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1
Yamashita, Kaoru, Akishi Murata, and Masanori Okuyama. "Miniaturized infrared sensor using silicon diaphragm based on Golay cell." Sensors and Actuators A: Physical 66, no. 1-3 (April 1998): 29–32. http://dx.doi.org/10.1016/s0924-4247(97)01702-0.
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Gaudreault, Charles, Joanny Salvas, and Joël Sirois. "Savitzky–Golay smoothing and differentiation for polymerase chain reaction quantification." Biochemistry and Cell Biology 96, no. 3 (June 2018): 380–89. http://dx.doi.org/10.1139/bcb-2016-0194.
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In quantitative PCR (qPCR), replicates can minimize the impact of intra-assay variation; however, inter-assay variations must be minimized to obtain a robust quantification method. The method proposed in this study uses Savitzky–Golay smoothing and differentiation (SGSD) to identify a derivative-maximum-based cycle of quantification. It does not rely on curve modeling, as is the case with many existing techniques. PCR fluorescence data sets challenged for inter-assay variations (different thermocycler units, different reagents batches, different operators, different standard curves, and different labs) were used for the evaluation. The algorithm was compared with a four-parameter logistic model (4PLM) method, the Cy0 method, and the threshold method. The SGSD method compared favourably with all methods in terms of inter-assay variation. SGSD was statistically different from the 4PLM (P = 0.03), Cy0 (P = 0.05), and threshold (P = 0.004) methods on relative error comparison basis. For intra-assay variations, SGSD outperformed the threshold method (P = 0.005) and equalled the 4PLM and Cy0 methods (P > 0.05) on relative error basis. Our results demonstrate that the SGSD method could potentially be an alternative to sigmoid modeling based methods (4PLM and Cy0) when PCR data are challenged for inter-assay variations.
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Huang, Yue, Yuting Yang, Hang Xu, Gang Zhao, Liwen Feng, Jinqiang Xu, Senlin Huang, and Kexin Liu. "The impact of modulation parameters on the responsivity of Golay cell measuring terahertz radiation." Infrared Physics & Technology 125 (September 2022): 104290. http://dx.doi.org/10.1016/j.infrared.2022.104290.
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Desmaris, Vincent, Hawal Rashid, Alexey Pavolotsky, and Victor Belitsky. "Design, simulations and optimization of micromachined Golay-cell based THz sensors operating at room temperature." Procedia Chemistry 1, no. 1 (September 2009): 1175–78. http://dx.doi.org/10.1016/j.proche.2009.07.293.
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Pei, Jie, and Gang Chen. "Design of Terahertz-Wave Transmission Imaging System." Applied Mechanics and Materials 347-350 (August 2013): 1940–44. http://dx.doi.org/10.4028/www.scientific.net/amm.347-350.1940.
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An imaging system based on the transmission and reflection modes in the terahertz (THz) region is developed by using a backward-wave oscillator (BWO) as source, a Golay-Cell as detector, and an oscilloscope as data acquisition unit. The system software based on the oscilloscope is designed to control the object movement, and the capture and display of the continuous THz wave image data. The imaging of different objects is tested at the frequencies of 450 GHz and 890 GHz to show the validity of the imaging system at room temperature. The influence of objects humidity and thickness, incident wavelength, and translation step on the THz imaging is discussed in details.
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Barton, Sinead, Salaheddin Alakkari, Kevin O’Dwyer, Tomas Ward, and Bryan Hennelly. "Convolution Network with Custom Loss Function for the Denoising of Low SNR Raman Spectra." Sensors 21, no. 14 (July 6, 2021): 4623. http://dx.doi.org/10.3390/s21144623.
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Raman spectroscopy is a powerful diagnostic tool in biomedical science, whereby different disease groups can be classified based on subtle differences in the cell or tissue spectra. A key component in the classification of Raman spectra is the application of multi-variate statistical models. However, Raman scattering is a weak process, resulting in a trade-off between acquisition times and signal-to-noise ratios, which has limited its more widespread adoption as a clinical tool. Typically denoising is applied to the Raman spectrum from a biological sample to improve the signal-to-noise ratio before application of statistical modeling. A popular method for performing this is Savitsky–Golay filtering. Such an algorithm is difficult to tailor so that it can strike a balance between denoising and excessive smoothing of spectral peaks, the characteristics of which are critically important for classification purposes. In this paper, we demonstrate how Convolutional Neural Networks may be enhanced with a non-standard loss function in order to improve the overall signal-to-noise ratio of spectra while limiting corruption of the spectral peaks. Simulated Raman spectra and experimental data are used to train and evaluate the performance of the algorithm in terms of the signal to noise ratio and peak fidelity. The proposed method is demonstrated to effectively smooth noise while preserving spectral features in low intensity spectra which is advantageous when compared with Savitzky–Golay filtering. For low intensity spectra the proposed algorithm was shown to improve the signal to noise ratios by up to 100% in terms of both local and overall signal to noise ratios, indicating that this method would be most suitable for low light or high throughput applications.
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Wang, Yuye, Gang Nie, Changhao Hu, Kai Chen, Chao Yan, Bin Wu, Junfeng Zhu, Degang Xu, and Jianquan Yao. "High-sensitive terahertz detection by parametric up-conversion using nanosecond pulsed laser." Chinese Physics B 31, no. 2 (February 1, 2021): 024204. http://dx.doi.org/10.1088/1674-1056/ac2d20.
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A high-sensitive terahertz detector operating at room temperature was demonstrated based on parametric up-conversion. A nanosecond 1064-nm Nd:YAG laser was used to pump the parametric up-conversion detector and the up-conversion from terahertz wave to NIR laser was realized in a lithium niobate crystal. The minimum detectable terahertz energy of 9 pJ was realized with the detection dynamic range of 54 dB, which was three orders of magnitude higher than that of commercial Golay cell. The detectable terahertz frequency range of the detection system was 0.90 Thz–1.83 THz. Besides, the effects of pump energy and effective gain length on the detection sensitivity were studied in experiment. The results showed that higher pump energy and longer effective gain length are helpful for improving the detection sensitivity of parametric up-conversion detector.
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Xu, Chenhua, Zhicheng Tu, Wenjie Zhang, Jian Cen, Jianbin Xiong, and Na Wang. "A Method of Optimizing Cell Voltage Based on STA-LSSVM Model." Mathematics 10, no. 24 (December 12, 2022): 4710. http://dx.doi.org/10.3390/math10244710.
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It is challenging to control and optimize the aluminum electrolysis process due to its non-linearity and high energy consumption. Reducing the cell voltage is crucial for energy consumption reduction. This paper presents an intelligent method of predicting and optimizing cell voltage based on the evaluation of modeling the comprehensive cell state. Firstly, the Savitzky–Golay filtering algorithm(SGFA) is adopted to denoise the sample data to improve the accuracy of the experimental model. Due to the influencing factors of the cell state, a comprehensive evaluation model of the cell state is established. Secondly, the model of the least squares supports vector machine (LSSVM) is proposed to predict the cell voltage. In order to improve the accuracy of the model, the state transition algorithm (STA) is employed to optimize the structure parameters of the model. Thirdly, the optimization and control model of the cell voltage is developed by an analysis of the technical conditions. Then, the STA is used to realize the optimization of the front model. Finally, the actual data were applied to the experiments of the above method, and the proposed STA was compared with other methods. The results of experiments show that this method is efficient and satisfactory. The optimization value of average cell voltage based on the STA-LSSVM is 3.8165v, and it can be used to guide process operation. The DC power consumption is 11,971 KW·h per tonne of aluminum, with a reduction in power consumption of 373 KW·h. This result guarantees the reduction of aluminum electrolysis energy consumption.
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Li, Lian, Baoyang Ding, Qi Yang, Shang Chen, Huaying Ren, Jinfeng Wang, Hengchang Zang, Fengshan Wang, and Lixuan Zang. "The relevance study of effective information between near infrared spectroscopy and chondroitin sulfate in ethanol precipitation process." Journal of Innovative Optical Health Sciences 07, no. 06 (October 21, 2014): 1450022. http://dx.doi.org/10.1142/s1793545814500229.
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Near infrared spectroscopy (NIRS) is based on molecular overtone and combination vibrations. It is difficult to assign specific features under complicated system. So it is necessary to find the relevance between NIRS and target compound. For this purpose, the chondroitin sulfate (CS) ethanol precipitation process was selected as the research model, and 90 samples of 5 different batches were collected and the content of CS was determined by modified carbazole method. The relevance between NIRS and CS was studied throughout optical pathlength, pretreatment methods and variables selection methods. In conclusion, the first derivative with Savitzky–Golay (SG) smoothing was selected as the best pretreatment, and the best spectral region was selected using interval partial least squares (iPLS) method under 1 mm optical cell. A multivariate calibration model was established using PLS algorithm for determining the content of CS, and the root mean square error of prediction (RMSEP) is 3.934 g⋅L-1. This method will have great potential in process analytical technology in the future.
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Черномырдин, Н. В., А. С. Кучерявенко, Е. Н. Римская, И. Н. Долганова, В. А. Желнов, П. А. Каралкин, А. А. Грядунова, et al. "Терагерцовый микроскоп на основе эффекта твердотельной иммерсии для визуализации биологических тканей-=SUP=-*-=/SUP=-." Журнал технической физики 126, no. 5 (2019): 642. http://dx.doi.org/10.21883/os.2019.05.47665.14-19.
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A novel method of terahertz (THz) microscopy was proposed for imaging of biological tissues with sub-wavelength spatial resolution. It allows for overcoming the Abbe diffraction limit and provides a sub-wavelength resolution thanks to the solid immersion effect – i.e. to the reduction in the dimensions of electromagnetic beam caustic, when the beam is focused in free space, at a small distance (smaller than the wavelength) behind the medium featuring high refractive index. An experimental setup realizing the proposed method was developed. It uses a backward wave oscillator, as a THz-wave emitter, and a Golay cell, as a THz-wave detector. In this setup, the radiation is focused behind the silicon hemisphere in order to realize the solid immersion effect. The spatial resolution of 0.15λ was demonstrated for the developed microscope, while the measurements were carried out at the wavelength of λ=500 μm, with the metal-air interface as a test object. Such a high spatial resolution represents a significant advantage over that of the previously reported arrangements of solid immersion microscopes. The solid immersion microscopy does not imply using any diaphragms or other near-field probes for achieving the sub-wavelength spatial resolution; thus, it eliminates the energy losses associated with such elements. The proposed methods were applied for imaging of biological tissues, and the observed results highlight its potential in biology and medicine.
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Shipilo, Daniil E., Nikolay A. Panov, Irina A. Nikolaeva, Alexander A. Ushakov, Pavel A. Chizhov, Kseniia A. Mamaeva, Vladimir V. Bukin, Sergey V. Garnov, and Olga G. Kosareva. "Low-Frequency Content of THz Emission from Two-Color Femtosecond Filament." Photonics 9, no. 1 (December 29, 2021): 17. http://dx.doi.org/10.3390/photonics9010017.
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We experimentally investigate the low-frequency (below 1 THz) spectral content of broadband terahertz (THz) emission from two-color femtosecond filament formed by the 2.7-mJ, 40-fs, 800+400-nm pulse focused into air. For incoherent detection, we screened the Golay cell by the bandpass filters and measured the THz angular distributions at the selected frequencies ν=0.5, 1, 2 and 3 THz. The measured distributions of THz fluence were integrated over the forward hemisphere taking into account the transmittance of the filters, thus providing the estimation of spectral power at the frequencies studied. The spectral power decreases monotonically with the frequency increasing from 0.5 to 3 THz, thus showing that the maximum of THz spectrum is attained at ν≤0.5 THz. The THz waveform measured by electro-optical sampling (EOS) based on ZnTe crystal and transformed into the spectral domain shows that there exists the local maximum of the THz spectral power at ν≈1 THz. This disagrees with monotonic decrease of THz spectral power obtained from the filter-based measurements. We have introduced the correction to the spectral power reconstructed from EOS measurements. This correction takes into account different focal spot size for different THz frequencies contained in the broadband electromagnetic pulse. The corrected EOS spectral power is in semi-quantitative agreement with the one measured by a set of filters.
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Dalla Pietà, A., E. Cappuzzello, P. Palmerini, R. Sommaggio, G. Astori, K. Chieregato, O. Perbellini, et al. "P09.01 Adoptive cell therapy of hematological malignancies using cytokine-induced killer cells retargeted with monoclonal antibodies." Journal for ImmunoTherapy of Cancer 8, Suppl 2 (October 2020): A51.2—A52. http://dx.doi.org/10.1136/jitc-2020-itoc7.101.
