ALBERT

Description: Brain death is a permanent loss of all brain function [1]. Current clinical organ transplantations mostly depend on the organs from brain-dead patients [2]. And of note, a lot of blood deases are easy to cause cerebral haemorrhage, which is quite of danger and usually induce brain death if not detected and treated in time. Thus prompt evaluation of brain death is of great significance for saving medical resources and reducing economic burden of the patients' families. Current guide for diagnosing brain death required to perform a list of 〉30 hours neurological examninations, some of which are even invasive, not in time and easily hampered by many confounding factors. An ideal ancillary test to assess brain death is highlighted to be noninvasive, sensitive, universally available, timely, and easy to perform at the bedside. Near infrared spectroscopy ( NIRS ) is capable of monitoring hemodynamics in response to brain activity noninvasively, conveniently, continually, and relatively inexpensively, evidented by a series of clinical cerebral studies recently. Weigl et al newly reported to use a time resolved NIRS to detect the fluorescence photons excited in the indocyanine green ( ICG ) for cerebral perfusion detection. It provided a novel optical ancillary tool to assess brain death, while its accuracy was only 69.2%, which did not reach the level of brain death confirmation. Plus, it was invasive, requiring injection of optical contrast agent. We attempted to assess brain death completely in nonivasive way with just a custom wearable NIRS device developed in our lab [3] ( fig.1 a ). We novelly incororate a protocol at markedly but safely varied fractions of oxygen respiration. Firstly, Monte Carlo modeling were carried out to test the difference in photon transport within human brain at different oxygen concentrations induced by varied fractions of oxygen respiration ( FIO2 ) [4]. 18 healthy subjects ( 41 ± 11 years old ) and 17 brain dead patients were recruited from the intensive care unit (ICU) in Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital. No significant difference in age was found between patients and healthy groups ( p 〉0.413 ). These patients were finally clinically diagnosed by the international standards of brain death. Two protocols were used ( fig.1 b). One is consisted of 1 hour resting, 3-minute baseline measure, half-hour measurement at 60% FIO2 ( phase I, high oxygen ),a half hour measure at 40% FIO2 ( phase II, low oxygen ), and a half hour measure at 60% FIO2 ( phase III, high oxygen ). The other is low, high, and low. The Δ[Hb] and Δ[HbO2] time courses were recorded by NIRS in real time with related signal processing ( fig.1 c ). Statistical analysis were focus on the sensitivity and specificiy of our proposed methodology at combination of NIRS and above protocol, as well as which protocol act better. Fig.1 ( c right ) showed that the detected light signal profile dramatically differed among varied oxygen concentrations in human brain. Plus the hemodynamic responses varied clearly between two subject groups among varied FIO2 in both protocols ( fig1. d ). The ' II-III ' phase act more distinct in differing two groups than ' I-II ' phase. And the low-high-low protocol acted almost perfect in accessing brain death with highest sensitivity and specificity. Over all, the novel incorporation of NIRS and a low-high-low varied FIO2 protocol was shown to a be most sensitive, highly specific, noninvasive and real time way to assess brain death and promptly offer quality assured donor organs. [1] E. F. M. Wijdicks, P. N. Varelas, G. S. Gronseth, D. M. Greer, Evidence-based guideline update: Determining brain death in adults report of the quality standards subcommittee of the American Academy of Neurology, Neurology, vol. 74, no. 23, pp. 1911-1918, 2010 [2] K. Singbartl, R. Murugan, A. M. Kaynar, D. W. Crippen, S. A. Tisherman, K. Shutterly, S. A. Stuart, R. Simmons, Intensivist-led management of brain-dead donors is associated with an increase in organ recovery for transplantation, J. M. Darby, Am. J. Transplant., vol. 11, no. 7, pp. 1517-1521, 2011 [3] T. Li, M. Duan, Y. Zhao, G. Yu, Z. Ruan. Bedside monitoring of patients with shock using a portable spatially-resolved near-infrared spectroscopy. Biomed. Opt. Express, vol. 6, no. 9, pp. 3431-3436, 2015 [4] B. Pan, C. Huang, X. Fang, X. Huang, T. Li*, Noninvasive and Sensitive Optical Assessment of Brain Death, J. Biophotonics, vol. 12, no. 3, pp. e201800240, 2018 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3440 Background: How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryocyte Leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, plays critical roles in muscle, neuron, and megakaryocyte differentiation. Regulation of MKL1 subcellular localization is one mechanism by which a cell can control SRF activity with MKL1 localization to the nucleus being critical for its function as a transcriptional activator. MKL1 subcellular localization is cell-type specific; MKL1 is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types until it is sequestered in the nucleus following actin polymerization. In contrast, MKL1 is constitutively localized to the nucleus in neuronal cells. Objective: We tested the hypothesis that MKL1 subcellular localization is tightly regulated in megakaryocytic cells during induction of maturation. Methods and Results: Using a human erythroleukemia (HEL) cell line, we systematically dissected the events that occur after 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation to assess the relationships between RhoA activation, actin polymerization, MKL1 subcellular localization, and upregulation of SRF-target genes essential for megakaryocyte differentiation. In response to treatment with TPA, the percentage of HEL cells with predominantly nuclear localization went from

