. A coal bag comprising a body shaped at its lower end to produce a continuous flange providing a restricted opening, a bottom consisting of s aced apart disconnected slats, the ends of W ich are attached to said flange, and a flexible strip extending across said bottom and transversely of said slats, said strip constituting additional means for securing said slats to said body.
Signed at Bridgeport in the county of Fairficld and State of Connecticut this 5th day of January A. D., 1921.
HARRY J O'SEPHSON Witnesses:
C(M. NEWMAN, LILLIAN M. ALLING.


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This week, US President Barack Obama and Chinese President Hu Jintao will both address the UN general assembly. Premier Hu expected to announce a carbon intensity target to show its commitment to international efforts to mitigate climate change. Xinhua, China's state controlled media service, warned that "Any attempts by a party in the UN negotiations to maximize its own interest at the cost of interest of others in the negotiation process is not conducive to powering green economy and protecting our planet." China and Europe have both criticized the US for insufficient commitments to climate change mitigation and both may be seeking to cement itself as the international leader on climate change issues.


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After visiting a construction exposition for high school students in St. Charles on Wednesday, Missouri Republican Gov. Mike Parson expressed disappointment that his proposal for increasing teacher salaries languished in the legislature.
Parson also wasn’t familiar with a proposed tax credit in the House and didn’t favor a one-time tax cut.
“I would say it’s unfortunate,” Parson said of the House’s removal of teacher salary increases during an interview with The Center Square. “We have an opportunity to do it right now. You look at the education system that we have at all levels and we’re paying teachers a base pay of $25,000, which is roughly $12 an hour. That’s not competitive in today’s market. If we are going to make sure that these are the best people forming our young people’s minds, we want to make sure we get good, qualified people. And we’re going to have to pay for that.”
Parson said any ripple effect of teacher salary increases on pensions and salaries of other teachers and administrators shouldn’t be a deterrent.
“I think moving that base up to what we’re trying to do is also going to help with some of the other salaries above that,” Parson said. “I think there’s a little bit of an effect on that. But look, we we want good qualified people taking care of our kids.”
Parson said the current political environment is targeting education, but the purpose of the sector and its institutions are essential to the state’s future.
“The bottom line of it is vast majority teachers get it right every day all across the state of Missouri,” Parson said. “The point of this is (students) are going to be the future of our state. We’re going to need good teachers and good classrooms, but you’re going to have to pay for them.”
Parson said there was no discussion in previous months about the tax-credit bill, and he first heard about it through media reports. House Bill 3021 was perfected and approved by the House on Wednesday and could face a final vote on Thursday.
“It’s one thing we don’t know very much about,” Parson said. “So until we look into that and see if it’s even feasible… I think it’s one thing you have to be careful with right now. I think the things we’ve done since I’ve been governor – cutting people’s income tax – that’s forever. I’m always concerned when it’s one time, whether it’s a, whatever you want to call it, a tax rebate, stimulus, a siphon, whatever it is.”
During the past few months, Parson continually emphasized the state should focus on reducing unemployment. The state’s unemployment rate decreased by one-tenth of a percentage point in March, declining to 3.6%. The rate was 1.2 percentage points below the March 2021 rate. The national unemployment rate in March was 3.6%, down two-tenths of a percentage point from the previous month.
Parson said job creation should continue to be the priority instead of the tax credit.
“We need to be working on getting people back to work and earning paychecks like we’re doing here today,” Parson said. “We’ll have to look at it, but I don’t know who all is involved, who’s affected, who’s not affected, and can it even be done with the amount of money that’s budgeted?”
This article was originally posted on Parson questions long-term cost of tax credit, still wants Missouri teacher raises