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BackgroundCytokine-Induced Killer (CIK) cells are a population of effector cells that represents a promising tool for adoptive cell therapy. They are easily expandable ex-vivo, safe, and exert cytotoxicity against a broad range of tumor histotypes.1 We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity.2 Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells can be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® (RTX) and Obinutuzumab® (OBI).Materials and MethodsCIK cells were obtained from peripheral blood mononuclear cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb and IL-2 for 14 days; fresh IL-2 was provided every 3–4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against B-cell lines, primary samples and patient-derived xenografts (PDX) obtained from B-cell lymphoma patients after written informed consent.ResultsThe combination with both RTX and OBI significantly increased specific CIK cells lysis against several CD20-expressing lymphoma B cell lines, primary tumors from B-cell lymphoma patients and an established PDX, compared to the combination with a control mAb (cetuximab, CTX). NK-depletion demonstrated that the mAb-mediated cytotoxicity is accountable to the CIK cells fraction within the bulk population since no difference in the lytic activity was detectd in the absence of NK cells. In addition, these results are further supported by in vivo preliminary experiments where the treatment with CIK cells in combination with OBI extensively reduced the growth of PDX and increased mice survival, compared to CIK cells or OBI administered alone.ConclusionsHere we proved that CIK cells can be retargeted with clinical-grade mAbs against CD20-expressing lymphomas. These data indicate that the combination of CIK cells with mAbs can represent a novel approach for the treatment of haematological malignancies.ReferencesFranceschetti M, Pievani A, Borleri G, Vago L, Fleischhauer K, Golay J, et al. Cytokine-induced killer cells are terminally differentiated activated CD8 cytotoxic T-EMRA lymphocytes. Exp Hematol 2009;37:616–28.Cappuzzello E, Tosi A, Zanovello P, Sommaggio R, Rosato A. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies. Oncoimmunology 2016 Aug;5(8):e1199311.The research leading to these results has received funding from Fondazione AIRC under IG 2018 - ID. 21354 project - P.I. Rosato AntonioDisclosure InformationA. Dalla Pietà: None. E. Cappuzzello: None. P. Palmerini: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. O. Perbellini: None. M. Tisi: None. C. Visco: None. M. Ruggeri: None. A. Rosato: None.
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Olesberg, Jonathon T., Mark A. Arnold, and Michael J. Flanigan. "Online Measurement of Urea Concentration in Spent Dialysate during Hemodialysis." Clinical Chemistry 50, no. 1 (January 1, 2004): 175–81. http://dx.doi.org/10.1373/clinchem.2003.025569.
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Abstract Background: We describe online optical measurements of urea in the effluent dialysate line during regular hemodialysis treatment of several patients. Monitoring urea removal can provide valuable information about dialysis efficiency. Methods: Spectral measurements were performed with a Fourier-transform infrared spectrometer equipped with a flow-through cell. Spectra were recorded across the 5000–4000 cm−1 (2.0–2.5 μm) wavelength range at 1-min intervals. Savitzky–Golay filtering was used to remove baseline variations attributable to the temperature dependence of the water absorption spectrum. Urea concentrations were extracted from the filtered spectra by use of partial least-squares regression and the net analyte signal of urea. Results: Urea concentrations predicted by partial least-squares regression matched concentrations obtained from standard chemical assays with a root mean square error of 0.30 mmol/L (0.84 mg/dL urea nitrogen) over an observed concentration range of 0–11 mmol/L. The root mean square error obtained with the net analyte signal of urea was 0.43 mmol/L with a calibration based only on a set of pure-component spectra. The error decreased to 0.23 mmol/L when a slope and offset correction were used. Conclusions: Urea concentrations can be continuously monitored during hemodialysis by near-infrared spectroscopy. Calibrations based on the net analyte signal of urea are particularly appealing because they do not require a training step, as do statistical multivariate calibration procedures such as partial least-squares regression.
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Paulish, Andrey G., Anatoly V. Gusachenko, Alexander O. Morozov, Vladimir A. Golyashov, Kirill V. Dorozhkin, and Valentin I. Suslyaev. "Sensitivity of the tetraaminodiphenyl based pyroelectric sensor from visible to sub-THz range." Sensor Review 40, no. 3 (June 15, 2020): 291–96. http://dx.doi.org/10.1108/sr-03-2020-0047.
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Purpose The purpose of this paper is to study the spectral sensitivity characteristics of new pyroelectric sensor based on tetraaminodiphenyl film within the wavelength range of 0.4-10 µm and 300-3,000 µm. Design/methodology/approach Mylar film with the thickness of about 70 µm was used as the input window. The MDR-41 monochromator-based spectrometric complex and the quasi-optical spectrometer with the set of backward-wave oscillators were used for measurements of the pyrodetector spectral characteristics within the 0.4-10 µm and 300-3,000 µm ranges, respectively. Findings Mylar was found to have absorption lines within the range of 0.4-10 µm, which must be taken into account when broadband detectors developing. The noise equivalent power in the visible and infrared ranges was less than 6 × 10–10 W/Hz1/2, which is about five times lower than for analogue ones. In the sub-THz range, the pyrodetector sensitivity is 2-8 times higher than the Golay cell. The sensitivity of such pyrodetector weakly depends on the wavelength in the total measured range. Practical implications The pyroelectric sensor has good prospects for use in super wide spectral range, from ultraviolet to millimeter radiation, in spectrometers for scientific research, in industry for the operational control of THz radiation sources, as well as in security THz-systems. Originality/value The spectral sensitivity characteristics of the pyroelectric photosensor based on TADPh in the visible, infrared and terahertz ranges were measured. The prospects for the use of such sensors were determined.
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Berdyugin, A. I., A. V. Badin, R. P. Gursky, E. A. Trofimov, and G. E. Kuleshov. "Terahertz Scanning Reflectometer for Structure Visualization of Polymer Constructions in Additive Manufacturing." Ural Radio Engineering Journal 5, no. 3 (2021): 207–24. http://dx.doi.org/10.15826/urej.2021.5.3.001.
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The results of the development and practical application of a quasi-optical system for reflectometry of objects in the terahertz frequency range for analyzing the structure in additive manufacturing of objects are presented. A backward-wave oscillator is used for continuous generation of electromagnetic radiation; an acousto-optic converter (Golay cell) is applied as a detector. The reflectometer is controlled by personal computer through the L-card E 154 input-output module and the standard digital-to-analog converter of the STD-21 spectrometer. The system is tested at the frequency of 874 GHz on the 3D-printed composite structure sample. Our paper is terahertz reflectometer with a source of continuous monochromatic electromagnetic radiation based on a backward wave oscillator is presented. The purpose of this work in creating a scanning THz reflectometer is considered to have been achieved. At the same time, the following tasks are solved: a quasi-optical scheme of the reflectometer is selected and assembled; the hardware part of the system (all mechanisms and components) is implemented; a program for controlling the radiation intensity registration system is adapted for this task; the test sample is manufactured using 3D printing technology, the THz reflectometer is tested. The obtained practical results of registration of the two-dimensional distribution of the reflection coefficient show that the use of THz radiation is promising for visualizing the structure of structures obtained by additive technology. Further development of the project is planned by changing the construction of the positioning mechanism, which will provide micrometric calibration of the sample holder relative to the diaphragm. The use of the quasi-optical scheme of the two-beam interferometer for recording the phase distribution and amplitude of reflected THz radiation will allow obtaining information about the spatial location of defects (inhomogeneities) of products obtained by additive technology.
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Guerini, Vittoria, Orietta Spinelli, Anna Salvi, Guido Finazzi, Tiziano Barbui, Martino Introna, Josee Golay, and Alessandro Rambaldi. "Potent Inhibition of EEC Colony Formation in JAK2V617F PV and ET by Low Doses of ITF2357, a New Histone Deacetylase Inhibitor." Blood 108, no. 11 (November 16, 2006): 2702. http://dx.doi.org/10.1182/blood.v108.11.2702.2702.
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Abstract Background In Polycythemia Vera (PV) and Essential Thrombocythemia (ET) hematopoietic progenitor cells can proliferate in vitro in the absence of exogenous growth factors. A somatic point mutation in the JAK2 gene (JAK2V617F) has been recently recognised as the key pathogenetic lesion of these diseases leading to constitutive tyrosine phosphorylation of JAK2, cytokine hypersensitivity and autonomous outgrowth of hematopoietic progenitor cells. Hystone-Deacetylase inhibitors (HDACi) are known inducers of cell differentiation, apoptosis and cell cycle arrest of neoplastic cells. ITF2357 is a new HDACi (Italfarmaco, Milano, Italy) which, at low micromolar concentration in vitro, inhibits the secretion of several cytokines such as IL-1, IL-6, VEGF and IFN-g and exerts a potent anti tumor activity against multiple myeloma (MM) and acute myeloid leukemia cells (AML) (Golay et al., submitted). ITF2357 is well tolerated when given to normal healthy volounteers and Phase II clinical trials are currently ongoing in AML and MM. Aim To investigate the ability of ITF2357 and the prototypic HDAC inhibitor Suberoyl Anilide Hydroxamic Acid (SAHA) used as control, to inhibit the spontaneous outgrowth of hematopoietic stem cells obtained from patients with PV (n= 6, all JAK2V617F ), ET (n= 13, 7 JAK2V617F ) and Idiopathic Erythrocytosis (IE, n= 6, all negative for JAK2V617F ). Results Endogenous erythroid colonies (EEC) assays were performed using mononuclear cells (MNC) from peripheral blood samples obtained from patients at the time of regular follow-up visits in our clinic. MNC obtained from IE or ET patients negative for JAK2V617F neither exhibited spontaneous EEC formation nor Epo hypersensitivity (from 0.1 UI/ml up to 10UI/ml). On the contrary, MNC from JAK2V617F PV and ET patients invariably sustained the spontaneous EEC outgrowth with a marked Epo hypersensitivity. When ITF2357 was added to the colony assay (ranging from 0.001 to 0.75 μM), a 90% inhibition of EEC formation was observed in all JAK2V617F PV and ET patients at 0.01 μM concentration, which corresponds to a blood level easily attained following oral administration of safe doses of ITF2357 to healthy individuals. By contrast, the prototypic HDAC inhibitor SAHA displayed a similar inhibitory activity on EEC formation only when used at 0.25 μM. By flow cytometry experiments performed on mature granulocytes isolated from PV patients we could show that ITF2357 does not modulate the overexpression of Leucocyte Alkaline Phospatase and CD177 (the PRV-1 gene product) thus suggesting that the inhibitory activity on hematopoietic cells is mainly due to a direct action on the stem cell compartment. Conclusion ITF2357, at concentration easily attained after low oral doses of the drug, show a potent inhibitory activity on the autonomous proliferation of hematopoietic stem cells of PV and TE carrying the JAK2 V617F mutation. This may provide the framework for a Phase II study of ITF2357 in these malignancies.
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Golay, Josée, Fabio Da Roit, Luca Bologna, Claudia Ferrara Koller, Jeanette H. W. Leusen, Alessandro Rambaldi, Christian Klein, and Martino Introna. "Glycoengineered CD20 Antibody Obinutuzumab Activates Neutrophils and Mediates Phagocytosis Through CD16B More Efficiently Than Rituximab." Blood 122, no. 21 (November 15, 2013): 4419. http://dx.doi.org/10.1182/blood.v122.21.4419.4419.
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Background Obinutuzumab (GA101) is a third generation, glycoengineered defucosylated anti-CD20 antibody which shows higher binding affinity for CD16A than fully glycosylated rituximab (RTX). This higher binding leads to stronger NK mediated antibody dependent cellular cytotoxicity activity (ADCC) by obinutuzumab compared to that induced by RTX. The GPI-anchored CD16B molecule is highly homologous to CD16A and is the major FcγR on polymorphonuclear neutrophils (PMN). We have therefore investigated the binding of obinutuzumab to CD16B and its functional activity on human PMN compared to parent rituximab (RTX) or to defucosylated rituximab (G2 antibody). Methods Binding to CD16B of glycoengineered or fully glycosylated anti-CD20 antibodies was measured by surface plasmon resonance (Biacore). For PMN activation and phagocytosis, we have used either purified PMN or analyzed PMN function in unmanipulated whole blood assays from normal donors or CLL patients. PMN activation was measured as CD11b upregulation and CD62L downmodulation by flow cytometry. Phagocytosis by PMN of chronic lymphocytic leukemia (CLL) cells was measured by triple fluorescence (PKH26, CD15-FITC and CD19-APC) and flow cytometry. Results Obinutuzumab or glycoengineered defucosylated rituximab (called G2) bound CD16B with about 7 fold higher affinity, compared to non-glycoengineered wild type parental antibodies. This was true either using surface plasmon resonance or measuring antibody binding to live PMN. Furthermore obinutuzumab activated PMN, either purified or in whole blood, more efficiently than RTX. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on PMN and in release of TNFα, IL-6 and IL-8. Activation was not accompanied by generation of reactive oxygen species or ADCC, but led to phagocytosis of anti-CD20 antibody opsonized CLL targets by purified PMN. Indeed up to 50% phagocytic PMN could be observed in presence of obinutuzumab or G2 antibodies after 6-24 hours incubation of purified PMN with CLL targets. Significant phagocytosis (15%) was also observed in whole blood, but only in presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using blocking F(ab) and F(ab’)2 fragments specific for CD16B and CD32A, that both these receptors are involved in PMN activation, phagocytosis and cell death induced by glycoengineered anti-CD20 antibodies. The possible effect of NA1 and NA2 polymorphisms of CD16B on obinutuzumab binding and phagocytosis is under further investigation. Conclusions We conclude that phagocytosis by PMN is an additional mechanism of action of obinutuzumab, mediated through its higher binding affinity for CD16B compared to RTX. Phagocytosis takes place in whole blood and is followed by PMN death. This effect may in part explain the neutropenia observed after treatment of B-CLL patients with GA101. Disclosures: Golay: Roche Glycart AG: Research Funding. Ferrara Koller:Roche Glycart AG: Employment. Rambaldi:Roche Italia: Consultancy, Honoraria. Klein:Roche Glycart AG: Employment. Introna:Roche Glycart AG: Research Funding.