Description: Background: MicroRNAs (miRNAs) are small non coding RNAs that have been shown to play a regulatory role in a number of different settings including development, hematopoiesis and lineage-selection. The expression patterns of miRNAs in various cellular processes and in various normal and malignant tissues are an area of active exploration. Bioinformatic analyses of the genome suggest that there might be thousands of miRNAs encoded in the genome. However, thus far only about 600 unique miRNAs have been identified in humans. The role of microRNAs in B cell malignancies is poorly understood. Mature B cells comprise naive, germinal center, memory and plasma cells. These B cell stages comprise the majority of leukemias and lymphomas. We have previously demonstrated a role for microRNAs in the regulation of key transcription factors and oncogenes including PRDM1, LMO2 and MYBL1. We hypothesized that microRNAs play a role in the pathogenesis of lymphomas and have applied high throughput sequencing to understand the pattern and function of microRNA expression in normal B cells and their malignant counterparts. Methods: CD19+ mature B cells were obtained from normal patients undergoing tonsillectomy and sorted using flow cytometry into naive, germinal center, memory and plasma cells. We also obtained cells from tumor cell lines derived from mantle cell lymphoma (Mino, JVM2), Burkitt lymphoma (Ramos, BL41) and multiple myeloma (H929, U266), as well as patient tumor samples derived from Burkitt lymphoma and diffuse large B cell lymphomas. From these cells, we purified small RNAs (18-25 nucleotides) and ligated sequencing adapters to these small RNAs and subjected them to 15 cycles of PCR amplification. The constructs were then subjected to massively parallel high throughput sequencing (Illumina) in picoliter wells to identify millions of sequences per sample. Sequences thus identified were matched to the genome and microRNAs were identified based on their characteristic stem-loop secondary structure, thermodynamic stability, and evidence of processing with the microRNA-related enzymes drosha and dicer. Results: Using massively parallel high-throughput sequencing of small RNAs isolated from these B cell subsets, we analyzed a total of 62,293,147 sequences (〉 1.6 billion bases). We found that 261 known microRNAs are expressed in normal and malignant B cells, a number that is three times higher than previously recognized. Our work also identified the expression of 86 novel miRNAs in normal and malignant B cells, many of which appear to target genes important in B cell differentiation including BCL6, NLK, EBF, as well as oncogenes including MYC, LMO2, and CCND1. We found no evidence of decreased expression of microRNAs in B cell malignancies, in contrast to the described down-regulation of microRNAs in tumors from other lineages. On the other hand, there were striking differences between the microRNA expression patterns in normal and malignant B cells, although B cell malignancies still frequently express miRNAs that are characteristic of their normal B cell counterpart. Each malignancy had a characteristic pattern of microRNA expression that was distinct from other malignancies and normal B cells. Conclusion: Through high throughput sequencing, we have discovered novel microRNAs that target important oncogenes including BCL6, MYC, LMO2, and CCND1, suggesting a role for microRNAs in B cell lymphomas.

