SCR Control Research Articles

A solving new method for the urea-selective catalytic reduction (SCR) system in a diesel engine using coupled hyperbolic-parabolic partial differential equations (PDEs)

To control the diesel engine urea SCR system with high accuracy, firstly, the partial differential equations of the SCR system are simplified through variable substitution and the method of characteristic lines to eliminate the partial derivative terms of the hyperbolic partial differential equations in the flow direction. The backward difference method is used to solve the problem, and the adaptive time step is adjusted to improve computational efficiency. Secondly, the Levenberg-Marquardt algorithm is applied to identify the model parameters per second based on the 1800-s test bench data. By combining the experimental data with the parameter identification results, this paper calculated the downstream NOx concentration with 99.5 % accuracy. Finally, the 1800s transient test data was applied to a commonly used single-state SCR control model, and cell numbers 1–4 of the cases were numerically simulated. It was found that the reduced-order model had a computation time of 1 s but was less accurate. When the test data was applied to the model presented in this study, the calculation time was 27s, and the model's calculation results show that the average error of the downstream NOx concentration is 16.95 ppm, which is 14.3 ppm lower than that of the two-cell one-state model.

Development of a Real-Time NOx Prediction Soft Sensor Algorithm for Power Plants Based on a Hybrid Boost Integration Model

Nitrogen oxides (NOxs) are some of the most important hazardous air pollutants from industry. In China, the annual NOx emission in the waste gas of industrial sources is about 8.957 million tons, while power plants remain the largest anthropogenic source of NOx emissions, and the precise control of NOx in power plants is crucial. However, due to inherent issues with measurement and pipelines in coal-fired power plants, there is typically a delay of about three minutes in NOx measurements, bringing mismatch between its control and measurement. Measuring delays in NOx from power plants can lead to excessive ammonia injection or failure to meet environmental standards for NOx emissions. To address the issue of NOx measurement delays, this study introduced a hybrid boosting model suitable for on-site implementation. The model could serve as a feedforward signal in SCR control, compensating for NOx measurement delays and enabling precise ammonia injection for accurate denitrification in power plants. The model combines generation mechanism and data-driven approaches, enhancing its prediction accuracy through the categorization of time-series data into linear, nonlinear, and exogenous regression components. In this study, a time-based method was proposed for analyzing the correlations between variables in denitration systems and NOx concentrations. This study also introduced a new evaluation indicator, part of R2 (PR2), which focused on the prediction effect at turning points. Finally, the proposed model was applied to actual data from a 330 MW power plant, showing excellent predictive accuracy, particularly for one-minute forecasts. For 3 min prediction, compared to predictions made by ARIMA, the R-squared (R2) and PR2 were increased by 3.6% and 30.6%, respectively, and the mean absolute error (MAE) and mean absolute percentage error (MAPE) were decreased by 9.4% and 9.1%, respectively. These results confirmed the accuracy and applicability of the integrated model for on-site implementation as a 3 min advanced prediction soft sensor in power plants.

Knockdown of Placental Major Facilitator Superfamily Domain Containing 2a in Pregnant Mice Reduces Fetal Brain Growth and Phospholipid Docosahexaenoic Acid Content.

Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid critical for fetal brain development that is transported to the fetus from the mother by the placenta. The lysophosphatidylcholine (LPC) transporter, Major Facilitator Superfamily Domain Containing 2a (MFSD2a), is localized in the basal plasma membrane of the syncytiotrophoblast of the human placenta, and MFSD2a expression correlates with umbilical cord blood LPC-DHA levels in human pregnancy. We hypothesized that placenta-specific knockdown of MFSD2a in pregnant mice reduces phospholipid DHA accumulation in the fetal brain. Mouse blastocysts (E3.5) were transduced with an EGFP-expressing lentivirus containing either an shRNA targeting MFSD2a or a non-coding sequence (SCR), then transferred to pseudopregnant females. At E18.5, fetuses were weighed and their placenta, brain, liver and plasma were collected. MFSD2a mRNA expression was determined by qPCR in the brain, liver and placenta and phospholipid DHA was quantified by LC-MS/MS. MFSD2a-targeting shRNA reduced placental mRNA MFSD2a expression by 38% at E18.5 (n = 45, p < 0.008) compared with SCR controls. MFSD2a expression in the fetal brain and liver were unchanged. Fetal brain weight was reduced by 13% (p = 0.006). Body weight, placenta and liver weights were unaffected. Fetal brain phosphatidyl choline and phosphatidyl ethanolamine DHA content was lower in fetuses with placenta-specific MFSD2a knockdown. Placenta-specific reduction in expression of the LPC-DHA transporter MFSD2a resulted in reduced fetal brain weight and lower phospholipid DHA content in the fetal brain. These data provide mechanistic evidence that placental MFSD2a mediates maternal-fetal transfer of LPC-DHA, which is critical for brain growth.

Model-based NH3 Adaptation Method for SCR Control

Model-based NH3 Adaptation Method for SCR Control

MicroRNA29B induces fetal hemoglobin via inhibition of the HBG repressor protein MYB in vitro and in humanized sickle cell mice.