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- Who is the Queen and King of Kpop 2020?
- Who is the King of Pop now?
- Who is the best dancer in BTS 2020?
- Who is the best performer in BTS?
- Who is the king of Kpop 2020?
- Who is the best dancer in Kpop?
- Who is the best singer and dancer in BTS?
- Who is the king of kpop?
- Who has best voice in Kpop?
- Is BTS good at dancing?
- Who is Jungkook wife?
- What voice type is Jungkook?
- Who is the richest member of BTS?
- Who is the richest Kpop group?
- Who is Jimin wife?
- Who is the Favourite singer of Jungkook?
- Who is the dance master of BTS?
- Does BTS choreograph their own dance?
- Who is the weakest vocal in BTS?
- Who has the sweetest voice in BTS?
Who is the Queen and King of Kpop 2020?
JisooBlackpink’s Jisoo was introduced ‘2020 QUEEN OF Ok-POP’ after she topped on King Choice’s ‘2020 Queen of Ok-pop’ voting ballot..
Who is the King of Pop now?
Justin Bieber1. Justin Bieber. So here he is, the new King of Pop and the Grand Monarch of Music as well. Justin Bieber is, according to our figures, the most-streamed artist, with the second-largest social media fanbase after Taylor Swift (160 million follows across the two networks).
Who is the best dancer in BTS 2020?
Hands down because of these skills, JIMIN is the BEST DANCER in BTS. His dance techniques are fluid, accentuated, and multi-dimensional. Jhope and JK are more catered to hard-popping.
Who is the best performer in BTS?
They are great solo performers whether it’s singing, rapping or dancing.Here’s proof:RM. RM has fab rapping skills & some fine moves😍❤He has a solo chill ‘Seoul’❤Jin. … Suga. … J-Hope. … Jimin. … V. … JK.
Who is the king of Kpop 2020?
JiminBTS’ Jimin crowned ‘King of Kpop 2020’ and breaks record.
Who is the best dancer in Kpop?
Keep reading to find out who the best dancers in the K-Pop industry are, as voted by idols themselves:EXO’s Kai and TWICE’s Momo.SEVENTEEN’s Hoshi.SHINee’s Taemin and BTS Jimin.SUPER JUNIOR’s Eunhyuk and BTS’ J-Hope.AB6IX’ Park Woojin.Dec 10, 2020
Who is the best singer and dancer in BTS?
JiminAs the lead vocalist and dancer of K-pop’s most popular boy band, Jimin has certainly hit a high note with his fans. Learn where he draws his inspiration from and what he has to say about his eyeliner. Jimin of K-pop boy band BTS is a 22-year-old singer and dancer from Busan, South Korea.
Who is the king of kpop?
BTS singer Jimin crowned King Choice’s ‘The King of Kpop’ for the second consecutive year with whopping votes.
Who has best voice in Kpop?
These Are The Top 50 Best Vocalists In K-Pop, According To FansJaejoong (JYJ)Changmin (TVXQ)Yunho (TVXQ)Daesung (BIGBANG)Onew (SHINee)Junsu (JYJ)Kihyun (MONSTA X)Hongki (F.T. Island)More items…•May 23, 2020
Is BTS good at dancing?
As a whole group they are undoubtedly very good. They strategically place Junkook, Jimin and J-Hope always in the front rows, and you will almost always find RapMon and Jin in the back. Therefore the worse dancers are being well concealed pretty well, and they blend in nicely.
Who is Jungkook wife?
In terms of actual girlfriends, Jungkook is currently single but has previously been rumoured to date K-pop stars Jeong Ye In of the group ‘Lovelyz’, Jung Chae Yeon from ‘DIA’ as well as a 2015 rumour that he dated CUBE trainee Ko So-hyun.
What voice type is Jungkook?
tenorJungkook has a very unique voice. His voice type (core) is a tenor.
Who is the richest member of BTS?
J-HopeWith an estimated net worth of $22 million, J-Hope is unofficially deemed the richest BTS member so far.
Who is the richest Kpop group?
Though, not all of them can earn as many as these 9 richest Kpop groups.GOT7.Red velvet. redvelvet.smtown. 9.8m followers. … MAMAMOO. mamamoo_official. 6.2m followers. … SEVENTEEN. SEVENTEEN (세븐틴) ‘HOME;RUN’ Official MV. … EXO.TVXQ.BLACKPINK. #BLACKPINK ‘Ice Cream (with @SelenaGomez)’ M/V HITS 400 MILLION VIEWS @Youtube. … TWICE.More items…•Nov 17, 2020
Who is Jimin wife?
Who is Jimin’s wife? BTS’ Jimin is single and unmarried till now. There have been a few link ups but he not married to anyone.
Who is the Favourite singer of Jungkook?
Jungkook shared his favorite collaborations with BTS, including ‘On feat. Sia’ and ‘Boy With Luv feat. Halsey’ BTS, as well as individual members of the boy band, did a number of collaborations since their debut.
Who is the dance master of BTS?
Meet Sienna LalauMeet Sienna Lalau, the 19-Year-Old Who Has Choreographed for K-Pop Superstars BTS. “Dionysus” by superstar boy group BTS arguably boasts some of the band’s most intense choreography. Performance and dance practice videos have garnered millions of YouTube views.
Does BTS choreograph their own dance?
The company’s performance director and main choreographer is Son Sungdeuk. He was with BTS since the very beginning and is still their performance director even up till now. Others choreographers include the members themselves such as Jimin and J-Hope for ‘Spring Day’.
Who is the weakest vocal in BTS?
I have no doubt that Jin is the weakest vocalist in BTS overall, at least, technically speaking. This doesn’t mean he is a bad vocalist, he isn’t just as strong as the other members. But looking at the bright side, it is incredible how much he has progressed since BTS debut.
Who has the sweetest voice in BTS?
Who has the sweetest voice in bts ?Jung kook. 33.3%Jimin. 31.0%V. 14.9%Jin. 20.8%Oct 4, 2018


