Modulation Of mRNA Expression Research Articles

MiRNA:RBP Interplay as a Key Regulatory Element in Health and Disease

Numerous crosstalk interactions between RNA-binding proteins (RBPs) and microRNAs (miRNAs) have been recently reported, unveiling the complexity and importance of gene expression modulation in health and disease. They control physiological processes such as stem cell maintenance, neuronal development or energetic metabolism, but are also responsible for pathological conditions, such as muscle waste and dystrophies, atherosclerosis, obesity and cancer. MiRNAs and RBPs are two of the well-studied post-transcriptional regulators and they may even reciprocally regulate themselves. MiRNAs can act on RBPs expression while RBPs modulate miRNA biogenesis, function and degradation. RBPs and miRNAs modulate mRNA expression at different levels, affecting their stability, splicing and translation efficiency through either competition for overlapping binding or modulation of mRNA structure by binding, but several other forms of interaction have been described. In this review, we will address the current bibliography regarding miRNA:RBP interactions and crosstalk events as well as their implications in health and disease.

Riproximin Exhibits Diversity in Sugar Binding, and Modulates some Metastasis-Related Proteins with Lectin like Properties in Pancreatic Ductal Adenocarcinoma.

Riproximin (Rpx) is a type II ribosome-inactivating protein with specific anti-proliferative activity. It was purified from Ximenia americana by affinity chromatography using a resin coupled with lactosyl residues. The same technique facilitated isolation of proteins with lectin-like properties from human Suit2-007 and rat ASML pancreatic cancer cells, which were termed lactosyl-sepharose binding proteins (LSBPs). The role of these proteins in cancer progression was investigated at mRNA level using chip array data of Suit2-007 and ASML cells re-isolated from nude rats. These data compared significant mRNA expression changes when relating primary (pancreas) and metastatic (liver) sites following orthotopic and intraportal implantation of Pancreatic Ductal Adenocarcinoma (PDAC) cells, respectively. The affinity of Rpx to 13 simple sugar structures was modeled by docking experiments, the ranking of which was principally confirmed by NMR-spectroscopy. In addition, Rpx and LSBPs were evaluated for anti-proliferative activity and their cellular uptake was assessed by fluorescence microscopy. From 13 monosaccharides evaluated, open-chain rhamnose, β-d-galactose, and α-l-galactopyranose showed the highest affinities for site 1 of Rpx’s B-chain. NMR evaluation yielded a similar ranking, as galactose was among the best binders. Both, Rpx and LSBPs reduced cell proliferation in vitro, but their anti-proliferative effects were decreased by 15–20% in the presence of galactose. The program “Ingenuity Pathway Analysis” identified 2,415 genes showing significantly modulated mRNA expression following exposure of Suit2-007 cells to Rpx in vitro. These genes were then matched to those 1,639 genes, which were significantly modulated in the rat model when comparing primary and metastatic growth of Suit2-007 cells. In this overlap analysis, LSBP genes were considered separately. The potential suitability of Rpx for treating metastatic Suit2-007 PDAC cells was reflected by those genes, which were modulated by Rpx in a way opposite to that observed in cancer progression. Remarkably, these were 14% of all genes modulated during cancer progression, but 71% of the respective LSBP gene subgroup. Based on these findings, we predict that Rpx has the potential to treat PDAC metastasis by modulating genes involved in metastatic progression, especially by targeting LSBPs.

Exosomes from MSCs overexpressing microRNA-223-3p attenuate cerebral ischemia through inhibiting microglial M1 polarization mediated inflammation