Therapeutic strategies aimed at reactivating HBG gene transcription and fetal hemoglobin (HbF) synthesis remain the most effective strategy to ameliorate the clinical symptoms of sickle cell disease (SCD). We previously identified microRNA29B (MIR29B) as a novel HbF inducer via targeting enzymes involved in DNA methylation. We provided further evidence that the introduction of MIR29B into KU812 leukemia cells significantly reduced MYB protein expression. Therefore, the aim of this study was to determine the extent to which MIR29B mediates HbF induction via targeting MYB in KU812 leukemia cells and human primary erythroid progenitors and to investigate the role of MIR29B in HbF induction in vivo in the humanized Townes SCD mouse model. Human KU812 were cultured and normal CD34 cells (n = 3) were differentiated using a two-phase erythropoiesis culturing system and transfected with MIR29B (50 and 100 nM) mimic or Scrambled (Scr) control in vitro. A luciferase reporter plasmid overexpressing MYB was transfected into KU812 cells. Luciferase activity was quantified after 48 h. Gene expression was determined by quantitative real-time PCR. In vivo studies were conducted using Townes SCD mice (6 per group) treated with MIR29B (2, 3, and 4 mg/kg/day) or Scr control by 28-day continuous infusion using subcutaneous mini osmotic pumps. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes at weeks 0, 2, and 4. Flow cytometry was used to measure the percentage of HbF-positive cells. In silico analysis predicted complementary base-pairing between MIR29B and the 3'-untranslated region (UTR) of MYB. Overexpression of MIR29B significantly reduced MYB mRNA and protein expression in KU812 cells and erythroid progenitors. Using a luciferase reporter vector that contained the full-length MYB 3'-UTR, we observed a significant reduction in luciferase activity among KU812 cells that co-expressed MIR29B and the full-length MYB 3'-UTR as compared to cells that only expressed MYB 3'-UTR. We confirmed the inhibitory effect of a plasmid engineered to overexpress MYB on HBG activation and HbF induction in both KU812 cells and human primary erythroid progenitors. Co-expression of MIR29B and MYB in both cell types further demonstrated the inhibitory effect of MIR29B on MYB expression, resulting in HBG reactivation by real-time PCR, Western blot, and flow cytometry analysis. Finally, we confirmed the ability of MIR29B to reduce sickling and induce HbF by decreasing expression of MYB and DNMT3 gene expression in the humanized Townes sickle cell mouse model. Our findings support the ability of MIR29B to induce HbF in vivo in Townes sickle cell mice. This is the first study to provide evidence of the ability of MIR29B to modulate HBG transcription by MYB gene silencing in vivo. Our research highlights a novel MIR-based epigenetic approach to induce HbF supporting the discovery of new drugs to expand treatment options for SCD.

MiRNA29B Induces Fetal Hemoglobin through Targeting DNMT3 and MYB in Vitro and In Vivo in Preclinical Townes Sickle Cell Mice

Introduction: Therapies aimed at inducing fetal hemoglobin (HbF) expression is an effective approach for ameliorating the clinical severity of sickle cell disease (SCD) in adults and children, however drug treatment options are limited. DNA methyltransferase (DNMT) inhibitors tested in clinical trials induce HbF expression by producing γ-globin gene promoter DNA hypomethylation. Alternative approaches to increase HbF involve development of small non-coding microRNAs (miR) to silence major repressors of γ-globin gene transcription. Studies from our group demonstrated that miR29b inhibits DNA methylation through binding the 3' UTR of DNMT3 genes and is associated with high HbF levels in SCD patients. Studies performed in KU812 cells showed concomitant DNMT3 silencing along with ү-globin gene activation and HbF induction. Moreover, miR29b decreased expression of the γ-globin repressor protein MYB in normal adult CD34+ stem cells. Therefore, we tested the hypothesis that miR29b activates γ-globin and HbF expression via DNA hypomethylation and MYB gene silencing in normal erythroid progenitors and in Townes SCD mice. Methods: Human KU812 were cultured and normal CD34+ cells (n=3) were differentiated using a two-phase erythropoiesis culturing system. KU812 cells were co-transfected with the MYB luciferase reporter plasmid and the effects of miR29b (50 nM and 100 nM) overexpression or negative Scrambled (Scr) mimic on luciferase activity was quantified after 48 hours. The effect of MYB knockdown on ү-globin activation and HbF induction in KU812 cells using a retroviral vector encoding a MYB shRNA was also assessed. Subsequent studies were conducted in CD34 cells electroporated with miR29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 hours. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the percentage of HbF positive cells (F-cells). Real-time PCR quantified expression of miR29b, γ-globin, and β-globin gene expression, and Western blot measured DNMT, HbF and HbA protein. In vivo studies were performed in Townes SCD mice (6 per group), treated by 28-day continuous infusion using subcutaneous mini-osmotic pumps. The treatment groups consisted of miR29b (2, 3, and 4 mg/kg/day) and corresponding doses of Scr control. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes percent by flow cytometry. Gene expression was determined by real-time PCR. Results: Co-transfection of miR29b and a luciferase reporter plasmid containing the MYB 3'-UTR into KU812 cells resulted in a >90% decrease in luciferase activity at both miR29b concentrations. MYB knockdown by shRNA significantly increased the ү/ү+β ratio and miR29b expression in KU812 cells and normal CD34 cells. We observed efficient erythroid differentiation at day 7, day 12, and day 14 of cell culture assessed by Giemsa stain. Treatment with miR29b increased the ү/ү+β mRNA ratio by 2.5-fold (p<0.01) and F-cell levels increased from ~6.0% (Scr) to 15% with miR29b (100 nM); we observed lower DNMT3 mRNA and protein levels in cells transfected with 100 nM miR29b. Analysis of HbF induction by miR29b revealed >85% increase in F-cells by flow cytometry in normal erythroid progenitors as compared to Scr cells. Subsequent in vivo studies were conducted with Townes SCD mice treated with miR29b for 28 days; treatment was well tolerated with good weight gain and no deaths. There was minimal effects on hematopoiesis with a decrease in total white blood count and increase in neutrophils. Hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a dose-dependent 2.6-fold, 2.8-fold, and 3.0-fold (p<0.05) increase in F-cells at the respective miR29b doses compared to Scr control. We also observed a significant decrease in the percent of sickle cells on peripheral smear (p<0.05) mediated by miR29b with 20% inhibition (p<0.05) at week 4. MiR29b significantly decreased DNMT3 mRNA expression at week 2 and MYB mRNA at week 4 with a concomitant increase in the ү/ү+β mRNA ratio at weeks 2 and 4. Conclusions: Our findings support the ability of miR-29b to induce HbF in sickle erythroid progenitors without significant toxicity in vitro and SCD mice in vivo. This research highlights a novel miRNA-based epigenetic approach to induce HbF supporting discovery of new drugs to expand treatment options for SCD.

