ALBERT

Description: Highly-conserved LIN28 proteins regulate certain developmentally-timed events in multicellular organisms by decreasing the levels of let-7 miRNAs. It was recently reported that increased expression of LIN28 proteins or decreased expression of the target let-7 miRNAs in human erythroblasts cultured from healthy adult humans causes increased fetal hemoglobin expression. Here LIN28A expression in sickle cell donors’ cells was explored for its potential to regulate fetal and sickle hemoglobin, and to affect the morphological sickling of the mature erythrocytes. After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT-PCR (control: 8.6E+00 ± 8.1E+00 copies/ng, LIN28A-OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A-OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control: 2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024). Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected. For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects’ cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009). These results demonstrate that transgenic expression of LIN28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points LIN28B regulates HbF expression in erythroblasts that are cultured from umbilical cord and adult human blood. LIN28B expression manifested a more fetal-like phenotype among adult human erythroblasts.

Description: Abstract 827 The highly-conserved Lin28 genes regulate cellular metabolism as well as the timing of developmental events and cell fates in multicellular organisms. Lin28 protein acts primarily by negatively regulating biogenesis of let-7 RNA, a microRNA family whose targets include growth-related signaling and transcription factor proteins. Published studies showed significantly increased expression of let-7 in purified adult blood reticulocytes compared to umbilical cord blood reticulocytes (1). This pattern correlates inversely with Lin28B expression. While present in the fetal liver and umbilical cord blood, Lin28B decreased to undetectable levels in adult bone marrow (2). Based upon the association of human ontogeny with hemoglobin switching, Lin28 was explored to identify novel mechanisms for hemoglobin regulation that may be useful for therapeutic application among patients with thalassemia or other hemoglobinopathies. To study the effects of Lin28B upon erythropoiesis and hemoglobin, ectopic expression of Lin28B was accomplished using retroviral transduction of human CD34+ cells cultivated ex vivo in erythropoietin-supplemented, serum-free cultures for 21 days. All experiments were performed in triplicate using cells from three separate adult volunteers. Lin28B over-expression (Lin28B-OE) was confirmed by Q-RT-PCR (control: 0.14 ± 0.37 copies/ng, Lin28B-OE: 1.8E+04 ± 353.8 copies/ng, p=0.01). Western analyses confirmed protein expression, and confocal microscopy revealed Lin28B predominantly in the cytoplasm of the transduced cells. Proliferation, maturation and morphology assays revealed that Lin28B-OE did not inhibit erythropoiesis when compared to control (empty vector) transductions. Terminal maturation with loss of CD71 from the erythroblast surface and enucleation by culture day 21 was detected in the control and Lin28B-OE samples. Expression levels of globin genes were evaluated upon Lin28B-OE by Q-RT-PCR. Lin28B-OE enhances gamma-globin mRNA expression (control: 5.14E+06 ± 2.6E+06 copies/ng, Lin28B-OE: 1.81E+07 ± 5.82E+06 copies/ng, p=0.038). Protein analysis confirmed the increased expression of gamma-globin. Fetal hemoglobin (HbF) levels were also increased in the Lin28B-OE cultures (control: 5.82 ± 4.54%, Lin28B-OE: 33.63 ± 9.38%; p=0.011). The increased HbF expression was maintained throughout differentiation including enucleated populations of culture-generated erythrocytes. Possible mechanism(s) for the increased expression of HbF caused by Lin28B-OE were investigated. Q-RT-PCR analyses demonstrated suppression of the let-7 microRNA family with greater-than 70% reductions of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. Expression patterns of several transcription factors including BCL11A, KLF1, SOX6 and GATA1 were explored. No major changes were detected with the exception of BCL11A. Lin28B-OE caused a 65% reduction in BCL11A expression (control: 3.07E+03 ± 1.5E+02 copies/ng, Lin28B-OE: 1.07E+03 ± 18 copies/ng; p=0.02). Western blot analyses of Lin28B-OE showed a consistent reduction of BCL11A protein. By comparison with Lin28B-OE, separately performed studies of BCL11A knockdown in adult CD34+ cells produced comparable increases in gamma-globin expression, but Lin28B expression in those cells was not affected. In addition to a more general role in development and metabolism, these experimental results suggest that Lin28B increases fetal hemoglobin and regulates BCL11A in human erythroblasts. Lin28B is thus identified as the first defined link between the regulation of a developmental clock and hemoglobin switching in humans. Disclosures: No relevant conflicts of interest to declare.