AimsTo explore the therapeutic effect and possible mechanism of exosomes from MSCs overexpressing miR-223 on cerebral ischemia and microglia polarization mediated inflammation. Main methodsRats after middle cerebral artery occlusion and reperfusion (MCAO/R) surgery and microglia BV-2 exposed to oxygen and glucose deprivation (OGD) and cysteinyl leukotrienes (CysLTs) stimulation were subject to exosomes from miR-223-3p transfected MSCs treatment, respectively. Behavioral tests were applied to assess the rats' neurological function. FACS was used to analyze M1/M2 microglia BV-2. production of cytokines in the ischemic hemisphere and BV-2 was detected by ELISA or qRT-PCR. Western blotting and qRT-PCR were also used to examine the expression of cysteinyl leukotriene receptor 2 (CysLT2R) in vivo and in vitro. Key findingsExosomes from MSCs over expressing miR-223-3p decreased MCAO/R induced cerebral infarct volume, improved neurological deficits, promoted learning and memorizing abilities. They suppressed pro-inflammatory factors expression and promoted anti-inflammatory factors secretion in the ischemic cortex and hippocampus. In vitro, exosomal miR-223-3p exhibited a more evident impact on modulating mRNA expression and protein production of cytokines. It promoted M2 microglia transformation of M1 microglia induced by NMLTC4 with a concentration-dependent manner. Western blot and qRT-PCR also revealed exosomal miR-223-3p decreased mRNA and protein expression of CysLT2R in vitro and in vivo. SignificanceExosomal miR-223-3p from MSCs attenuated cerebral ischemia/reperfusion injury through inhibiting microglial M1 polarization mediated pro-inflammatory response, which may be related with inhibitory effect of exosomal miR-223-3p on CysLT2R.

Role of long noncoding RNA-mediated competing endogenous RNA regulatory network in hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are noncoding RNAs (ncRNAs) that occupy over 90% of the human genome, and their main function is to directly or indirectly regulate messenger RNA (mRNA) expression and participate in the tumorigenesis and progression of malignances. In particular, some lncRNAs can interact with miRNAs as competing endogenous RNAs (ceRNAs) to modulate mRNA expression. Accordingly, these RNA molecules are interrelated and coordinate to form a dynamic lncRNA-mediated ceRNA regulatory network. Mounting evidence has revealed that lncRNAs that act as ceRNAs are closely related to tumorigenesis. To date, numerous studies have established many different regulatory networks in hepatocellular carcinoma (HCC), and perturbations in these ceRNA interactions may result in the initiation and progression of HCC. Herein, we emphasize recent advances concerning the biological function of lncRNAs as ceRNAs in HCC, with the aim of elucidating the molecular mechanism underlying these HCC-related RNA molecules and providing novel insights into the diagnosis and treatment of HCC.

Lactobacillus paracasei treatment modulates mRNA expression in macrophages

Macrophage metabolic pathways show changes in response to various external stimuli. Especially, increased lipopolysaccharide, an important bacterial component and Toll-like receptor 4 agonist, can induce activity in various macrophage metabolic pathways, including energy production and biosynthesis, as well as high immune responses due to increase in differentiated M1 macrophages. In this study, we confirmed that Lactobacillus paracasei (L. paracasei) KBL382, KBL384 and KBL385, isolated from the feces of healthy Koreans, can modulate various enzymes and membrane transporters related to glycolysis or macrophage polarization including hypoxia-inducible factor 1-alpha (HIF1A), inducible nitric oxide synthase (iNOS) and arginase in stimulated macrophages at the mRNA level, using the in vitro rodent bone‐marrow‐derived macrophage (BMDM) model. All L. paracasei exhibited significant down-regulatory effects on mRNAs for glycolysis-related enzymes, including lactate dehydrogenase A, solute carrier family 2 member 1, and triosephosphate isomerase. Moreover, L. paracasei treatment could lead to significant reductions in HIF1A or iNOS mRNA, and induced arginase mRNA in the BMDM model. Therefore, further extensive studies should be performed to support the application of L. paracasei, such as in probiotics or therapeutics, in controlling abnormal immune responses related to macrophage.

Hsa_circ_0041795 contributes to human retinal pigment epithelial cells (ARPE 19) injury induced by high glucose via sponging miR-646 and activating VEGFC.