Investigating performance of compound heating furnace subject to heating by two elements and (Silicon Controlled Rectifier) SCR control at 1600ᴼC

The present research examines the performance of a resistance heating furnace using two different heating elements, Silicon Carbide (SiC) heating rods and Molybdenum Di-Silicide (MoSi2) to raise working chamber temperature to 1600ᴼC. SiC rods are used first, starting from the beginning (ambience) temperature 35ᴼC up to 1300ᴼC, followed by MoSi2 heating elements to raise chamber temperature from 1300ᴼC to set temperature at 1600ᴼC. Transition from SiC to MoSi2, heating system is uninterrupted, and swift in heating element effected by inter-locking system (an electronic device or an electro-magnetic system) without any drop in effect. The system under analysis consists of Programmable (Proportional-Integral-Derivative) PID, (Silicon Controlled Rectifier) Thyristor power pack, recrystallised alumina tubes, sensing elements: thermo-couple, Pt-Pt/13%.Rh, semiconductor based circuit that controls power and current to the system requirement (step down) and thereby control voltage automatically with transformer (depending on size of working area, and 53 amp (I), 220 V for single phase, reduced to 60V by a step down transformer) and auto current limiting facilities. Present analysis is designed for programmable and also for non-programmable type of cycles of operations set before starting the furnace within maximum working temperature of 1600ᴼC to achieve objectives, like saving of amperage (current consumption 53 amp) and power at reduced voltage (40V), long life of the heating elements (2 years and more) and optimization of thermal efficiency (60%) for high working temperature 1600ᴼC for long hours of operation in a Compound Heating Resistance (CHR) furnace.

Markers of kidney tubular and interstitial injury and function among sugarcane workers with cross-harvest serum creatinine elevation

ObjectivesSerum creatinine (SCr) is a routine marker of kidney injury but also increases with dehydration and muscular work. This study was to elucidate whether increase in SCr is associated with...

Improving the NOx reduction performance of an Euro VI d SCR System in real-world condition via nonlinear model predictive control

This paper deals with the possibility of improving the urea dosage control for the Selective Catalytic Reduction Systems (SCR) of an Euro VI d diesel light commercial vehicle in order to increase [Formula: see text] after-treatment reduction performance. To this aim, first, we assess the effective emissions abatement performance for the appraised diesel vehicle via real-world experimental campaign, carried out according to the Real Driving Emissions (RDE) tests on urban, extra-urban and motorway road sections in Naples, Italy. Based on these real-world data, we derive a parameterized control-oriented model for the SCR system which is, then, exploited for the designing of an alternative urea injection logic which could be able to maximize the [Formula: see text] reduction efficiency while minimizing tailpipe ammonia slip. Specifically, the optimal AdBlue injection rate is designed according to a Nonlinear Model Predictive Control Approach which allows obtaining a proper trade-off between the [Formula: see text] abatement and the urea overdosing problem. The effectiveness of the proposed controller is evaluated by comparing the performance assessed for the appraised SCR system during the experimental tests with the ones achievable if the Euro VI diesel would be equipped with the proposed control strategy. Numerical simulation discloses the effectiveness of the NMPC controller in ensuring improved [Formula: see text] reduction with performance complying with the emissions norms, main in avoiding excessive ammonia slip and in guaranteeing a reduced feed ratio w.r.t. to the standard industrial SCR controller mounted on the vehicle.