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Galli, Monica, Silvia Salmoiraghi, Josée Golay, Antonella Gozzini, Alberto Bosi, Claudia Crippa, Giuseppe Rossi, et al. "A Phase II Multiple Dose Clinical Trial of Histone Deacetylase Inhibitor ITF2357 in Patients with Relapsed or Progressive Multiple Myeloma: Preliminary Results." Blood 110, no. 11 (November 16, 2007): 1175. http://dx.doi.org/10.1182/blood.v110.11.1175.1175.
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Abstract Multiple myeloma (MM) is an incurable disease due to the neoplastic proliferation of plasma cells. Histone deacetylase (HDAC) inhibitors are a novel class of agents that can induce tumour cell growth arrest, differentiation and/or apoptosis in vitro and inhibit tumour growth in animals. ITF2357, an orally effective member of the family of HDAC inhibitors, is a potent inducer of apoptosis and death of MM cells (Golay et al. Leukemia, 2007). It has also a potent inhibitory activity on secretion of pro-inflammatory cytokines by stromal and mesenchimal stem cells. For these properties, ITF2357 has been given safely to patients with Crohn’s disease and plaque psoriasis as an anti-inflammatory drug. We report the preliminary results of a phase II multiple dose clinical trial of oral ITF2357 in patients with relapsed or progressive MM, who had received at least two previous lines of treatment. Primary endpoint was to determine the maximum tolerated dose of ITF2357 administered every 12 hours for 4 consecutive days followed by 3 days of rest every week during the first cycle (i.e., first 4 weeks). Up to 12 weeks of treatment with ITF2357 were scheduled. Concomitant oral dexamethasone was given at a maximum dose of 20 mg every week. Fifteen patients (aged 52–77 years) have been enrolled so far. The first 6 patients received 150 mg ITF2357 every 12 hours for 4 consecutive days every week. Because 2 of them experienced a grade 3 gastro-intestinal toxicity (diarrhea) during the first cycle, the subsequent 9 patients received 100 mg ITF2357 every 12 hours with the same weekly schedule. None of them experienced a dose-limiting toxicity during the first cycle. The median duration of treatment was 7 weeks, range from 2 (1 patient) to 12 weeks (5 patients). Thrombocytopenia(≥ grade 2) was the most common side effect, which we observed in 11 cases: grade 4 thrombocytopenia occurred in 4 patients. Grade 3–4 gastro-intestinal toxicity was observed in 4 patients. No grade 4 neutropenia was registered. Three patients experienced 4 serious adverse events: pneumonia in one, severe deterioration of general conditions requiring hospitalization in another and both events in the last one. Two months after completing the 12 weeks of treatment one patient developed atrial fibrillation which required therapy. Two other patients had transient ECG abnormalities without need of hospitalization or therapy. We observed 1 partial remission, 4 stable diseases (SD) and 10 disease progressions. At last follow-up, 6 patients remain alive (3 in SD and 3 with disease progression) and 9 are dead (all for progressive MM). Although ITF2357 is tolerable when administered orally at the dose of 100 mg every 12 hours for 4 consecutive days every week, it is unlikely that it may play a significant therapeutic activity when used alone in advanced MM. However, we noted some evidence of anti-myeloma activity in a group of patients with deteriorated clinical conditions and advanced disease. Therefore, further clinical investigation is warranted to clarify how best to exploit this drug when given in combination with other active drugs to MM patients.
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Guerini, Vittoria, Valentina Barbui, Orietta Spinelli, Anna Salvi, Chiara Dellacasa, Martino Introna, Tiziano Barbui, Josée Golay, and Alessandro Rambaldi. "Selective Targeting of the JAK2V617F Mutation in Polycythemia Vera and Essential Thrombocythemia by ITF2357, a Novel Histone Deacetylase Inhibitor." Blood 110, no. 11 (November 16, 2007): 555. http://dx.doi.org/10.1182/blood.v110.11.555.555.
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Abstract A somatic point mutation in the JAK2 gene (JAK2V617F) is the key pathogenetic lesion of Polycythemia Vera (PV) and Essential Thrombocythemia (ET) and a significant effort is now paid to identify drugs which may be able to interfere with the JAK2V617Fmutated protein. Among others, one potentially interesting drug family is represented by the Histone Deacetylase Inhibitors (HDACi), which may modify the chromatin structure and ultimately the transcription of many genes, the cell cycle progression and the programmed cell death. ITF2357 is a new HDACi (Italfarmaco, Milan, SpA) that shows a potent anti-proliferative and pro-apoptotic activity against acute myeloid leukemia and multiple myeloma cells and little toxicity against normal hematopoietic and mesenchymal stem cells (Golay J et al.: Leukemia 2007). The most common side effects after its administration to normal volounteers and MM patients are represented by thrombocytopenia and gastrointestinal toxicity. These observations prompted us to investigate the inhibitory activity played by ITF2357 on the autonomous proliferation of cells obtained by PV and ET patients carrying the JAK2V617F mutation and to elucidate the mechanism of action of this inhibition. We first investigated the effect of ITF2357 on the clonogenic activity of cell lines carrying or not the JAK2V617F mutation. ITF2357 inhibited colony formation of HEL cells (an erythroleukemia cell line carrying a JAK2V617F homozygous mutation) with an IC50 of about 0.001 μM. In contrast, the doses of drug required to block colony formation by K562, KG1, NB4 and GF-D8 (all negative for the JAK2V617F mutation) were 100–500 fold higher (IC50 ranging from 0.1 to 0.5 μM). Clonogenic assays were then performed using blood mononuclear cells obtained from 4 PV and 7 ET patients, all carrying the JAK2V617F mutation. Either in the presence or absence (EEC assay) of exogenous growth factors, colonies obtained from JAK2V617F mutated progenitor cells were inhibited at much lower doses of ITF2357 (IC50 0.001 μM) as compared to colonies obtained from JAK2 wild type progenitor cells (IC50 0.1–0.25 μM). When single colonies were picked randomly and analyzed by PCR for the presence of wild type or mutated JAK2V617F alleles, a striking reduction of mutated colonies was detected when ITF2357 was added at 0.001 μM and 0.01 μM, confirming that low doses of ITF357 allow the preferential outgrowth of unmutated over mutated colonies from the peripheral blood mononuclear cells of PV patients bearing JAK2V617F. By Western blotting we also showed that ITF2357 treatment for 24 hours, led to virtual disappearance of total and phosphorylated JAK2V617F in HEL cells whereas it did not affect the wild type JAK2 protein in the control K562 cell line, even after 48 hours in the same conditions. Down-modulation of mutated JAK2V617F was accompanied by specific disappearance of p-STAT5 protein. Finally, by Real time PCR analysis of PV cells treated with ITF2357 for 24 hours, we could demonstrate that this drug does not affect JAK2 mRNA but rather it induces a significant decrease of the PRV1 gene, a known JAK2 target. These data suggest that ITF2357 down-modulates the mutated JAK2V617F protein by post-transcriptional mechanisms and that is followed by inhibition of p-STAT5 protein and PRV1 gene expression. The specific inhibition induced by ITF2357 on cells bearing the JAK2V617F mutation underlines its therapeutic potential as a new drug for PV and ET patients.
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Taylor, Ronald P., Paul V. Beum, Margaret A. Lindorfer, and Elizabeth M. Peek. "Examination of the Relative Rates of Rituximab-Mediated Loss of CD20 From B Cells Via Trogocytosis Versus Internalization. Implications for Rituximab Therapy of CLL." Blood 116, no. 21 (November 19, 2010): 2404. http://dx.doi.org/10.1182/blood.v116.21.2404.2404.
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Abstract Abstract 2404 The use of the anti-CD20 monoclonal antibody (mAb) rituximab (RTX) in the immunotherapy of cancer has led to substantial advances in the treatment of B cell malignancies. However, many patients are refractory to RTX therapy and a substantial fraction of responding patients suffer relapses. In some patients the loss or lack of efficacy can be explained because levels of CD20 are substantially reduced on target B cells as a consequence of RTX therapy. This phenomenon may be due to growth and proliferation, over a period of at least several months, of a CD20-negative B cell clone. In contrast, we have reported that levels of CD20 expressed on circulating CLL cells can be reduced > 90% in less than one hour following RTX infusion. These clinical observations, as well as our results seen in vitro and in mouse models, suggest that after the body's effector mechanisms that promote mAb-mediated cell killing are exhausted or saturated, an alternative reaction predominates. RTX-CD20 complexes on targeted B cells are rapidly removed from the B cells by monocytes and macrophages through an Fc receptor-mediated endocytic process known as trogocytosis, or the shaving reaction. Beers et al. recently reported (Blood, 2010) that human CD20 transgenic mouse B cells opsonized with Rit m2a, a mouse IgG2a mAb with RTX activity, internalized both bound mAb and up to half of cell-surface CD20 after incubation for 2 hrs at 37C. They also reported that on average 40% of cell-surface CD20 was internalized when CLL cells opsonized with RTX were similarly treated. It would thus appear that as a consequence of RTX therapy, both trogocytosis by effector cells and direct internalization by B cells can promote loss of CD20 from B cells targeted by RTX. Both of these reactions will reduce the efficacy of RTX therapy, and a key question must focus on the relative rates of trogocytosis and internalization in promoting CD20 loss in vivo. To address these questions we examined human CD20 transgenic mouse 38C13 B cells (a kind gift from Dr. Josee Golay) for RTX-mediated CD20 internalization versus shaving. To test for internalization of cell-bound RTX, CD20+ 38C13 cells were reacted with Al488- or biotinylated RTX for 10 min to 2 hours at 37C, then washed and secondarily probed with the following reagents: 1, Al647 mAb HB43, specific for the Fc region of human IgG; 2, Al488-streptavidin to detect surface-bound biotinylated RTX; 3, Anti-Al488 antibodies that induce quenching of surface-bound Al488 RTX; or 4, The cells were subjected to an acid wash, re-equilibrated at neutral pH, and re-tested for Al488 RTX binding. Our flow cytometry results reveal rapid and stable binding of Al488-labeled or biotinylated RTX to the CD20+ 38C13 cells. After two hours at 37C, the amount of cell-bound RTX that had been internalized, defined by the 4 different assays, was much less than that reported by Beers, and ranged between 5 and 25%, suggesting that the degree of internalization may be model-dependent. Moreover, reaction of acceptor THP-1 cells (a monocytic line) with the RTX-opsonized cells leads to rapid shaving and removal of ~ 90% of bound RTX and CD20 from the 38C13 cells. After just 45 min at 37C in the presence of adhered THP-1 monocytes, there is a 10-fold reduction in fluorescence intensity signal (molecules of equivalent soluble fluorochrome) on the 38C13 cells attributable to either bound Al488 RTX or due to secondary development with Al647 anti-human IgG mAb HB43. Alternatively, Al488 RTX-opsonized CD20+ 38C13 cells were reacted in solution with THP-1 monocytes at varying THP-1/38C13 cell ratios. Within 45 min at 37C at a 3/1 ratio more than half of cell bound Al488 RTX and CD20 were removed from the 38C13 cells, and large amounts of Al488 RTX were found on the acceptor THP-1 cells. Our results indicate that the shaving reaction is considerably more rapid and indeed leads to the removal of much more bound anti-CD20 mAb RTX (and CD20) than the internalization reaction, suggesting that the shaving reaction is most likely primarily responsible for the rapid down-modulation of CD20 that is observed when CLL patients are treated with RTX. Disclosures: No relevant conflicts of interest to declare.
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O’Donnell, Marie Anne. "Erin Goley: Catching the bug for studying the cytoskeleton." Journal of Cell Biology 216, no. 3 (February 24, 2017): 528–29. http://dx.doi.org/10.1083/jcb.201702053.
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Ningsih, Hesty Wulandari, Indriyani Safitri, and Abdul Yusuf. "Pengaruh e-Service Quality dan Kepuasan terhadap e- Loyalty (Survey pada Pengguna Gopay)." Business Management Journal 18, no. 1 (April 21, 2022): 51. http://dx.doi.org/10.30813/bmj.v18i1.3068.