Description: Abstract 3652 Poster Board III-588 SRF is a MADS-box transcription factor first identified as an inducer of immediate-early gene expression, such as c-fos, Junb, Fosb and Egr1, in response to cytokines. It plays a critical role in cardiac and smooth muscle development and differentiation by regulating cell cycle, apoptosis, cell growth, and differentiation. SRF regulates expression of contractile and cytoskeletal genes. Its function in hematopoiesis has not yet been revealed. While no hematopoietic disease specific mutations of SRF have been identified, the region on chromosome 6 where the SRF gene is located, is a site of frequent chromosomal aberrations in MDS. MKL1, a target of the t(1;22) translocation in acute megakaryoblastic leukemia, is a cofactor for SRF mediated gene activation. In published work (Cheng EC et al., Blood 2009;113:2826), we showed that MKL1 expression increases with normal megakaryocytic (Mk) differentiation, that it promotes Mk differentiation by increasing the expression of Mk specific genes such as CD42b, and that mice lacking MKL1 have decreased platelet counts while showing increased numbers of low ploidy Mk in the BM. In addition, we showed that the effects of MKL1 on Mk differentiation require SRF. In order to test the role of SRF in Mk development, we crossed PF4-Cre mice (Tiedt R et al., Blood 2007;109:1503), which express Cre recombinase in cells committed to the Mk lineage, to SRFF/F mice (Miano JM et al., PNAS 2004;101:17132) in which functional SRF is no longer expressed after Cre-mediated excision. Our findings are quite surprising, as knockout of SRF in the Mk lineage leads to a far more profound phenotype than MKL1 KO. PF4-Cre x SRFF/F (PF4-Cre/SRF) mice are born with a normal mendelian frequency, but have significant macrothrombocytopenia. The platelet counts in WT and KO mice are 703 ± 33 × 103/μl vs 460 ± 23 × 103/μl, respectively. Despite the decreased platelet number, the BM has increased numbers and percentages of CD41+ Mk (WT: 0.41 ± 0.06% and KO: 1.92 ± 0.12%) with a predominance of Mk with hypolobated nuclei. Spleens of PF4-Cre/SRF mice show significantly increased numbers of Mk (mean of 4 and 15 Mk per high power field for WT and KO spleens, respectively). Ploidy as an assay of Mk maturation in the BM is significantly reduced in SRF −/− Mk. The percentage of CD41+ cells in SRF KO BM that is 2N is nearly 2-fold higher, and the percentage with greater than 8N ploidy is decreased by 70%. Hematopoietic stem and progenitor populations in PF4-Cre/SRF BM are unaffected, as expected due to stage and lineage specific knockout of SRF. In contrast, there is an increase in Mk progenitors in vivo in PF4-Cre/SRF mice, reflected both by FACS analysis and CFU-Mk in vitro analysis. The mechanism by which SRF disrupts Mk maturation was studied by assessment of Mk morphology, and gene expression analysis. Mk lacking SRF expression have abnormal stress fiber formation based on phalloidin staining, a phenotype parallel to that observed in mice lacking myosin heavy chain 9 (MYH9) expression, and MYH9 expression was decreased in SRF KO Mk. In summary, the data show that SRF is critical for normal Mk maturation including polyploidization and platelet production, and the mechanism by which loss of SRF disrupts megakaryocytopoiesis is, at least in part, by loss of normal expression of known targets of SRF, such as cytoskeletal genes including MYH9. Disclosures: No relevant conflicts of interest to declare.