Investigation of miRNA-29b As a Fetal Hemoglobin Inducer in Sickle Cell Disease

Introduction: Therapeutic intervention aimed at inducing fetal hemoglobin (HbF) expression is an effective approach for ameliorating the clinical severity of sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved drug with proven efficacy for inducing HbF in SCD. Recently, DNA methyltransferase (DNMT) inhibitors were shown to reactivate γ-globin gene expression via DNA hypomethylation. However, alternate approaches involve development of small non-coding microRNAs (miR) to silence major repressors of γ-globin transcription. Previous studies from our group showed that miR-29b inhibits DNA methylation by binding the 3' untranslated region of DNMT3A/B (Starlard-Davenport A et al., J Carcinog 12:15; 2013). Subsequent, studies demonstrated that increased levels of miR-29b are associated with high HbF levels in patients with SCD. To gain insights into mechanisms, studies performed in KU812 cells demonstrated DNMT3A/B silencing with ү-globin gene activation. Moreover, miR29b increased HbF expression in erythroid progenitors generated from normal adult CD34 + stem cells (Starlard-Davenport A et al., Br J Haematol 186:91-100; 2019). Therefore, we tested the hypothesis that miR-29b activates γ-globin transcription via DNA hypomethylation in normal and sickle erythroid progenitors and Townes SCD mouse model.Methods: Normal CD34 + cells (n=3) and peripheral blood mononuclear cells (n=2) isolated from SCD patients, were cultured using a two-phase erythroid culturing system. Cells were electroporated with miR-29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 h. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the % HbF positive cells (F-cells). Real-time PCR was used to quantify expression of miR-29b, γ-globin, and β-globin genes, and Western blot was performed to measure DNMT, HbF and HbS protein levels. In vivo studies were performed in Townes SCD mice (3 per group) treated for 28 days by continuous infusion with subcutaneous mini-osmotic pumps. The treatment groups included miR-29b (2mg/kg/day and 3mg/kg/day) and corresponding doses of Scr control mimics.Results: We observed erythroid differentiation of untreated normal and SCD erythroid progenitors at day 7, 12, and 14 of culture assessed by Giemsa stain. Treatment with miR-29b increased the ү/ү+β mRNA ratio by 2.5‐fold (p<0.01) and F‐cell levels increased from ~6.0% (Scr) to 15% with miR-29b (100nM); we observed lower DNMT3 mRNA and protein levels after 100nM miR-29b treatment. To confirm HbF induction by miR-29b in sickle progenitors we treated cells on day 8 with 50nM and 100nM miR-29b and observed >85% increase in %F-cells compared to Scr cells. Subsequent miR-29b treatment was conducted for 28 days in Townes SCD mice, which was well tolerated documented by good weight gain and no deaths. There were minimal effects on hematopoiesis with a decrease in total white blood count and an increase in neutrophils. Total hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a 0.5 fold and 2.3-fold (p<0.005) increase in %F-cells at the 2mg/kg and 3 mg/kg miR-29b doses respectively compared to corresponding Scr controls. By week 4, miR29b increased HbF levels 2.1-fold by Western blot, and sickle cell levels under hypoxic conditions decreased 32% (p<0.01).Conclusions: Our findings support the ability of miR-29b to induce HbF in normal and sickle erythroid progenitors without significant toxicity in vitro and in SCD mice in vivo. This research highlights a novel miRNA-based epigenetic approach to induce HbF supporting discovery of new drugs to expand treatment options for SCD. DisclosuresPace: Imara Inc.: Consultancy.

The Involvement of STAT/SOCS Signaling in miRNA-Induced T Cell Exhaustion in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS) arises from CD4+ T cells in a background of chronic inflammation. The chronic inflammation fosters the growth of CTCL cells and may facilitate T-cells exhaustion that is characterized by deprived effector function and sustained expression of inhibitory receptors. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. At present, the roles of miRNAs or signaling pathways involved in the expression of immune checkpoints in CTCL has yet to be elucidated. Therefore, this project is aimed to elucidate how immune checkpoints are regulated by miRNAs, and how this regulation contributes to T-cell exhaustion and the development of CTCL. Methods: We first conducted miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. The sequencing data analysis was performed at the City of Hope Integrative Genomics Core. Next, we verified the expression of 3 highly upregulated miRNAs (miR-155, -21 and -130) from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Myla 2059 and HuT 78 with anti-miR-155, -21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against SOCS, PTEN, pSTAT3 and GAPDH as a loading control. Results: The data analysis revealed that miR-155 had the highest correlation with CTLA-4 (r = 0.59, P &amp;lt; 0.0001), PD1 (r = 0.42, P = 0.0021), PD-L1 (r = 0.42, P = 0.0025), TIM3 (r = 0.63, P &amp;lt; 0.0001), LAG3 (r = 0.57, P &amp;lt; 0.0001), and ICOS (r = 0.74, P &amp;lt; 0.0001) mRNA; -21 had the highest correlation with PD-L1 (r = 0.44, P = 0.0012), TIM3 (r = 0.52, P &amp;lt; 0.0001), and ICOS (r = 0.37, P &amp;lt; 0.0073) mRNA; and -130 had the highest correlation with CTLA-4 (r = 0.5, P = 0.0002), PD-L1 (r = 0.54, P &amp;lt; 0.0001), TIM3 (r = 0.69, P &amp;lt; 0.0001), LAG3 (r = 0.49, P = 0.0003), and ICOS (r = 0.68, P &amp;lt; 0.0001) mRNA. qRT-PCR and ISH revealed that miRs-155, -21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a dramatic increase in SOCS proteins and significant decrease in pSTAT3 expression in Myla 2059 and HuT 78 cells transfected with anti-miRs-155, -21 or -130, compared to cells transfected with Scr. Conclusions: Immune checkpoints and ligands like PD-L1 expression in CTCL are regulated by miRs-155, -21 and -130. The SOCS family of proteins, negative regulators of STAT signaling, are involved in miRs-155, -21 and -130-induced immune checkpoints expression in CTCL. Taken together, these results demonstrate the mechanisms of miRNA-induced T cell exhaustion and pave the way for the development of miRNA therapeutics in CTCL. Disclosures Rosen: Seattle Genetics: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy. Querfeld:Celgene: Research Funding; Stemline: Consultancy; Trillium: Consultancy; MiRagen: Consultancy; Kyowa Kirin: Consultancy; Bioniz: Consultancy; Helsinn: Consultancy.