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Advances in technology and information that are growing rapidly require all companies to be able to create innovations to adapt to the progress of today's world. With advances in technology, competition in the increasingly fierce business world, many companies take advantage of an opportunity to create innovations that provide convenience for humans. One of them is a company in the transportation sector that creates digital-based service applications, namely GoJek, which provides payments through the GoPay feature. GoPay is one of the fintech breakthroughs from PT. GoJek utilizes advances in technology and information. The problem in this research is how e-service quality and satisfaction affect the e-loyalty of GoPay users. This study uses a casual analysis design with quantitative methods. The test equipment in this study uses the SPSS Statistic 25 and EViews 11 data processing applications involving 100 respondents as samples from GoPay users throughout Indonesia. Methods of data collection using questionnaires and literature study. The research instrument uses an interval scale. The results show that e-service quality and satisfaction have a positive and significant effect on GoPay customer loyalty.<p align="center"> </p>
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Rambaldi, Alessandro, Chiara Maria Dellacasa, Silvia Salmoiraghi, Orietta Spinelli, Maria Luisa Ferrari, Elisabetta Gattoni, Paola Guglielmelli, Alessandro M. Vannucchi, Giovanni Barosi, and Tiziano Barbui. "A Phase 2A study of the Histone-Deacetylase Inhibitor ITF2357 in Patients with Jak2V617F Positive Chronic Myeloproliferative Neoplasms." Blood 112, no. 11 (November 16, 2008): 100. http://dx.doi.org/10.1182/blood.v112.11.100.100.
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Abstract ITF2357 (Italfarmaco SpA, Cinisello Balsamo, Italy) is a new synthetic class I and II HDAC inhibitor with a potent anti-proliferative and pro-apoptotic activity against several hematologic malignancies both in vitro and in vivo. It also inhibits the production/release of several cytokines including TNFa, IL-1b, IL-12, IFNg, IL-6 and VEGF by neoplastic as well as endothelial and mesenchymal cells (Golay J et al., Leukemia 2007). Recently, we have shown that ITF2357 inhibits the autonomous proliferation of hematopoietic cells from patients with Polycythemia Vera (PV) and Essential Thrombocythemia (ET) through a specific down modulation of the JAK2V617F mutated protein and inhibition of its downstream signaling (Guerini et al., Leukemia, 2008). For these latter properties we planned to evaluate the safety and efficacy of ITF2357 in the treatment of PV, ET as well as Myelofibrosis, both primary (PMF) or secondary to a previous PV or ET (post PV/ET MF). Eligible to this non-randomized, open label, phase II A study, were patients refractory to hydroxyurea or young patients in need of cytoreductive therapy. ITF2357, was given orally at a starting daily dose of 50 mg b.i.d that could be escalated to 50 mg t.i.d., in the absence of toxicity. The dose was reduced at 50 mg/daily or withdrawn in case of toxicity. The total duration of drug administration could not exceed 6 months. In PV and ET a complete response (CR) was defined as HCT< 45%, no need of phlebotomy, platelets < 450 x109/L, WBC <10 x109/L, no splenomegaly, no systemic symptoms (pruritus, fever, weight loss and micro circulatory symptoms). A partial response (PR) was defined if hematocrit was less than 45% without phlebotomy or if response of at least 3 of the above-mentioned criteria was achieved. A no response was defined if the above criteria were not met. For PMF and post PV/ET MF patients, responses were defined according to EUMNET criteria. A total of 26 patients (male/female, 12/14), with a median age of 56 (range 36–70) and a confirmed diagnosis of JAK2V617F positive PV (n= 12), ET (n= 1), PMF (n= 4) or post PV/ET MF (n= 9) were enrolled into this study. At the time of this analysis, all patients received at least 1 month of treatment but only 13 patients have completed at least 12 weeks of treatment. ITF2357 was well tolerated and no patient experienced grade III-IV toxicity. Reasons for dose reduction or transient suspension included on-target side effects (grade 2 thrombocytopenia and anemia were observed in 1 and 4 patients, respectively) and other grade 1 or 2 toxicities (diarrhea, gastric pain, liver enzymes elevation, fatigue, and transient QTc elongation) in 9 patients. In PV/ET patients, 3 complete, 8 partial and 3 no responses were documented. Noteworthy, a significant reduction of palpable spleen was observed in 6 of 8 splenomegalic patients and the disappearance of pruritus in most of them. Two major and 2 moderate responses (EUMNET criteria) were registered among the 13 MF patients. Molecular monitoring of the JAK2V617F mutated allele performed by quantitative PCR showed a median reduction of 8% (range 1–17%) in 10/17 patients and 12% (range 2–29%) in 7/8 patients analyzed after 12 and 24 weeks of treatment, respectively. In conclusion, treatment with ITF2357, a drug with a pleiotropic activity on chromatin structure and gene transcription, was followed by a rapid improvement of constitutional symptoms, spleen size and hematologic response in the great majority of PV/ET and in some MF patients. Different drug dosing and combinations are now under investigation.
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Pozzo, Federico, Tamara Bittolo, Pietro Bulian, Erika Tissino, Francesca Maria Rossi, Paolo Macor, Riccardo Bomben, et al. "NOTCH1 Mutations Are Associated with Low CD20 Expression in Chronic Lymphocytic Leukemia: Evidences for a NOTCH1-Mediated Epigenetic Regulatory Mechanism." Blood 124, no. 21 (December 6, 2014): 296. http://dx.doi.org/10.1182/blood.v124.21.296.296.
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Abstract Background. NOTCH1 mutations are found in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases and in Richter Syndrome transformations (Rossi et al. Blood, 2013). In CLL, NOTCH1 mutations have been recently associated with clinical resistance to anti-CD20 immunotherapy (Stilgenbauer et al. Blood, 2014, Dal Bo et al. AHO, 2014). In lymphoproliferative disorders, susceptibility to rituximab is determined by CD20 levels (Golay et al. Blood, 2001), which are in turn epigenetically modulated via HDAC (Shimizu et al., Leukemia, 2010). Aim. To investigate whether NOTCH1 mutations could affect CD20 expression in CLL. Methods. NOTCH1 mutations and NOTCH1 mutational burden were evaluated by ARMS PCR, QRT-PCR, conventional and next generation sequencing. NOTCH1 protein levels were evaluated by western blot. CD20 expression was evaluated by flow cytometry. Transcript levels of MS4A1, encoding for CD20 protein, were evaluated by QRT-PCR. CLL cells and CLL-like cell line MEC-1 cells were treated with the HDAC inhibitor valproic acid (VPA) at a concentration of 3mM. Susceptibility to rituximab was evaluated by complement dependent cytotoxicity (CDC) assay. MEC-1 cells were transfected with a vector containing a NOTCH1 intracellular domain (NICD) carrying either the 7544-7545delCT or a stop codon at the beginning of NICD sequence (c.5304G>A), as control. Results. i) In a cohort of 452 CLL, 54 cases carried NOTCH1 mutations. NOTCH1-mutated cases (NOTCH1-mut) with high mutational burden showed 3.0- fold higher transmembrane NOTCH1 and 2.1- fold higher NICD protein expression compared to NOTCH1 wild-type cases (NOTCH1-wt). NOTCH1-mut showed a lower Mean Fluorescent Intensity (MFI) of CD20 expression than NOTCH1-wt both in trisomy 12 (tris12, 18 NOTCH1-mut vs. 44 NOTCH1-wt, p<0.001) and in non-tris12 (36 NOTCH1-mut vs. 354 NOTCH1-wt, p=0.005) cases. MS4A1 transcript levels were lower in NOTCH1-mut than in NOTCH1-wt (tris12 CLL, p=0.024; non-tris12 CLL, p=0.002). By performing cell sorting to isolate CD20low and CD20high subpopulations in 4 cases with subclonal NOTCH1 mutations (range 26%-45%), the CD20low subpopulation always showed an enrichment in NOTCH1 mutational burden when compared to the CD20high counterpart, along with lower MS4A1 expression levels. NICD mutated MEC-1 cells, expressing higher NICD transcript (2.2 over the control) and NICD (1.3 over the control) protein, were characterized by lower CD20 (0.1 over the control) and MS4A1 transcript (0.77 over the control) expression. ii) In keeping with CD20 levels, primary NOTCH1-mut CLL cells showed lower relative lysis induced by rituximab than NOTCH1-wt CLL cells (mean relative lysis: NOTCH1-mut, 5% vs. NOTCH1-wt, 30%, p=0.008). This phenomenon was confirmed by using the MEC-1 cell model (mean relative lysis: NICD mutated, 3% vs. control, 30%, p=0.006). iii) NICD mutated MEC-1 cells expressed higher levels of both HDAC1 (2.0 over the control) and HDAC2 (1.4 over the control) transcripts. Moreover, in co-immunoprecipitation experiments, NICD mutated MEC-1 cells were characterized by lower levels of HDAC1/HDAC2 bound to the transcriptional factor CBF-1, due to a competition for the binding to CBF1 with the high NICD levels present in these cells, and suggesting a nuclear interplay between NOTCH1 and HDAC where CBF-1 is the connecting element. The dependency of low CD20 expression by HDAC activity was demonstrated by the capability of VPA to increase CD20 expression in primary NOTCH1-mut CLL cells (1.3 over the control) and NICD mutated MEC-1 transfectants (1.4 over the control). Conclusions. CLL cases carrying NOTCH1 mutations are characterized by low CD20 expression levels that may confer resistance to anti-CD20 immunotherapy. The low CD20 expression, at least in part due to HDAC-dependent repression mechanism(s), can be reverted by HDAC inhibitor therapy. Disclosures No relevant conflicts of interest to declare.
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Thompson, Roger J., Hillary C. S. R. Akana, Claire Finnigan, Kathryn E. Howell, and John H. Caldwell. "Anion channels transport ATP into the Golgi lumen." American Journal of Physiology-Cell Physiology 290, no. 2 (February 2006): C499—C514. http://dx.doi.org/10.1152/ajpcell.00585.2004.
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Anion channels provide a pathway for Cl− influx into the lumen of the Golgi cisternae. This influx permits luminal acidification by the organelle's H+-ATPase. Three different experimental approaches, electrophysiological, biochemical, and proteomic, demonstrated that two Golgi anion channels, GOLAC-1 and GOLAC-2, also mediate ATP anion transport into the Golgi lumen. First, GOLAC-1 and -2 were incorporated into planar lipid bilayers, and single-channel recordings were obtained. Low ionic activities of K2ATP added to the cis-chamber directly inhibited the Cl− subconductance levels of both channels, with Km values ranging from 16 to 115 μM. Substitution of either K2ATP or MgATP for Cl− on the cis, trans, or both sides indicated that ATP is conducted by the channels with a relative permeability sequence of Cl− > ATP4− > MgATP2−. Single-channel currents were observed at physiological concentrations of Cl− and ATP, providing evidence for their importance in vivo. Second, transport of [α-32P]ATP into sealed Golgi vesicles that maintain in situ orientation was consistent with movement through the GOLACs because it exhibited little temperature dependence and was saturated with an apparent Km = 25 μM. Finally, after transport of [γ-32P]ATP, a protease-protection assay demonstrated that proteins are phosphorylated within the Golgi lumen, and after SDS-PAGE, the proteins in the phosphorylated bands were identified by mass spectrometry. GOLAC conductances, [α-32P]ATP transport, and protein phosphorylation have identical pharmacological profiles. We conclude that the GOLACs play dual roles in the Golgi complex, providing pathways for Cl− and ATP influx into the Golgi lumen.
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Golay, Josee, Luca Bologna, Elisa Gotti, Alessandro Rambaldi, Renato Bassan, and Martino Introna. "Predominant Complement Mediated Lysis of B-CLL Cells by Therapeutic MAbs Rituximab and Campath-1H in Whole Blood Assays." Blood 114, no. 22 (November 20, 2009): 3445. http://dx.doi.org/10.1182/blood.v114.22.3445.3445.
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Abstract Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit (<0.1%) in most cases analysed, suggesting that ADCC in the circulation is not a major mechanism of lysis in this disease subtype. Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.
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Adaye, Kouassi Albert, N’Guessan Simon Andon, and Quonan Christian Yao-Kouassi. "Facteurs Géographiques Explicatifs des Inondations en Milieu Urbain: Cas du Bassin Versant du Cours d’eau Goley a Sinfra (Centre-Ouest Côte d’Ivoire)." European Scientific Journal, ESJ 18, no. 26 (August 31, 2022): 163. http://dx.doi.org/10.19044/esj.2022.v18n26p163.
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Les inondations urbaines du Goley de Sinfra sont-elles liées à la forme de son bassin versant ou aux usages socio-économiques de cet espace ? Cette étude veut saisir les facteurs explicatifs des inondations du bassin versant urbain du Goley. La méthodologie a associé les SIG, les enquêtes socioculturelles et administratives. Les résultats montrent que les inondations ne sont pas liées à la forme du bassin versant mais aux usages de l’espace urbain lié au fonctionnement hydrologique du Goley. Ces usages se traduisent par les installations d’habitats hors lotissement et d’autres formes d’anthropisation dans les servitudes et lits du réseau hydrographique urbain. A cela, s’ajoutent l’absence d’aménagement des lits urbains contre les inondations et la présence de divers aménagements obstacles aux libres écoulements des eaux dans les lits dont les voiries aux canaux des ponts obstrués par la matière plastique formant ainsi des digues de barrages dans l’espace urbain. En outre, la sacralisation d’une partie du tronçon du réseau hydrographique urbain montre encore la mainmise des populations sur cet espace dédié au fonctionnement hydrologique qui empêche d’avance tout projet d’aménagement urbain du Goley contre les inondations.