Description: Although the maintenance of HSC quiescence and self-renewal are critical for controlling stem cell pool and transplantation efficiency, the mechanisms by which they are regulated remain largely unknown. Understanding the factors controlling these processes may have important therapeutic potential for BM failure and cancers. Here, we show that Skp2, a component of the Skp2 SCF complex, is an important regulator for HSC quiescence, frequency, and self-renewal capability. Skp2 deficiency displays a marked enhancement of HSC populations through promoting cell cycle entry independently of its role on apoptosis. Surprisingly, Skp2 deficiency in HSCs reduces quiescence and displays increased HSC cycling and proliferation. Importantly, loss of Skp2 not only increases HSC populations and long-term reconstitution ability but also rescues the defect in long-term reconstitution ability of HSCs on PTEN inactivation. Mechanistically, we show that Skp2 deficiency induces Cyclin D1 gene expression, which contributes to an increase in HSC cycling. Finally, we demonstrate that Skp2 deficiency enhances sensitivity of Lin− Sca-1+ c-kit+ cells and leukemia cells to chemotherapy agents. Our findings show that Skp2 is a novel regulator for HSC quiescence and self-renewal and that targeting Skp2 may have therapeutic implications for BM transplantation and leukemia stem cell treatment.

Description: A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Description: The role of CXCR3 in mediating the angiostatic activity of IFN-inducible ELR−CXC chemokines has been further defined with the finding that CXCR3 exists as two alternatively spliced variants, termed CXCR3A and CXCR3B respectively. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, but only binds to its specific receptor CXCR3B for the angiostatic activity. CXCR3B, a novel chemokine receptor, is 51 amino acid residues longer at the NH2-terminus than CXCR3A. Unlike activation of CXCR3A, which is pertussis toxin-sensitive and activates cellular proliferation, activation of CXCR3B is pertussis toxin-resistant and leads to inhibit cellular proliferation. Thus, the 51 amino acid residues at CXCR3B NH2-terminus are very important for the CXCL4/PF4 binding and for the CXCR3B inhibitory effect. In the present studies, a GST-tagged fusion protein of CXCR3B (a.a 1-51) was used as the immunogen for generation of monoclonal antibodies (mAbs) against CXCR3B (a.a. 1–51), and three peptides derived from CXCR3B NH2-terminus (a.a. 1–19, a.a. 17–35, and a.a. 33–51) for epitope mapping of the established mAbs. Nine hybrodoma cell lines secreting mAbs against CXCR3B were established, and we found that two mAbs reacted specifically with the CXCR3B a.a. 1–19, and one antibody with the CXCR3B a.a. 33–51. Three of these antibodies could also bind to CXCR3B on the surface of human intestinal epithelial Caco2 cells, and CXCR3B recognized by the antibodies were localized in the vascular endothelial cells of human fetal heart tissues. The data from Cell proliferation assay showed that three mAbs could inhibit the growth of U937 cells, which were found to express CXCR3B as well. We conclude that the novel panel of CXCR3B mAbs could be useful tools for the CXCR3B functional studies. Meanwhile, CXCR3B may be not only the inhibitory target on endothelial cells, but also the new directly reactive site of CXCR3B-positive tumor cells such as promonocytic U937 cells.

Description: Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and D (VEGF-D), are the major molecules involved in the development of the embryonic vascular system and pathological lymphangiogenesis. Throughout embryogenesis, VEGFR-3 is expressed in most endothelial cells, whilst being restricted to lymphatic vessels later in development. This receptor plays a significant role in the normal development of blood and lymphatic vessels. In the present studies, we generated a novel panel of 17 monoclonal antibodies (mAbs) against the human VEGFR-3 and characterized their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. In CAM angiogenesis experiments, the angiogenesis induced by recombinant GST-VEGF-D was decreased in the presence of antibody BDD073. These data indicate that this novel neutralizing antibody against human VEGFR-3 not only might be a potential compounds in blocking the VEGF-D-induced angiogenesis and lymphangiogenesis, but also be a tool for the investigations of biology of VEGFR-3 and analysis of lymphatic vessels in malignant tumors and their metastases.

Description: Key Points RhoA-induced actin polymerization promotes nuclear accumulation of MKL1 and transcriptional activation. Thrombopoietin activates nuclear accumulation of MKL1 and transcriptional activation in primary megakarocytes.

Description: Serum response factor (Srf) is a MADS–box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41+ megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.