NOx sensor cross sensitivity model and simultaneous prediction of NOx and NH3 slip from automotive catalytic converters under real driving conditions

This work presents the development of a model to capture the NOx sensors cross sensitivity behavior based on [Formula: see text] sensor cell temperature, as well as a model do predict the slip of the NOx and NH3 after the SCR catalyst, as a way to reduce the error in the exhaust emissions estimation needed for feedback SCR control strategies. The emissions prediction model is based on the different cross sensitivity behavior of two distinct NOx sensors. The proposed models were tested and compared on a fully instrumented engine test bench when applied in a Worldwide harmonized Light vehicles Test Cycle (WLTC) and a full map cycle. As a result, the proposed model showed for NOx sensors cross sensitivity estimation an overall improvement of 34.8% for sensor 1 and 31.0% for sensor 2, and in terms of emissions prediction an overall improvement of 36.3% for NOx and 45.5% for NH3 slip.

Development of an EKF Observer for an automotive SCR system

Abstract In the recent years, engine developer attention is focused on the application of advanced control strategies for the SCR system, considered as the leading technology for the abatement of NOX emissions in Diesel engines. The most challenging task in SCR control is the optimization of the urea dosing strategy while reaching the balance between high NOX reduction efficiency and low NH3 slip in the tailpipe, especially during transients. In order to accomplish this task, an Extended Kalman Filter (EKF) estimator is developed, to be integrated in the closed loop control of SCR urea dosing. The EKF estimator is applied to a one-state model, in which the ammonia coverage ratio θ is the only dynamic process taken into account. To improve the model accuracy, an additional dynamics, related to total ammonia adsorption capacity Ω, is considered while conceiving the EKF. The recursive EKF algorithm allows achieving enhanced estimation accuracy with a slight increase of the computational burden that is still suitable with on-board application.

Effects of Vitamin D Supplementation on Renal Function, Inflammation and Glycemic Control in Patients with Diabetic Nephropathy: a Systematic Review and Meta-Analysis

Background/Aims: Vitamin D (VD) is widely recognized as renal protective. However, whether VD supplementation provides benefit to patients with diabetic nephropathy (DN) remains controversial. Here, we performed a meta-analysis to systematically evaluate the impact of VD supplementation on indexes of renal function, inflammation and glycemic control in DN patients, and to explore the potential renal protective mechanism of VD. Methods: We searched Pubmed, Embase, Cochrane Library, and three major Chinese biomedical databases (CNKI, WANGFANG and VIP) for randomized controlled trials (RCTs) examining the effects of VD or its analogs in DN patients, published between September 2007 and July 2018. Quality assessment and data extraction were performed independently by two authors, according to the Cochrane systematic review methods. Meta-analysis based on the extracted results were performed via Revman 5.2 software. Results: We included 20 RCTs representing 1,464 patients with DN in this meta-analysis. VD supplementation significantly reduced 24-hour urine protein [MD = -0.26; 95% CI (-0.34, -0.17); P < 0.00001; I² = 95%], UAER [MD = -67.36; 95% CI (-91.96, -42.76); P < 0.00001; I² = 97%], hs-CRP [MD = -0.69; 95% CI (-0.86,-0.53); P < 0.00001; I² = 0%], TNF-α [MD = -56.79; 95% CI (-77.05, -36.52); P < 0.00001; I² = 89%] and IL-6 [MD = -0.73; 95% CI(-1.03, -0.44); P < 0.00001; I² = 0%]. However, VD supplementation failed to decrease SCr [MD = -0.83; 95% CI (-3.67,2.02); P = 0.57; I² = 0%] or increase eGFR [MD = 2.13; 95% CI (-2.06, 6.32); P = 0.32; I² = 0%]. In addition, VD supplementation showed no impact on indexes of glycemic control, such as HbA1c [MD = 0.01; 95% CI (-0.09, 0.11); P = 0.84; I² = 0%] and FBG [MD = -0.05; 95% CI (-0.29, 0.20); P = 0.70; I² = 0%]. Analysis of 24-hour urine protein, SCr, eGFR, hs-CRP or HbA1c revealed no difference between subgroups based on the type of VD supplementation, including calcitriol, alfacalcidol and vitamin D3, and the dose or duration of calcitriol usage. Conclusion: In patients with DN, VD supplementation provides beneficial effects on 24-hour urine protein and inflammation indexes, but not on SCr, eGFR or glycemic control indexes. More RCTs that comprehensively evaluate the impact of VD supplementation on indexes of renal function, inflammation and glycemic control in DN atients are required in order to reach conclusive results.