Are the urban floods of the Sinfra Goley linked to the shape of its catchment area or to the socio-economic uses of this space? This study aims to understand the explanatory factors of flooding in the Goley urban watershed. The methodology combined GIS, socio-cultural and administrative surveys. The results show that the floods are not linked to the shape of the watershed but to the uses of the urban space linked to the hydrological functioning of the Goley. These uses result in the installation of habitats outside subdivisions and other forms of anthropization in the easements and beds of the urban hydrographic network. Added to this is the lack of development of the urban beds against flooding and the presence of various structures that impede the free flow of water in the beds, including the roads to the canals of the bridges obstructed by the plastic material thus forming dykes of dams in the urban space. In addition, the sanctification of part of the section of the urban hydrographic network further shows the control of the populations on this space dedicated to hydrological functioning which prevents in advance any urban development project of the Goley against floods project to develop the Goley watercourse against flooding in the urban catchment area.
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Golan, Karin, and Tsvee Lapidot. "Daily light-and-darkness onset regulates mouse hematopoietic stem cells." Blood Advances 3, no. 4 (February 26, 2019): 704. http://dx.doi.org/10.1182/bloodadvances.2018027722.
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Abstract In this issue’s Blood Advances Talk, Lapidot and Golan discuss how changes in daily light regulate hematopoiesis. This fascinating mechanism helps control the process of maintaining the hematopoietic stem cell pool while promoting sufficient differentiation to supply adequate numbers of functional blood cells. We hope you enjoy listening to this interesting topic.
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Badin, A. V., A. I. Berdyugin, V. D. Moskalenko, K. V. Simonova, and R. P. Gursky. "Two-dimensional THz reflectometry of a periodic structure obtained by additive technology." Journal of Physics: Conference Series 2140, no. 1 (December 1, 2021): 012015. http://dx.doi.org/10.1088/1742-6596/2140/1/012015.
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Abstract This paper considers the development and application of a system of reflectometry for the analysis of the homogeneity of structures manufactured by additive technologies. A system of reflectometry based on a backward wave oscillator, a two-dimensional object positioning system and an optoacoustic detector (Goley cell) is described. The results of reflectometry of the hexagonal periodic structure of cells based on acrylonitrile butadiene styrene at a wavelength of 343 microns are presented.
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Zuo, Bin, Junsheng Cheng, and Zehui Zhang. "Degradation prediction model for proton exchange membrane fuel cells based on long short-term memory neural network and Savitzky-Golay filter." International Journal of Hydrogen Energy 46, no. 29 (April 2021): 15928–37. http://dx.doi.org/10.1016/j.ijhydene.2021.02.069.
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Martín Mosquero, Mª Ángeles, Rocío Juan, and Julio Pastor. "Estudio morfológico)' anatómico en miculas de Nepeta L. (Lamiaceae) del suroeste de España." Acta Botanica Malacitana 27 (December 1, 2002): 15–25. http://dx.doi.org/10.24310/abm.v27i0.7305.
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RESUMEN. Estudio morfológico)' anatómico en miculas de Nepeta L. (Lamiaceae) del suroeste de España. Se describe la micromorfología y la anatomía de las núculas en las cinco especies de Nepeta L. presentes en el suroeste de España: N. catana L., N. amethystina var. anticaria Ladero & Rivas Goday, N. multibracteata Desf., N. tuberosa L. subsp. tuberosa y N. apuleii Ucria, tanto al microscopio óptico (M.O.) como al microscopio electrónico de barrido (M.E.B.). Los resultados han permitido diferenciar tres tipos estructurales atendiendo básicamente a la ornamentación de la núcula. Además, los caracteres anatómicos han permitido delimitar los taxones estudiados fundamentalmente a nivel del epicarpo, mesocarpo y capa en empalizada. La excreción de mucílago en las núculas de N. amethystina var. anticaria ha permitido diferenciar claramente este taxón. Así mismo, atendiendo a los caracteres carpológicos estudiados, se propone una clave para diferenciar los citados taxones.Palabras clave. Núcula, morfología, anatomía, mucílago, Nepeta, Lamioideae.ABSTRACT. Morphological and anatomical studies on nutlets of Nepeta L. (Lamiaceae) from South-West Spain. The micromorphology and anatomy of nutlets of the five Nepeta L. species from South-West Spain: N. ca/aria L., N. amethystina var. anticaria Ladero & Rivas Goday, N. multibracteata Desf., N. tuberosa L. subsp. tuberosa y N. apuleii Ucria, are described with light and scanning electron microscopy. The results have showed the possibility to differentiate three structural nutlet types basically attending of nutlet ornamentation. Anatomical characters have allowed distinguish the examined taxa using epicarp, mesocarp and sclerenchymatic cell layer characters. The mucilage secretion of N. atnethystina var. anticaria nutlet has allowed easily define this taxon. Also, a key to differentiate the five taxa using carpological characters is provided.Key words. Nutlet, morphology, anatomy, mucilage, Nepeta, Lamioideae.
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Shafique, Muhammad, Ahmad Sattar Khan, Aman Ullah Malik, and Muhammad Shahid. "Exogenous Application of Oxalic Acid Delays Pericarp Browning and Maintain Fruit Quality of Litchi cv. “Gola”." Journal of Food Biochemistry 40, no. 2 (October 22, 2015): 170–79. http://dx.doi.org/10.1111/jfbc.12207.
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Maharani, Anggita Putri, and Agung Triayudi. "Sentiment Analysis of Indonesian Digital Payment Customer Satisfaction Towards GOPAY, DANA, and ShopeePay Using Naïve Bayes and K-Nearest Neighbour Methods." JURNAL MEDIA INFORMATIKA BUDIDARMA 6, no. 1 (January 25, 2022): 672. http://dx.doi.org/10.30865/mib.v6i1.3545.
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The utilization of different results from the pace of technological development provides a lot of convenience, benefits, and time efficiency. Now, many related companies or organizations are engaged in technology, offering various services that they have developed to meet the needs of a highly consumptive society, with different terms and conditions. There are many descriptions related to the experience of using the platform that it developed on social media, where everyone can express their feelings and opinions about something since people commonly use Twitter. This study used sentiment analysis and opinion mining to see public satisfaction with digital payment services available in Indonesia by focusing on several available services such as GOPAY, DANA, and ShopeePay. The dataset that became the source of this research was taken from Twitter data through several preparation stages, including data crawling, data cleaning, feature selection, and classification with two machine learning approaches (K-Nearest Neighbour and Naïve Bayes). The raw data held is pre-processed until making the clean data. According to the classification algorithm, a feature search is implemented in this study so that the classification process and data modeling validation provide significant results.
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Rajopadhye, Anagha, and Anuradha S. Upadhye. "Estimation of Bioactive Compound, Maslinic Acid by HPTLC, and Evaluation of Hepatoprotective Activity on Fruit Pulp ofZiziphus jujubaMill. Cultivars in India." Evidence-Based Complementary and Alternative Medicine 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/4758734.
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Fruits ofZiziphus jujubaMill. (family: Rhamnaceae), known as Indian jujube or “Ber,” are of potential nutritional and medicinal value. The objectives of the present study were to estimate bioactive compound maslinic acid by HPTLC method and to evaluatein vitroantioxidant and hepatoprotective activity of eight cultivars of Indian jujube. Maslinic acid and the fruit pulp of various cultivars of Indian jujube, namely,Gola,Sannur,Umaran,Mehrun, andChhuhara, exhibited significantly high antioxidant and hepatoprotective activity. HPTLC-densitometric method was developed for quantification of maslinic acid from fruits of Indian jujube cultivars. The trend of occurrence of maslinic acid in fruits pulp extracts was as follows:Gola>Sannur>Umaran>Mehrun>Chhuhara> Wild >Kadaka>Apple. A significant correlation was shown by maslinic acid content and prevention of oxidative stress induced by CCl4in liver slice culture cell treated with extract. Maslinic acid along with its other phytoconstituents like phenols, flavonoids, and ascorbic acid may act as a possible therapeutic agent for preventing hepatotoxicity caused by oxidative stress generated due to the prooxidants like CCl4. This is the first report of fruit pulp extracts ofZ. jujubecultivars in India and maslinic acid preventing CCl4induced damage in liver slice culture cell of mice.
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Huang, Ying, Allan B. Okey, and Patricia A. Harper. "Aromatic hydrocarbon receptor in cultured fetal cells from C57BL/6J and DBA/2J mice: similarity in molecular mass to receptors in adult livers." Canadian Journal of Physiology and Pharmacology 73, no. 1 (January 1, 1995): 18–26. http://dx.doi.org/10.1139/y95-003.
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In liver of adult responsive C57BL/6J (B6) mice the aromatic hydrocarbon receptor (AHR) has high affinity for specific halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as nonhalogenated aromatic hydrocarbons (PAHs), such as benz[a]anthracene (BA) or 3-methylcholanthrene (MC). In livers of adult nonresponsive DBA/2J (D2) mice TCDD binds to a low-affinity variant form of AHR. Both TCDD and MC induce aryl hydrocarbon hydroxylase (AHH) in adult B6 mice, whereas adult D2 mouse liver is nonresponsive to MC. In fetal cell cultures derived from D2 mice AHH is induced by PAHs such as MC or BA, and these PAHs bind to cytosolic AHR (P.A. Harper, C.L. Golas, and A.B. Okey. Mol. Pharmacol. 40: 818–826, 1991). We compared AHR from fetal cell cultures with AHR from adult livers to determine whether there was some structural difference in receptors expressed in fetal cell culture that might permit cells from "nonresponsive" mice to respond to PAHs. The apparent molecular mass of AHR from cells cultured from 18-day fetuses is identical with that from adult liver within each strain of inbred mice tested (Mr ~ 95 kDa in B6 and ~ 105 kDa in D2 mice). The AHR in D2 fetal cells was able to activate a transfected chloramphenicol acetyltransferase linked to a dioxin-responsive element nucleotide sequence (DRE–CAT) when the cells were treated with TCDD or MC. The potency of CAT expression in D2 fetal cells was similar to that in B6 fetal cells. Our data suggest that the responsiveness of fetal cells from "nonresponsive" mice is likely mediated by AHR in these cells but is not due to expression of a different allelic form of AHR ligand-binding subunit in fetal cells versus adult liver.Key words: aromatic hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin, cultured fetal cells, C57BL/6J mice, DBA/2J mice.
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Wheeler, Caroline, Yuanquan Yang, Daniel Spakowicz, Rebecca Hoyd, and Mingjia Li. "942 The tumor microbiome correlates with response to immune checkpoint inhibitors in renal cell carcinoma." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A988—A989. http://dx.doi.org/10.1136/jitc-2021-sitc2021.942.