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The Dow Jones dipped by more than 450 points on Thursday. Investors, meanwhile, focused on some notable insider trades.
When insiders purchase or sell shares, it indicates their confidence or concern around the company’s prospects. Investors and traders interested in penny stocks can consider this a factor in their overall investment or trading decision.
Below is a look at a few recent notable insider transactions for penny stocks. For more, check out Benzinga’s insider transactions platform.3
- The Trade: IN8bio, Inc. INAB Director Cavu Advisors, LLC bought a total of 25,000 shares at an average price of $2.02. To acquire these shares, it cost around $50.53 thousand.
- What’s Happening: HC Wainwright & Co., last month, initiated coverage on IN8bio with a Buy rating and announced a price target of $14.
- What IN8bio Does: IN8bio Inc is a clinical-stage biotechnology company.
- The Trade: VerifyMe, Inc. VRME Director Marshall S Geller acquired a total of 2,006 shares at an average price of $1.18. The insider spent around $2.37 thousand to buy those shares.
- What’s Happening: The company’s stock has dropped around 9% over the past month.
- What VerifyMe Does: VerifyMe Inc is a technology solutions company based in the United States. It markets a broad patent portfolio that includes patents in the field of authenticating products, people, and financial transactions.
Tilray To Crater Over 30%? Here Are 5 Other Price Target Changes For Friday
Don’t forget to check out our premarket coverage here .
- The Trade: Nexalin Technology, Inc. NXL Leonard Osser acquired a total of 9,500 shares at an average price of $1.12. To acquire these shares, it cost around $10.64 thousand.
- What’s Happening: Nexalin Technology recently priced its IPO at $4.15 per share.
- What Nexalin Technology Does: Nexalin Technology Inc design and develop innovative neurostimulation products to uniquely and effectively help combat the ongoing global mental health epidemic.
- The Trade: PEDEVCO Corp. PED 10% owner SK Energy LLC acquired a total of 38,742 shares at an average price of $1.06. The insider spent $41.07 thousand to buy those shares.
- What’s Happening: Pedevco, last month, posted Q2 EPS of $0.04.
- What PEDEVCO Does: PEDEVCO Corp is an energy company. It is engaged in the acquisition, exploration, development, and production of oil and natural gas shale plays in the Denver-Julesberg Basin (D-J Basin) in Colorado.
Image and article originally from www.benzinga.com. Read the original article here.

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