PD-L1 Expression Is Regulated By microRNAs-21 and -130 in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS), is characterized by the accumulation of neoplastic memory T- cells in the skin in a background of chronic inflammation. This observation suggests that chronic inflammation fosters the growth of CTCL cells, and may impair successful anti-tumor responses mediated by the CD8+ tumor-infiltrating T-cells. T-cell exhaustion plays an important role in the pathogenesis of CTCL. PD-1 and other inhibitory immune checkpoints are overexpressed in exhausted T-cells (Cancer Immunol Res 6; 2018). Their binding to corresponding ligands like PD-L1 on dendritic cells (DCs), macrophages and tumor cells in the microenvironment directly reduces the functional and proliferative capabilities of T-cells by repressing T-cell receptor signaling and inducing genes that impair T-cell function. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. The miRNA profile of CTCL correlates with a dysfunctional/exhausted immunophenotype, suggesting that epigenetic regulation is involved in the regulation of immune checkpoints. Signaling pathways such as JAK-STAT are constitutively active in CTCL and may also be involved in immune checkpoint regulation. At present, the exact role of miRNAs in regulating the expression of PD-L1 in CTCL has yet to be elucidated. Therefore, the goal of this project is to understand how PD-L1 is regulated, how this regulation contributes to T-cell exhaustion, and how this regulation is altered to contribute to the development of CTCL. To this end, we set out to test the hypothesis that PD-L1 is regulated by oncogenic miRNAs in CTCL.Methods: We first conducted a high-throughput miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. Library preparation, miRNA sequencing, and correlation calculation of miRNAs with PD-L1 mRNA expression was performed by the City of Hope Integrative Genomics Core. Next, we verified the expression of 2 highly upregulated miRs-miRs-21and -130- from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Hut78 with anti-miR-21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against PD-L1, pSTAT3 and GAPDH as a loading control.Results: The miRNAseq data revealed that miRs-21 and -130 had the highest correlation with PD-L1 mRNA. qRT-PCR and ISH revealed that miRs-21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a drastic decrease in PD-L1 and pSTAT3 expression in Hut78 cells transfected with anti-miRs-21 or -130, compared to cells transfected with Scr.Conclusions: PD-L1 expression in CTCL is regulated by miRs-21 and -130. miRs-21 and -130 may be regulating PD-L1 indirectly by silencing negative regulators of STAT signaling such as the SOCS family of proteins. This would explain the decrease in pSTAT3 levels in Hut78 cells transfected with anti-miRs. Taken together, these results demonstrate the ability of miRNAs to regulate immune checkpoints and ligands like PD-L1, and pave the way for the creation of miRNA therapeutics to reverse T-cell exhaustion and slow CTCL disease progression. DisclosuresQuerfeld:Acelion: Membership on an entity's Board of Directors or advisory committees; Kyowa: Membership on an entity's Board of Directors or advisory committees; Bioniz: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract 1999: Study of CD44 variant 6 (CD44v6) in prostate cancer chemo-/radio resistance in vivo