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BackgroundImmune checkpoint inhibitor therapy, or ICI, is currently the most successful treatment option for patients with renal cell carcinoma (RCC). However, only 20% of patients have a durable response,1 driving a significant need to improve treatment outcomes. The tumor microbiome has recently been shown to play a role in chemotherapy-based treatment outcomes, but, to our knowledge, no study has explored its role in response to ICIs.2–4MethodsTumor samples were collected from 22 patients with RCC as a part of the Total Cancer Care program at The Ohio State University Comprehensive Cancer Center. Raw RNA-seq reads from these biopsies, as well as data on the responses to ICI therapy were collected. Response evaluation was based on RECIST v1.1 criteria with complete or partial response, or stable disease classified as ”Responders,”, and progressive disease classified as ”Non-responsders”. The RNA-seq reads were processed through a pipeline developed by the Spakowicz lab, known as ExoTIC (Exogenous sequences in Tumor and Immune Cells), to carefully identify exogenous sequences.5 6 Reads that don’t align to the human reference genome are meticulously filtered of (1) common laboratory contaminants, (2) taxa that inversely correlate with input RNA quantity, and (3) taxa commonly found in the negative controls of microbiome experiments. DESeq2 was used to perform a differential abundance analysis on the comparison groups at every taxonomic level.ResultsThe 22 patients with RCC range from 22 to 74 years of age at diagnosis, are 72.7% male, and 54.5% responded to ICIs. Exogenous taxa are identified in the tumor RNAseq, including bacteria, fungi, and viruses (figure 1). Within the tumors responsive to immunotherapy, there was found to be a significant enrichment of certain microbial species, including Bacillus thuringiensis, Comamonas testosteroni, Colletotrichum higginsianum, and Elaeis guineesis. Comparatively, the cohort of non-responsive tumors was found to have a significant enrichment of Candidatus Promineofilum breve, Clostridioides difficile, Nocardia cyriacigeorgica, Streptomyces sp. CdTB01, and Streptomyces venezuelae (figure 2).Abstact 942 Figure 1Relative abundances of exogenous taxa found in tumor RNAseq are shown in a stacked bar plotAbstact 942 Figure 2Differential abundance analysis of taxa found within tumor RNAseq data by the exotic pipeline. Colored points represent significantly (pvalue < 0.05) enriched taxa with a high (>2.5) fold-difference in abundance between the groupsConclusionsWe found that prior to ICI treatment the tumor microbiome of patients with RCC whose tumors responded to immunotherapy vary from those that did not respond to treatment. This implies that a therapeutic target to modify the tumor microbiome to improve treatment outcomes. Future research will evaluate whether these correlations are causally associated with outcomes and will evaluate their effect on the tumor microenvironment including immune cell infiltration.AcknowledgementsThe authors acknowledge the support and resources of the Ohio Supercomputing Center (PAS1695).ReferencesCiccarese C, Di Nunno V, Iacovelli R, Massari F. Future perspectives for personalized immunotherapy in renal cell carcinoma. Expert opinion on biological therapy. Taylor & Francis. 2017;17(9):1049–1052.Geller LT, Barzily-Rokni M, Danino T, Jonas OH, Shental N, Nejman D, Gavert N, Zwang Y, Cooper ZA, Shee K, Thaiss CA, Reuben A, Livny J, Avraham R, Frederick DT, Ligorio M, Chatman K, Johnston SE, Mosher CM, Brandis A, Fuks G, Gurbatri C, Gopalakrishnan V, Kim M, Hurd MW, Katz M, Fleming J, Maitra A, Smith DA, Skalak M, Bu J, Michaud M, Trauger SA, Barshack I, Golan T, Sandbank J, Flaherty KT, Mandinova A, Garrett WS, Thayer SP, Ferrone CR, Huttenhower C, Bhatia SN, Gevers D, Wargo JA, Golub TR, Straussman R. Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine. Science 2017 September 15;357(6356):1156–1160. PMID: 28912244.Nejman D, Livyatan I, Fuks G, Gavert N, Zwang Y, Geller LT, Rotter-Maskowitz A, Weiser R, Mallel G, Gigi E, Meltser A, Douglas GM, Kamer I, Gopalakrishnan V, Dadosh T, Levin-Zaidman S, Avnet S, Atlan T, Cooper ZA, Arora R, Cogdill AP, Khan MAW, Ologun G, Bussi Y, Weinberger A, Lotan-Pompan M, Golani O, Perry G, Rokah M, Bahar-Shany K, Rozeman EA, Blank CU, Ronai A, Shaoul R, Amit A, Dorf-man T, Kremer R, Cohen ZR, Harnof S, Siegal T, Yehuda-Shnaidman E, Gal-Yam EN, Shapira H, Baldini N, Langille MGI, Ben-Nun A, Kaufman B, Nissan A, Golan T, Dadiani M, Levanon K, Bar J, Yust-Katz S, Barshack I, Peeper DS, Raz DJ, Segal E, Wargo JA, Sandbank J, Shental N, Straussman R. The human tumor microbiome is composed of tumor type–specific intracellular bacteria. Science 2020 May 29;368(6494):973–980.Poore GD, Kopylova E, Zhu Q, Carpenter C, Fraraccio S, Wandro S, Kosciolek T, Janssen S, Metcalf J, Song SJ, Kanbar J, Miller-Montgomery S, Heaton R, Mckay R, Patel SP, Swafford AD, Knight R. Microbi-ome analyses of blood and tissues suggest cancer diagnostic approach. Nature 2020;579(7800):567–574. PMID: 32214244.Malalur, Pannaga, Mo, Xiaokui, Hoyd, Rebecca, Hays, John, Carbone, David, Spakowicz, Daniel. Investigating intra-tumor microbes, blood microbes, and CEA for development of non-invasive biomarkers in colorectal cancer. Journal of Clinical Oncology 2021;39(15_suppl): 3551–3551.Malalur PG, Mo X, Hoyd R, Carbone DP, Spakowicz D. Intra-tumoral microbes and overall survival in colorectal cancer patients. Journal of Clinical Oncology 2020;38(15_suppl):4083–4083.Ethics ApprovalData were obtained through an IRB-approved Honest Broker protocol (2015H0185) supporting the Total Cancer Care protocol 2013H0199.
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TONG, Jiefei, Guo Guang DU, S. R. Wayne CHEN, and David H. MACLENNAN. "HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine." Biochemical Journal 343, no. 1 (September 24, 1999): 39–44. http://dx.doi.org/10.1042/bj3430039.
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Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca2+ homoeostasis, the properties of intracellular Ca2+ stores and the methods used for measuring intracellular Ca2+ release in HEK-293 cells were evaluated. Ca2+ imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca2+ release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca2+ release in untransfected HEK-293 cells. Intracellular Ca2+ release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca2+ release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [3H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.
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Khalid, Zheen, and Ardawan Ali. "Assessment of Serum Podocalyxin as a Biomarker for Diabetic Nephropathy in Type 2 Diabetes patients in Duhok City." Journal of Life and Bio Sciences Research 3, no. 02 (November 4, 2022): 71–75. http://dx.doi.org/10.38094/jlbsr30275.
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Podocalyxin, a glycosylated cell surface sialomucin of the CD34 family, is a kidney podocyte membrane negatively charged protein and the essential constituent of the glomerular basement membrane charge barrier. Additionally, it has a crucial role in maintaining the glomerular filtration barrier permeability. It has been considered that one of the most important factors in diabetic nephropathy is podocyte injury. In the natural history of diabetic nephropathy and macrovascular complications, podocytes have been shown to be structurally and functionally affected. The current study aimed to determine serum podocalyxin levels in patients with type 2 diabetes mellitus and to analyse its relation to glomerular filtration rate and albuminuria. This cross-sectional study was performed at the diabetic center in Azadi Teaching Hospital and Golan Hospital in Dohuk city from September 2021 to March 2022. Consecutive sampling was applied to select 200 subjects (case group) with T2DM aged 30-65 years and 93 healthy subjects (control group). BMI, eGFR, albuminuria, HbA1c, and serum podocalyxin levels were measured for all study subjects. Serum podocalyxin levels were significantly higher in the case group (P =0.019). There was no significant difference in serum podocalyxin levels between case groups (normoalbuminuria, microalbuminuria, and macroalbuminuria). A significant negative correlation between eGFR and serum podocalyxin was found in both control and case group (P =0.010 r =-0.267, P =0.030 r =-0.154, respectively). Although S.PCX levels had a significant difference between the case and control group and a significant negative correlation with eGFR, it may not be considered as a diabetic nephropathy biomarker.
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Shevtsov, Sofia, Omer Murik, Hagit Zer, Ofir Weinstein, Nir Keren, Ori Fragman-Sapir, and Oren Ostersetzer-Biran. "The complete plastid genome sequence and the photosynthetic activity of the putative mycoheterotrophic orchid Limodorum abortivum." Israel Journal of Plant Sciences 66, no. 1-2 (March 11, 2019): 69–88. http://dx.doi.org/10.1163/22238980-00001075.
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The sparsely distributed Limodorum abortivum is a European-Mediterranean orchid species, which grows on decomposing plant material. Although some chlorophyll-pigmentation is observed in the degenerated scales-shaped leaf and stems regions of the plant, its photosynthetic capacity is assumed to be insufficient to support the full energy requirements of an adult plant. In Israel, L. abortivum shows a patchy distribution patterns in the Galilee, Golan, Carmel and Judean regions. To gain more insights into the physiology and photosynthetic activity of L. abortivum, we analyzed the organellar morphologies, photosynthetic activities the chloroplast-DNA sequence by Illumina-HTS. Microscopic analyses indicated to the presence of mature chloroplasts with well-organized grana-thylakoids in the leaves and stems of L. abortivum. However, the numbers of chloroplasts per cell and the grana ultrastructure density within the organelles were notably lower than those of model plant species and fully photosynthetically-active orchids. The cpDNA of L. abortivum (154,954 bp) encodes 60 proteins, 34 tRNAs and 4 rRNAs. The coding-regions of 24 genes are interrupted by 26 group-II intron-sequences. While many genes related to photosynthesis (RuBisCo, PSI, PSII and cytochrome b 6 /f subunits) have remained intact in the cpDNA, the majority of the NADH-dehydrogenase (ndh) subunits were either lost or became nonfunctional (i.e. pseudogenized). In agreement with previous reports, the photosynthetic-rates of adult Limodorum plants were found to be very low, further indicating that carbon-assimilation activity is insufficient to support the energy requirements of an adult plant, and may suggest that L. abortivum have adopted nutritional strategies similar to that of mycoheterotrophic orchid species.
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Szydlowski, Maciej, Filip Garbicz, Ewa Jabłońska, Patryk Górniak, Beata Pyrzynska, Kamil Bojarczuk, Dorota Komar, et al. "Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies." Blood 136, Supplement 1 (November 5, 2020): 33–34. http://dx.doi.org/10.1182/blood-2020-134778.
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R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.
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Sharma, Aditi, A. K. Gupta, K. Khosla, Rishi Mahajan, Bharti, and P. K. Mahajan. "Antagonistic potential of native agrocin-producing non-pathogenic Agrobacterium tumefaciens strain UHFBA-218 to control crown gall in peach." Articles scientifiques 97, no. 1 (July 13, 2017): 1–11. http://dx.doi.org/10.7202/1040509ar.
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A non-pathogenic agrocin-producing native isolate ofAgrobacterium tumefaciensstrain UHFBA-218 was tested as a biological control agent against the peach crown gall. This strain was compatible with all the recommended pesticides used in stone fruits in the integrated pest management (IPM) module, except for copper oxychloride, which was detrimental to its growth. Upon artificial co-inoculation of 4-wk-old plants of tomato var. Solan Gola withA. tumefaciensstrain UHFBA-218 and tumorigenicA. tumefaciensstrain Peach 2E-10, out of the 27 isolates recovered, six were transconjugants showing selective acquisition of tumorigenic factors as made evident by amplification withiptandvirD2primers, whereas the rest of the isolates did not acquire any of these tumorigenic factors. A white stone powder-based formulation of this isolate (103.3 × 108cfu g-1) retained appreciable viability for up to 6 months at room temperature. When peach roots and seeds were soaked in cell suspensions of different doses of a white stone powder-based bioformulation of UHFBA-218 before planting in the field, the number of plants with tumours was reduced, with the lowest incidence of crown gall being observed in the 0.1% UHFBA-218 root dip treatment, i.e. 1.48% and 0.80% during the years 2013 and 2014, respectively. No incidence of crown gall was observed in the three seed dip treatments, i.e. 30-min dip in UHFBA-218 followed by 1 h of shade drying, stratified seeds dipped for 30 min in 0.1% suspensions of strains UHFBA-218 or K84 followed by 1 h of shade drying before sowing, as compared with 14.76% incidence in untreated plants.
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Piya, Sujan, Huaxian Ma, Sujan Piya, Vivian Ruvolo, Mahesh Basyal, Aniela Golas, Renata Windak, Tomasz Rzymski, Michael Andreeff, and Gautam Borthakur. "Inhibition of Cyclin Dependent Kinase 8(CDK8): A Novel Approach to Target the Leukemia Initiating Cells (LICs) in T-Cell Acute Lymphoblastic Leukaemia (T-ALL)." Blood 138, Supplement 1 (November 5, 2021): 2250. http://dx.doi.org/10.1182/blood-2021-148853.