Description: Epigenetic modification of chromatin in erythroid cells represents an active field of study aimed, in part, toward increased expression of fetal hemoglobin in patients with beta-thalassemia. The homologous methyltransferases G9a and GLP regulate globin gene transcription by catalyzing mono- and dimethylation at Lys 9 and dimethylation at Lys 27 of histone H3. Inhibition of these methyltransferases by the small molecule named UNC0638 was recently shown to increase gamma-globin gene expression in adult human hematopoietic precursor and stem cells. Here UNC0638 was explored further to include fetal hemoglobin expression among more mature erythroid cells cultured from CD34(+) cells of three healthy adult human donors in a serum-free culture medium. According to this culture model, the main erythroblast population on culture days 0-7 consists of CD36(+), CD45(+), CD71(moderate), CD235a(-) erythroid progenitor cell. On culture days 7-14, the progenitor cells differentiate in the presence of erythropoietin to become CD36(+), CD45(-), CD71(high), CD235a(+) precursor cells. During the final week in culture, the erythroblasts undergo nuclear condensation, enucleation, and loss of RNA combined with the loss of CD36 and CD71 on the plasma membrane to become mature erythrocytes. To investigate different stages of erythroblast maturation, the cells were cultured in medium containing 1µM UNC0638 for periods of seven days (culture days 0-7, 7-14, or 14-21) and compared to control cultures without UNC0638. The effects of UNC0638 were determined by flow cytometry, Q-RT-PCR and hemoglobin chromatography (HPLC). Unexpectedly, fetal hemoglobin expression was highly-dependent upon the differentiation stage of the cells in the presence of UNC0638. When cultured in UNC0638 supplemented medium on culture days 0-7 or 14-21, the cells underwent terminal maturation, but there was no significant increase in the fetal hemoglobin content of the mature cells (see abstract figure). In contrast, UNC0638 added on culture days 7-14, caused a significant increase in fetal hemoglobin (HbF; control: 3.9 ± 3.5% vs. day 7-14 UNC0638: 32.6 ± 0.95%, p=0.007). The increase in HbF was associated with a similar increase in gamma-globin mRNA (control: 1.5E+06 ± 1.7E+05 copies/ng vs. day 7-14 UNC0638: 7.5E+06 ± 1.4E+06 copies/ng, p=0.021). Additionally, terminal maturation and enucleation were partially inhibited when compared to the other conditions or controls. These data suggest that UNC0638 causes a robust increase in fetal hemoglobin as the cells undergo maturation. Fetal hemoglobin increases were more pronounced after exposure to UNC0638 during the erythropoietin-dependent transition from CD235a(-) to CD235a(+) erythroblasts. The results suggest that fetal hemoglobin regulation by G9a and GLP may be differentiation stage dependent. Disclosures: No relevant conflicts of interest to declare.

Description: In this paper, we propose an intrusion detection system based on the estimation of the Rényi entropy with multiple orders. The Rényi entropy is a generalized notion of entropy that includes the Shannon entropy and the min-entropy as special cases. In 2018, Kim proposed an efficient estimation method for the Rényi entropy with an arbitrary real order α . In this work, we utilize this method to construct a multiple order, Rényi entropy based intrusion detection system (IDS) for vehicular systems with various network connections. The proposed method estimates the Rényi entropies simultaneously with three distinct orders, two, three, and four, based on the controller area network (CAN)-IDs of consecutively generated frames. The collected frames are split into blocks with a fixed number of frames, and the entropies are evaluated based on these blocks. For a more accurate estimation against each type of attack, we also propose a retrospective sliding window method for decision of attacks based on the estimated entropies. For fair comparison, we utilized the CAN-ID attack data set generated by a research team from Korea University. Our results show that the proposed method can show the false negative and positive errors of less than 1% simultaneously.

Description: To investigate pharmacokinetic and pharmacodynamic differences of zolpidem between males and females and their causes, including CYP3A4 activity. A single oral dose of zolpidem (10 mg) was administered to 15 male and 15 female healthy subjects. Blood samples were collected up to 12 h post-dose to determine plasma zolpidem concentrations. Pharmacokinetic parameters were obtained using non-compartmental analysis. Digit symbol substitution test, choice reaction time, and visual analog scale of sleepiness were used to evaluate pharmacodynamics. We measured CYP3A4 activity using 4β-hydroxycholesterol, an endogenous metabolite. Mean maximum plasma concentration and area under the plasma concentration–time curve were higher for females than for males (9.9% and 32.5%, respectively); other pharmacokinetic parameters showed no significant differences. Pharmacodynamic scores for females showed delayed recovery compared with that for males. CYP3A4 activity was higher in females than in males (p = 0.030). There was no serious adverse event, and adverse event incidence was not different between the sexes. Zolpidem exposure was about 30% higher in females than in males. Delayed pharmacodynamic score recovery in females could be related to higher zolpidem concentrations. Although apparent clearance was lower in females, systemic clearance might not be the cause of the different exposures to zolpidem.