Abstract Background: Chemo-/radio-resistance is an important reason for prostate cancer (CaP) progression and metastasis. CD44 is a well-documented cancer stem cell (CSC) biomarker, and one of its variants, CD44 variant 6 (CD44v6) is closely associated with aggressive behaviour and correlates with poor prognosis in a variety of human cancers. Our previous study has demonstrated increased expressions of CD44v6 in metastatic CaP cell lines and human CaP tissues, which was associated with CaP progression and chemo-/radio-resistance in vitro. However, the role of CD44v6 in CaP progression and therapeutic resistance in vivo is still uncertain. Aim: The aim of this study was to investigate the role of CD44v6 in CaP development and chemo-/radioresistance as well as underlying pathways in vivo, and find whether it is a suitable therapeutic target for CaP therapy. Methods: CD44v6 gene was knocked down (KD) in PC-3M CaP cell line using short hairpin RNA (shRNA). Subcutaneous (s.c.) and orthotopic CaP animal models were established using PC-3M-CD44v6-KD and PC-3M-CD44v6-scramble (scr) control cells, to assess the effect of CD44v6 on CaP tumourigenesis, chemotherapy (docetaxel) response and radiation response. Signal transduction proteins in PI3K/Akt/mTOR pathway (Akt, p-Akt, mTOR and p-mTOR) as well as proliferation and apoptosis makers were assessed by immunohistochemistry in xenografts from both animal models. Results: Both s.c. and orthotopic xenografts from PC-3M-CD44v6-KD cells displayed supressed tumour growth and increased responsiveness to docetaxel (40mg/kg) and radiation (2Gy/day for 4 consecutive days) compared to control xenografts from PC-3M-CD44v6-scr cells in NOD/SCID male mice. Down-regulation of the PI3K/Akt/mTOR pathway proteins (p-Akt and p-mTOR), proliferation marker (Ki-67) and angiogenesis marker (CD31), as well as up-regulation of apoptotic responses (cleaved Caspase-3) to chemotherapy, DNA damage responses (γ-H2AX) to radiation were found in PC-3M-CD44v6-KD s.c. xenografts, respectively. Conclusions: CD44v6 is actively involved in CaP tumorigenesis and treatment responses to chemo-/radio-therapy via PI3K/Akt/mTOR signalling pathway in in vivo mouse models and holds promise as a therapeutic target for the treatment of CaP. Key words: CD44v6, prostate cancer, chemo-/radio-resistance, animal model, metastasis Citation Format: Jie Ni, Paul Cozzi, Julia Beretov, Joseph Bucci, Peter Graham, Yong Li. Study of CD44 variant 6 (CD44v6) in prostate cancer chemo-/radio resistance in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1999.

Abstract 2068: Single-cell RNA-seq profiling of the transcriptional response to Rb loss in the retinoblastoma cell of origin

Abstract Retinoblastoma is a rare childhood tumor initiated by biallelic inactivation of the RB1 gene and loss of functional retinoblastoma (Rb) protein. The tumors are thought to originate in cone photoreceptor precursors since Rb knockdown induces cone precursor proliferation and elicits cone-precursor-derived retinoblastoma-like tumors in orthotopic grafts. The identification of the retinoblastoma cell of origin and the authentic initiating mutation presents an opportunity to define the detailed molecular mechanisms controlling malignant transformation of a human cancer. In this project, we seek to define the transcriptional and epigenetic cell state transitions that follow Rb depletion as post-mitotic human cone precursors are induced to re-enter the cell cycle. Prospectively isolated cone precursors were transduced with lentivirus carrying either of three RB1 shRNAs or a SCR control shRNA and then re-isolated by FACS. Single cell RNA-sequencing (scRNA-seq) was performed at multiple time points over 15 days by generating full-length cDNAs corresponding to polyadenylated RNAs followed by sequencing. Among the 572 individual cells that passed quality control, an average of 6.6 million mapped reads and ~21,000 genes (~93,000 transcript isoforms) were detected per transcriptome. In addition, 300 cells per shRNA group were evaluated for genome-wide open chromatin using uATAC-seq at day 0 and 15. Cell transcriptomes were pseudotemporally ordered using “Waterfall” in order to define cellular trajectories and corresponding transcriptomic responses to Rb loss. Using transcriptomic, epigenetic, and network analyses, we define cell state transitions and identify potential mediators of such transitions, that are associated with the initiation of retinoblastoma development. Citation Format: Sunhye Lee, Hardeep P. Singh, Martin Triska, Kevin T. Stachelek, Maxwell M. Bay, Brendan H. Grubbs, Matthew E. Thronton, Haoze Hu, Neil I. Segil, Michael A. Bonaguidi, David Cobrinik. Single-cell RNA-seq profiling of the transcriptional response to Rb loss in the retinoblastoma cell of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2068.

The design of SCR controller for national fifth emission standard in light diesel vehicle

The design of SCR controller for national fifth emission standard in light diesel vehicle

Abstract 2833: Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer progression and chemo-/radio-resistance in vitro and in vivo