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Abstract Background: T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy characterized by an aberrant proliferation of immature thymocytes affecting preferentially children and adolescents. Approximately 70% of T-ALL cases have activating mutations of the NOTCH1 followed by oncogenic pathways PI3K/Akt, the anti-apoptotic BCL-2 family, and CDKN2A/2B cell cycle regulators 1, 2. Unlike other leukemias such as chronic myeloid leukemia (CML) and Philadelphia-positive ALL, which are kinase-driven malignancies 3, 4, the initiating events in T-ALL cause the ectopic expression of transcription factors that drive leukemogenesis, hence targeted therapies needed to focused on this field. Cyclin Dependent Kinase 8 (CDK8) and its paralog CDK19, restrain activation of super-enhancer-associated tumor suppressors and lineage commitment genes in leukemia. Moreover CDK8 phosphorylates intracellular domain (ICD) and regulates activation and turnover of NOTCH1 5, 6, indicating that targeting CDK8 with specific inhibitors may be considered as a novel therapeutic strategy for T-ALL. RVU120 is a first-in-class, specific and selective inhibitor of CDK8/CDK19, currently in phase Ib dose-escalation trial in patients with relapsed or refractory (R/R) AML and HR-MDS (NCT04021368). Objectives: The goals of these studies were to investigated the effectiveness of RVU120 and other non-related CDK8/19 inhibitors against T-ALL cell lines/PDXs, including GSI-resistant lines. Comprehensive analysis of target inhibition and apoptotic signatures were evaluated. The clinical relevance of RVU120 in LICs was confirmed in Patient derived xenografts (PDXs). Results: T-ALL cell lines and PDXs were treated with RVU120 and other chemically non-related CDK8/19 inhibitors in different concentration for 12-18 days, and the changes in cell number and apoptosis using flow cytometry analysis were determined. High differential efficacy in double-digit nM range has been observed for NOTCH1 PEST domain mutant cell lines including GSI-sensitive HPB-ALL cells and GSI-resistant cells PF-382, KOPT-K1, and MOLT4, where both inhibition of cell proliferation and induction of apoptosis were observed. GI-resistant cell lines SUP-T1, SUP-T11, MOLT16 and JURKAT were considered as moderately sensitive and LOUCY (early T-cell phenotype ALL), and CCRF-CEM devoid those phenotype, were considered as resistant to RVU120. Results form etsblished cell lines were further corroborated in patient-derived xenograft cells (PDX) treated with RVU120. In T-ALL PDX 6522288 (Notch1 HD /PEST mut, TET2 mut, U2AF1 mut, WT1 mut and CUL76- CDKN2A/B mut, PTEN-/-) treatment with RVU120 was followed by a reduction in cell number and induction of apoptosis in phenotypically defined leukemia initiating cells (LICs) CD34+CD7+CD19-, with calculated IC50s value for bulk and LICs as 92.8 nM and 141.8 nM, respectively. The clinical relevance of these findings and impact on LICs are under the evaluation in PDX mice model. Mechanistically, treatment with RVU120 reduced the phosphorylation of STAT5 and STAT1 as well as the level of MYC and anti-apoptotic proteins MCL1, but induced expression of BAX in sensitive cells. Hence, inhibition of CDK8 is a novel approach to target the LICs in T-ALL. Conclusion: Inhibition of CDK8 by RVU120 triggers inhibition of cell proliferation and apoptosis in cell lines and LICs in T-ALL. Further clinical study is warranted to study the efficacy of RVU120 and CDK8 inhibition in T-ALL. Keywords: CDK8, RVU120, apoptosis, T-ALL, LICs in PDX model Reference 1 Girardi, T., Vicente, C., Cools, J. & De Keersmaecker, K. Blood129, 1113-1123, doi:10.1182/blood-2016-10-706465 (2017). 2 Sanchez-Martin, M. & Ferrando, A. Blood 129, 1124-1133, doi:10.1182/blood-2016-09-692582 (2017). 3 Braun, T. P., Eide, C. A. & Druker, B. J. Cancer cell 37, 530-542, doi:10.1016/j.ccell.2020.03.006 (2020). 4 Gazeau, N. et al. Leukemia 34, 2230-2233, doi:10.1038/s41375-020-0715-2 (2020). 5 Pelish, H. E. et al. Nature526, 273-276, doi:10.1038/nature14904 (2015). 6 Fryer, C. J., White, J. B. & Jones, K. A. Molecular cell 16, 509-520, doi:10.1016/j.molcel.2004.10.014 (2004). Disclosures Golas: Ryvu Therapeutics: Current Employment. Windak: Ryvu Therapeutics: Current Employment. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Andreeff: ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Glycomimetics: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Amgen: Research Funding; Aptose: Consultancy; Medicxi: Consultancy; Oxford Biomedica UK: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy; Senti-Bio: Consultancy. Borthakur: GSK: Consultancy; Astex: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees.
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PANARETOU, Christina, and Sharon A. TOOZE. "Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1." Biochemical Journal 363, no. 2 (April 8, 2002): 289–95. http://dx.doi.org/10.1042/bj3630289.
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Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive Gi/G0 proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913–16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on Gi/G0 activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1—GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280–287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962–13170], whereas ARF1—GTP does regulate the production of PtdIns(4,5)P2. Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.
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Golan, Karin, Aya Ludin, Tomer Itkin, Shiri Cohen-Gur, Orit Kollet, Xin-Jiang Lu, Alexander Kalinkovich, and Tsvee Lapidot. "Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling." Blood 122, no. 21 (November 15, 2013): 217. http://dx.doi.org/10.1182/blood.v122.21.217.217.
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Abstract Hematopoietic stem and progenitor cells (HSPC) are mostly retained in a quiescent, non-motile mode in the bone marrow (BM), shifting to a cycling, differentiating and migratory state on demand. How HSC replenish the blood with new mature leukocytes on a daily basis while maintaining a constant pool of primitive cells in the BM throughout life is not clear. Recently, we reported that the bioactive lipid Sphingosine 1-Phosphate (S1P) regulates HSPC mobilization via ROS signaling and CXCL12 secretion (Golan et al, Blood 2012). We hypothesize that S1P influences the daily circadian egress of HSPC and their proliferation. We report that S1P levels in the blood are increased following initiation of light at the peak of HSPC egress and are reduced towards the termination of light when circulating HSPC reach a nadir. Interestingly, mice with constitutively low S1P plasma levels due to lack of one of the enzymes that generates S1P (Sphingosine kinase 1), do not exhibit fluctuations of HSPC levels in the blood between day and night. We report that HSPC numbers in the BM are also regulated in a circadian manner. Unexpectedly, we found two different daily peaks: one in the morning, following initiation of light, which is accompanied by increased HSPC egress and the other at night after darkness, which is associated with reduced HSPC egress. In both peaks HSPC begin to cycle and differentiate via up-regulation of reactive oxygen species (ROS) however, the night peak had lower ROS levels. Concomitant with the peak of primitive stem and progenitor cells, we also observed (to a larger extent in the night peak), expansion of a rare activated macrophage/monocyte αSMA/Mac-1 population. This population maintains HSPC in a primitive state via COX2/PGE2 signaling that reduces ROS levels and increases BM stromal CXCL12 surface expression (Ludin et al, Nat. Imm. 2012). We identified two different BM peaks in HSPC levels that are regulated by the nervous system via circadian changes in ROS levels. Augmented ROS levels induce HSPC proliferation, differentiation and motility, which take place in the morning peak; however, they need to be restored to normal levels in order to prevent BM HSPC exhaustion. In the night peak, HSPC proliferate with less differentiation and egress, and activated macrophage/monocyte αSMA/Mac-1 cells are increased to restore ROS levels and activate CXCL12/CXCR4 interactions to maintain a HSPC primitive phenotype. Additionally, S1P also regulates HSPC proliferation, thus mice with low S1P levels share reduced hematopoietic progenitor cells in the BM. Interestingly S1P is required more for the HSPC night peak since in mice with low S1P levels, HSPC peak normally during day time but not at darkness. We suggest that the first peak is initiated via elevation of ROS by norepinephrine that is augmented in the BM following light-driven cues from the brain (Mendez-Ferrer at al, Nature 2008). The morning elevated ROS signal induces a decrease in BM CXCL12 levels and up-regulated MMP-9 activity, leading to HSC proliferation, as well as their detachment from their BM microenvironment, resulting in enhanced egress. Importantly, ROS inhibition by N-acetyl cysteine (NAC) reduced the morning HSPC peak. Since norepinephrine is an inhibitor of TNFα, upon light termination norepinephrine levels decrease and TNFα levels are up-regulated. TNFα induces activation of S1P in the BM, leading to the darkness peak in HSPC levels. S1P was previously shown also to induce PGE2 signaling, essential for HSPC maintenance by the rare activated αSMA/Mac-1 population. Indeed, in mice with low S1P levels, we could not detect a peak in COX2 levels in these BM cells during darkness. We conclude that S1P not only induces HSPC proliferation via augmentation of ROS levels, but also activates PGE2/COX2 signaling in αSMA/Mac-1 population to restore ROS levels and prevent HSPC differentiation and egress during the night peak. We hypothesize that the morning HSPC peak, involves proliferation, differentiation and egress, to allow HSPC to replenish the blood circulation with new cells. In contrast, the second HSPC night peak induces proliferation with reduced differentiation and egress, allowing the renewal of the BM HSPC pool. In summary, we identified two daily circadian peaks in HSPC BM levels that are regulated via light/dark cues and concomitantly allow HSPC replenishment of the blood and immune system, as well as maintenance of the HSPC constant pool in the BM. Disclosures: No relevant conflicts of interest to declare.
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Rahman, Md Mustafizur, Md Mahmudul Kabir, Md Abdur Rahman, Amir Hossain, and Md Golam Hossain. "Abstract 2216: Impact of EXO1 (K589E) single nucleotide polymorphism on breast cancer risk in Bangladeshi women." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2216. http://dx.doi.org/10.1158/1538-7445.am2022-2216.
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Abstract Background and aim: Exonuclease 1 (EXO1) is an essential member of the RAD2 nuclease family which is involved in mismatch repair system and contributes to the maintenance of genomic stability, modulation of DNA recombination and cell cycle arrest mediation. Potential polymorphism in EXO1 may have the ability to control cancer risks by influencing the repair activity. K589E (rs1047840) is a potentially functional polymorphism in EXO1 gene and we hypothesized that it might be associated with risk of breast cancer in Bangladesh. Method: We designed a case-control study consisting of 184 subjects with breast cancer and 200 healthy controls to identify the effect of K589E polymorphism on breast cancer susceptibility and clinicopathological features in Bangladeshi women. Genotype determination was performed by PCR-RFLP method. Results: After the adjustment of age and weight, a significant association was found between EXO1 K589E alleles and genotypes and risk of breast cancer. The frequencies of the three genotypes G/G, G/A, and A/A were 49, 42, and 9% in cases whereas 69, 27.5, and 3.5% in controls, respectively. The heterozygote G/A genotype and combined G/A+A/A genotypes showed 2.12-fold (OR=2.12, 95% CI=1.3733 - 3.2822 p=0.0007) and 2.27-fold (OR=2.27, 95% CI=1.5001 - 3.4493, p=0.0001) increased risk of breast cancer, respectively when compared with homozygous G/G genotypes. The variant A allele also was associated with increased risk of breast cancer (OR= 3.46, 95% CI=1.3720 - 8.7571, p=0.0086). Analysis of clinicopathological characteristics and genotype distribution also showed a significant correlation. Conclusion: Our results revealed that EXO1 K589E polymorphism could be a potential predisposing risk factor in genetic susceptibility to breast cancer in Bangladesh. Citation Format: Md. Mustafizur Rahman, Md. Mahmudul Kabir, Md. Abdur Rahman, Amir Hossain, Md. Golam Hossain. Impact of EXO1 (K589E) single nucleotide polymorphism on breast cancer risk in Bangladeshi women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2216.
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Oh, Grace, Annie Wang, Lidong Wang, Jiufeng Li, Gregor Werba, Daniel Weissinger, Ende Zhao, et al. "Abstract B065: Polymerase theta inhibition activates the cGAS-STING signaling pathway and elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma." Cancer Research 82, no. 22_Supplement (November 15, 2022): B065. http://dx.doi.org/10.1158/1538-7445.panca22-b065.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination (HR) repair genes in up to 25% of cases. Defects in HR impart to tumor cells a specific vulnerability to poly-ADP ribose polymerase inhibitors and platinum-based chemotherapy. However, not all patients respond, and some who initially respond develop resistance and progress. Thus, new therapies that are effective against HR-deficient PDAC are needed. Here, we show that inactivation of the HR pathway in PDAC is associated with overexpression of polymerase theta (Polθ, or POLQ), a key enzyme that regulates the alternative non-homologous end-joining (alt-NHEJ) pathway of double-strand break (DSB) DNA repair. Using both human and murine HR-deficient PDAC models, we find that POLQ knockdown is synthetically lethal with mutations in HR genes (BRCA1, BRCA2, and PALB2) as well as the DNA damage repair gene ATM. Further, we present the first findings that POLQ knockdown enhances cytosolic micronucleus formation and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling. In an in vivo murine model, POLQ inhibition also leads to STING-dependent recruitment of immune cells and enhanced CD8+ T cell infiltration in the BRCA2-deficient tumor microenvironment (TME). Through its role in the alt-NHEJ pathway, we thus find that POLQ is critical for DSB repair in HR-deficient PDAC, and its inhibition presents a novel synthetic lethal approach to suppress tumor growth while simultaneously stimulating an immune response in the PDAC TME. Citation Format: Grace Oh, Annie Wang, Lidong Wang, Jiufeng Li, Gregor Werba, Daniel Weissinger, Ende Zhao, Surajit Dhara, Rosmel Hernandez, Amanda Ackermann, Despoina Kalfakakou, Emily A. Kawaler, Talia Golan, Theodore H. Welling, Agnel Sfeir, Diane M. Simeone. Polymerase theta inhibition activates the cGAS-STING signaling pathway and elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B065.
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Abraham, Michal, Galia Oberkovitz, Baruch Bulvik, Klein Shiri, Hanna Wald, Orly Eizenberg, Katia Beider, et al. "The CXCR4 Antagonist BL-8040 Induces a Robust Mobilization of CD34+CD38−CD45RA−CD90+ CD49f+ HSCs with Long-Term and Secondary Myeloid and Lymphoid Repopulating Activity." Blood 130, Suppl_1 (December 7, 2017): 660. http://dx.doi.org/10.1182/blood.v130.suppl_1.660.660.
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Abstract Introduction: Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. The bulk of HSCs are CD34+, however, most CD34+ cells are lineage-restricted progenitors and HSCs remain rare. HSCs can be enriched further based on CD45RA, Thy1 (CD90), and CD38 expression. Loss of CD90 expression was proposed to be sufficient to separate CD34+CD38−CD45RA− CD90+ HSCs from CD34+CD38−CD45RA− CD90- multipotent progenitors (MPPs). Recently, it was demonstrated that the expression CD49f is a specific HSC marker. Single CD49f(+) cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting MPPs. Results: CD34+ cells were purified from BL-8040 and G-CSF mobilized grafts and stained for CD38, CD45RA, CD90, and CD49f. The percentage of CD34+CD38- hematopoietic stem and progenitors was similar in both grafts (Figure 1A). However, whereas 23.2 % of BL-8040 mobilized CD34+ CD38- cells did not express CD45RA; only 1.6% of G-CSF mobilized CD34+ CD38- cells did not express CD45RA (Figure 1B). The percentages of CD34+CD38−CD45RA−CD49f+CD90+/-, CD34+CD38−CD45RA−CD49f+ CD90+, and CD34+CD38−CD45RA− CD90+ HPCS were increased significantly by 45, 25 and 12 -fold in the BL-8040 graft compared to G-CSF graft derived CD34+CD38- cells (Figure 1C). To assess the long-term engraftment potential of the BL-8040 mobilized CD34+ cells, engraftment was allowed for 4, 8, and 22 weeks after transplantation. Successful and robust long-term human engraftment of CD45+ and CD45+CD34+ cells was observed at week 22 (Figure 2A, 2B). The % of human CD45 cells remained stable in the BM whereas the percentage of CD45 cells in the blood and spleen increased at week 22 (Figure 2C). At 4 weeks, human CD3+CD4+ T cells were only observed at a low percentage in the spleen but not in the BM, whereas no significant percentage of CD3+CD8+ cells were found neither in the BM nor in the spleen (Figure 2D, 2E). 22 weeks after transplantation, the percentage of human CD3+CD4+ and CD3+CD8+ T cells was significantly increased in the spleen (30% vs. 5%, respectively) and to much lower levels in the BM (Figure 2D, 2E). Furthermore, successful and robust long-term human engraftment of secondary recipient was observed 14 weeks following the second transplantation (Figure 2A and 2B). Conclusion: In association with the high percentage of HPCs in the BL-8040-derived graft, we found a robust myeloid and lymphoid long-term engraftment (week 22) of BL-8040 mobilized human CD34+ cells in NSG mice. The ability of BL-8040 to collect high numbers of HPCs may be beneficial for a variety of HPCs dependent therapeutics. Disclosures Abraham: Biokine: Employment. Oberkovitz: BiolineRx: Employment. Eizenberg: Biokine: Employment. Vainstein: BiolineRx: Employment. Benami: BiolineRx: Employment. Golan: BiolineRx: Employment. Or: Bioline: Consultancy. Peled: Biokine: Consultancy; Biosight: Consultancy.
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Faughnan, Patrick, Golam Mohi, and Chandrajeet Singh. "Abstract 149: The roles of PIM1 and PIM2 kinases in the progression and metastasis of triple negative breast cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 149. http://dx.doi.org/10.1158/1538-7445.am2022-149.
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Abstract Breast cancers are often evaluated by their expression of estrogen, progesterone and HER2/Neu receptors. Triple negative breast cancers (TNBC), which do not have upregulation of any of these receptors, lack directed therapies and therefore, are associated with a worse prognosis. The lack of directed therapies for TNBC and high incidence of relapse and drug resistance found in all breast cancers underscores the need for identification of additional therapeutic targets. The analysis of gene expression data revealed that PIM1 and PIM2 kinases are upregulated in triple negative breast cancer. PIM kinases are a family of serine/threonine kinases that activate cell division, promote cell growth, and inhibit apoptosis. Due to PIM1/2’s effect on these cellular processes and PIM1/2 being upregulated in TNBC, we hypothesized that PIM1/2 play an important role in progression of triple negative breast cancer. Knockdown of PIM1 significantly inhibited the proliferation, migration, and invasion of TNBC cells, including MDA-231, BT-20, HCC-1806, and MDA-468 cells. To study the in vivo roles of PIM1/2 in breast cancer progression and metastasis, we crossed the highly metastatic MMTV-PyMT breast cancer mouse model with PIM1/2 knockout mice. Deletion of PIM1 or PIM2 alone significantly inhibited tumor growth and reduced lung metastasis in these mice. Dual deletion of PIM1 and PIM2 significantly reduced tumor growth and almost completely blocked lung metastasis in these mice. A major advantage of the MMTV-PyMT model over xenograft models is that the immune system is intact in these mice, and that enabled us to perform immune cell profiling on the tumors. Tumor associated macrophages (TAMs) have been shown to promote tumor metastasis and are a poor prognostic marker for TNBC. Deletion of PIM1 alone and combined PIM1 and PIM2 deletion resulted in a significant reduction in the number of TAMs, indicating PIM1/2 play a significant role in regulating the immune milieu of these tumors. To understand the underlying mechanism by which PIM1 and PIM2 contribute to these processes, we performed biochemical analysis. Western blot analysis of PIM1 knockdown in MDA-231 cells showed a decrease in phosphorylation of CXCR4, S6 ribosomal protein, and 4EBP1 and increased expression of cell cycle regulator p27. Additionally, we performed RNA-seq analysis to identify which pathways were affected by PIM1/2 deletion in MMTV-PyMT tumors. GSEA analysis showed a significant enrichment of genes related to inflammatory response, extracellular matrix, EMT and metastasis in PyMT tumors and those were significantly reduced in PIM1/2 double knockout tumors. These results suggest that PIM1 and PIM2 play a significant role in the progression and metastasis of triple negative breast cancer and indicate that PIM1/2 inhibition could be a useful therapeutic approach for TNBC. Citation Format: Patrick Faughnan, Golam Mohi, Chandrajeet Singh. The roles of PIM1 and PIM2 kinases in the progression and metastasis of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 149.
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Westin, Shannon Neville, Kathleen N. Moore, Els Van Nieuwenhuysen, Amit M. Oza, Linda R. Mileshkin, Aikou Okamoto, Akiko Suzuki, et al. "DUO-E/GOG-3041/ENGOT-EN10: a randomized phase III trial of first-line carboplatin (carb) and paclitaxel (pac) in combination with durvalumab (durva), followed by maintenance durva with or without olaparib (ola), in patients (pts) with newly diagnosed (nd) advanced or recurrent endometrial cancer (EC)." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): TPS6108. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.tps6108.
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TPS6108 Background: There is a high unmet need for advances in EC treatment that provide progression-free survival (PFS) and overall survival (OS) benefits. EC tumors are sensitive to carb/pac (Pectasides et al. Gynecol Oncol 2008). Maintenance therapy with the poly(ADP-ribose) polymerase inhibitor (PARPi) ola (with or without bevacizumab) led to significant PFS benefits in advanced ovarian cancer pts with either nd (SOLO1, Moore et al. NEJM 2018; PAOLA-1, Ray-Coquard et al. NEJM 2019) or recurrent (SOLO2, Pujade-Lauraine et al. Lancet Oncol 2017; Study 19, Friedlander et al. Br J Cancer 2018) platinum-sensitive disease, regardless of BRCA mutation status (PAOLA-1; Study 19), and in BRCA-mutated metastatic pancreatic cancer pts (POLO, Golan et al. NEJM 2019). Molecular features of EC could predict sensitivity to PARPi (de Jonge et al. Clin Cancer Res 2019; Auguste et al. Mod Pathol 2018). PARPi has been shown to prime the immune microenvironment in a preclinical BRCA1 mutant ovarian model (Higuchi et al. Cancer Immunol Res 2015). Clinical trials have demonstrated antitumor activity of the anti-programmed cell death ligand-1 (anti-PD-L1) blocker durva (Antill et al. J Clin Oncol 2019) and anti-programmed cell death-1 (anti-PD-1) antibody therapies (Makker et al. ESMO 2019; Oaknin et al. SGO 2019) in EC pts. The DUO-E trial (EUDRACT 2019-004112-60, D9311C00001, NCT04269200) will investigate whether the addition of durva to carb/pac, followed by durva (with or without ola) maintenance treatment, improves PFS in pts with nd advanced or recurrent EC. Methods: Eligible pts for this multicenter, double-blind, Phase III trial must have nd Stage III/IV or recurrent EC and be naïve to first-line chemotherapy. Pts will be randomized (1:1:1; n=~233 per arm) to: arm A) carb/pac + placebo (pbo) (q3w for six cycles) followed by pbo maintenance treatment; arm B) carb/pac + durva (1120 mg; q3w for six cycles) followed by maintenance treatment with durva (1500 mg q4w) + pbo (tablets bid); or arm C) carb/pac + durva (1120 mg; q3w for six cycles) followed by maintenance treatment with durva (1500 mg q4w) + ola (300 mg bid tablets). Pts received maintenance treatment until disease progression. Primary endpoint: investigator-assessed PFS (RECIST 1.1) of arm B vs. arm A. Key secondary endpoints: PFS of arm C vs. arm A; OS of arm B vs. arm A, and of arm C vs. arm A. Enrollment began in Q1 2020. Clinical trial information: 2019-004112-60.
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Golan, Karin, Aya Ludin, Tomer Itkin, Shiri Cohen-Gur, Orit Kollet, Xin-Jiang Lu, Alexander Kalinkovich, Maria Maryanovich, Atan Gross, and Tsvee Lapidot. "Hematopoietic Stem Cells and Their BM Stromal Microenvironment Share a Dynamic Inverse Metabolic State During Quiescence and Proliferation Via ROS Transfer Between The Two Populations." Blood 122, no. 21 (November 15, 2013): 587. http://dx.doi.org/10.1182/blood.v122.21.587.587.
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Abstract Hematopoietic stem and progenitor cells (HSPC) are mostly retained in the bone marrow (BM) in a ROSlow quiescent mode via adhesion interactions with stromal cells, while mature blood cells are generated on demand via ROShigh stem cell proliferation and differentiation. The metabolic state of BM stromal cells and their cross-talk with quiescent and cycling HSPC is poorly understood. The bioactive lipid Sphingosine 1-Phosphate (S1P) is an important inducer of ROS in HSPC, increasing their motility (Golan et al, Blood 2012). We report that by increasing S1P levels in the murine BM, we also increased HSPC levels via augmented ROS production and cell cycling. Unexpectedly, we discovered an opposite effect of S1P on BM stromal cells, which contained less mesenchymal stem and progenitor cells (MSPCs) and also lower ROS levels. To further test the opposite metabolic relationship between BM HSPC and stromal cells, we utilized 3 repeated (once every 24h) G-CSF stimulations, which induce HSPC proliferation without mobilization. As expected, we found increased HSPC levels in the BM accompanied by augmented ROS levels. Importantly, we found again the opposite effect between HSPC and BM stromal cells, i.e. reduced MSPC levels that were less cycling and contained lower levels of ROS. These results suggest an inverse metabolic state between BM HSPC and their stromal microenvironment. While HSPC utilize oxidative phosphorylation and ROS generation during proliferation and differentiation, their stromal microenvironment share a low metabolic state, most probably due to anaerobic glycolysis. Mimicking alarm situations by clinical G-CSF treatment blocks bone remodeling to allow HSPC development and mature blood replenishment. As expected, mice with reduced S1P levels due to lack of one of its biosynthetic enzymes (Sphingosine kinase 1), exhibit lower levels of BM HSPCs accompanied by reduced ROS levels. More importantly, these mice share an augmented population of undifferentiated, primitive ROSlow, quiescent non-motile HSCs identified as EPCR+/LSK cells, which results in higher levels of long-term HSC repopulation in transplanted wild type mice (with normal S1P levels). Concomitantly, S1P low mice have BM MSPCs with increased ROS levels and higher colony-forming unit fibroblasts (CFU-F) potential in vitro. We conclude that when HSPC are maintained quiescent due to a low energy metabolic state of anaerobic glycolysis, the BM stromal microenvironment utilizes a high energetic metabolism by oxidative phosphorylation and ROS generation. Our data suggests that the dynamic, inverse metabolic state between HSPC and the stromal microenvironment is induced by energy transfer between the two populations. HSPC can eliminate excess ROS post 5-FU treatment, by transferring ROS to BM stromal cells via connexin43 (Cx43) gap-junctions (Taniguchi et al, PNAS 2012). In agreement, we found that mice treated with a broad inhibitor of connexin gap-junctions (CBX), induced BM HSPC proliferation via ROS elevation, concomitant with down regulation of ROS in MSPCs. Interestingly, in mice with low S1P levels, CBX inhibition of connexins did not elevate HSPC ROS levels or their numbers, implying that cell contact blockage is essential, but not sufficient to induce proliferation. To induce HSPC proliferation and differentiation, an activator of ROS, such as S1P, is also needed to initiate cell cycle entry and blockage of Cx43, further inducing CXCL12 secretion to the circulation and HSPC migration. Our results imply that once ROS reaches a certain level in HSPC, it is scavenged by the stromal microenvironment to prevent exhaustion of the hematopoietic stem cell pool and in parallel to initiate new bone development and remodeling. Altogether, we have discovered a dynamic, opposite metabolic state between BM HSPCs and their supporting stromal microenvironment during quiescence, proliferation and differentiation of the two populations, which are both ROS regulated on a one population at a time basis. Thus, either blood cell production or bone development takes place at the expense of the other. This metabolic seesaw is regulated via ROS transfer from HSCs to their supportive microenvironment via Cx43 gap-junctions, which also regulates surface CXCL12 expression that is essential for stem cell quiescence. Disclosures: No relevant conflicts of interest to declare.