Abstract Aims: Prostate cancer (CaP) is the most common cancer in males in Australia which caused more than 3000 deaths in 2015. EpCAM is a transmembrane protein that is expressed at low levels in a variety of human epithelial tissues, but overexpressed in most solid tumors. Our previous study indicated that EpCAM was strongly expressed in metastatic CaP cell lines, primary human CaP tissues and lymph node metastasis and is a biomarker involved in CaP progression, and chemo-/radio-resistance. However, the role of EpCAM in CaP progression and therapeutic resistance is still uncertain. The aim of this study was to investigate the role of EpCAM in CaP progression and chemo-/radio-resistance as well as underlying mechanisms in vitro and in vivo. Methods: EpCAM gene was knocked down (KD) in PC-3, DU145 and LNCaP CaP cell lines using shRNA. Proliferation assay, colony formation assay, docetaxel (DTX) and radiation dose-response assay were carried out to evaluate the effect of KD of EpCAM on proliferation and therapeutic response of CaP cells in vitro. Subcutaneous (s.c) and orthotopic CaP animal models were established using PC-3-EpCAM-KD and PC-3-EpCAM-scramble (scr) control cells in NOD/SCID mice, to assess the effect of EpCAM on CaP tumourigenecity, chemotherapy (DTX) and radiation response. Signal transduction proteins in PI3K/Akt/mTOR signaling pathway, as well as proliferation, apoptotic and radiation response markers were evaluated by immunohistochemistry in xenografts. Results: KD of EpCAM reduced CaP proliferative potential and enhanced DTX and radiation sensitivity in three CaP cell lines. Both s.c and orthotopic EpCAM-KD xenografts displayed suppressed tumor growth and increased DTX and radiation responsiveness compared to EpCAM-scr control xenografts in NOD/SCID mice. Marked down-regulation of PI3K/Akt/mTOR pathway proteins (p-Akt and p-mTOR) and proliferation marker (Ki-67) and significant up-regulation of apoptotic (Caspase-3) and radiation (γ-H2AX) responses to chemo-/radio-therapies were found in EpCAM-KD xenografts, compared with control xenografts. In addition, Kaplan-Meier curve analysis demonstrated that KD of EpCAM improved median survival (MS) of tumor-bearing mice by 21.5 days compared with the control group (HR=26.94, CI95% 4.317-168.1, p=0.0004) and that KD of EpCAM improved MS of tumor-bearing mice which received docetaxel (50mg/kg, single dose, i.p.) by 11 days (HR=20.95, CI95% 3.599-121.9, p=0.0007), and radiotherapy (2Gy/day for 4 days) by 12 days (HR=11.00, CI95% 2.11-57.36, p=0.0044) compared with the control group, respectively. Conclusions: EpCAM plays an important role in CaP progression and chemo-/radio-resistance via PI3K/Akt/mTOR signaling pathway in vitro and in vivo and it is a promising therapeutic target for the treatment of CaP. EpCAM targeted therapy combined with chemo-/radio-therapy could be a novel modality for treatment-resistant CaP. Citation Format: Jie Ni, Paul Cozzi, Julia Beretov, Junli Deng, Joseph Bucci, Peter Graham, Yong Li. Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer progression and chemo-/radio-resistance in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2833. doi:10.1158/1538-7445.AM2017-2833

ENOS Regulation of Nrf2 and BNIP3 in Primary Murine Hepatocytes

It is well known that endothelial nitric oxide synthase (eNOS)‐derived nitric oxide (NO) stimulates mitochondrial biogenesis in a cGMP‐PGC1α dependent manner in various tissues. However, the importance of eNOS in regulating mitophagy, the clearance of poorly functioning mitochondria, in hepatocytes remains unknown. We hypothesized that eNOS induces mitochondrial turnover through nuclear factor‐E2‐related factor‐2 (Nrf2) mediated BCL2/E1B 19 kDa protein‐interacting protein 3 (BNIP3) induction of mitophagy. To examine the relationship between eNOS, Nrf2, and BNIP3 in primary hepatocytes, we isolated primary hepatocytes from C57/BL6 wild‐type (WT) mice followed by siRNA treatment [Scramble control (Scr), eNOS, or Nfr2] for six hours. Growth media was applied for 12–18 hours after siRNA treatment, then 24 hours of either control media or 500 μM FFA (250 μM oleate + 250 μM palmitate); the 500 μM FFA condition also included one of the following treatments for 16 hours: 1 μM carbonyl cyanide 3‐chlorophenylhydrazone (CCCP, to depolarize mitochondria and induce mitophagy)/10 μM cholorquine (CQ, lysosomal inhibitor), 30 nM CDDO‐Methyl Ester (CDDO‐Me, NRF2 activator), or 50 μM diethylenetriamine (DETA) NONOate/CCCP/CQ. Silencing of eNOS in primary hepatocytes (50% reduction vs Scramble control, p&lt;0.01) knocked down Nrf2 mRNA expression by ~40% and Nrf2 activation (NQO1 mRNA; target gene of Nrf2) and BNIP3 mRNA expression by ~40% following FFA challenge (p&lt;0.05 vs Scr control). In contrast, treating primary hepatocytes with the NO donor DETA NONOate induced Nrf2 activation (NQO1 mRNA) and BNIP3 mRNA by 50% and 300%, respectively (p&lt;0.05 vs Scr control). In support of Nrf2 regulating BNIP3, we provide data, that to our knowledge, is the first evidence that the direct knockdown of Nrf2 in primary hepatocytes by an siRNA approach (−80% knockdown of Nrf2 vs Scr control, p&lt;0.001) not only reduces Nrf2 activity (−80% knockdown of NQO1 mRNA), but also blunts induction of mitophagy flux (BNIP3 induction with CCCP/CQ reduced 50% vs Scr) in primary hepatocytes. In addition, Nrf2 activation with CDDO‐Me treatment (3.5‐fold increase in NQO1 mRNA) induced a ~2‐fold increase in BNIP3 accumulation (w/CCCP+CQ), an effect completely ablated with Nrf2 siRNA. These preliminary observations point to a regulatory role of hepatocyte‐specific eNOS in Nrf2 and BNIP3, and a strong rationale for the further examination of the role of Nrf2 in the regulation of BNIP3 and mitophagy.Support or Funding InformationFunding: This work was supported by grants from the Missouri Foundation for Veterans Medical Research, the Richard Wallace Faculty Incentive, Department of Medicine Research Council, and a Veterans Affairs VHA‐CDA2 Grant (IK2BX001299).