Diabetic retinopathy (DR)is a common diabetes complication, resulting in the loss of vision. circRNAshave been reported to serve as ceRNA via targeting corresponding miRNAs and modulating mRNA expression in various diseases. Recently, increasing reports has indicated circRNAs can exert a significant role inDR progression. However, the expression and mechanism of hsa_circ_0041795 in human retinal pigment epithelial cells ARPE-19 treated by high glucose remains poorly known. Hence, we aimed to work figure out the effect of hsa_circ_0041795 in high glucose (HG)-induced ARPE-19 cell damage and study its molecular mechanisms. In our current research, we found that hsa_circ_0041795 was obviously up-regulated in HG-treated ARPE-19 cells. High dose of glucose greatly depressed ARPE-19 cell survival and contributed to cell apoptosis. In addition, we observed that loss of hsa_circ_0041795 enhanced cell proliferation and inhibit ARPE-19 cell apoptosis, after HG incubation. Furthermore, data of ELISA indicated that hsa_circ_0041795 siRNA significantly restrained inflammatory factors expression, such as TNF-α, IL-1β and IL-6 in ARPE-19 cells treated with HG. miR-646 has been recognized in multiple diseases and currently, we predicted that miR-646 acted as a target of hsa_circ_0041795. Moreover, we found that miR-646 inhibitors dramatically reversed the effect of hsa_circ_0041795 siRNA on ARPE-19 cells. Additionally, a dual-luciferase reporter assay proved that VEGFC was a direct target of miR-646. Our results demonstrated that hsa_circ_0041795 might exhibit a novel therapeutic potential in the treatment of DR.

Elderberry (Sambucus nigra L.) Fruit Extract Alleviates Oxidative Stress, Insulin Resistance, and Inflammation in Hypertrophied 3T3-L1 Adipocytes and Activated RAW 264.7 Macrophages.

Oxidative stress and inflammation in hypertrophied adipose tissue with excessive fat accumulation play a crucial role in the development of obesity and accompanying metabolic dysfunctions. This study demonstrated the capacity of elderberry fruit (EDB) extract to decrease the elevated production of reactive oxygen species in hypertrophied 3T3-L1 adipocytes. Treatment with the EDB extract resulted in modulation of mRNA expression and protein secretion of key adipokines in hypertrophied adipocytes. Expression of leptin and adiponectin was, respectively, down- and up-regulated. Moreover, glucose uptake stimulation was noticed in mature adipocytes, both sensitive to insulin and insulin resistant. This may suggest a positive effect of EDB extract on insulin resistance status. The extract was also found to alleviate the inflammatory response in activated RAW 264.7 macrophages by down-regulating the expression of proinflammatory genes (TNF-α, IL-6, COX-2, iNOS) and suppressing the enhanced production of inflammatory mediators (TNF-α, IL-6, PGE2, NO). In vitro experiments showed that the EDB extract could inhibit digestive enzymes, including α-amylase, α-glucosidase, and pancreatic lipase, leading to reduced intestinal absorption of dietary lipids and carbohydrates. Further in vivo studies could be postulated to support EDB as a functional food component for the prevention and treatment of obesity and metabolic-immune comorbidities.

Toll-like receptors control p38 and JNK MAPK signaling pathways in rat astrocytes differently, when cultured in normal or high glucose concentrations

Astrocytes play a vital role in regulating central nervous system inflammation, energy metabolism and brain homeostasis. Unlike macrophages and microglia, which are cells of myeloid ancestry, astrocytes are of ectodermal origin. However, regulatory specificities of signaling pathways connecting inflammatory and metabolic processes are still largely unknown. We analyzed firstly cellular responses to toll-like receptor (TLR) agonists and secondly, modulation of the mRNA of the three isoforms of the transcription factors PPARs (peroxisome proliferator-activated receptors) in primary rat astrocytes exposed to normal glucose (5.5 mM) and high glucose (25 mM). Cell culturing of rat brain astrocytes for 2 days in high glucose did not alter cellular morphology, but i) enhanced the release of TNFα that was induced by TLR4 agonist LPS or TLR3 agonist PIC and the synthesis of prostaglandin E2 (PGE2), ii) changed the signaling pathways of TLR4/MAPK (increase in p38 MAPK, and decrease in JNK activities at early stages of TLR activation) and iii) modulated mRNA expression of PPARs. High glucose cultivation reduced PPARα and PPARβ mRNA levels, without altering PPARγ mRNA level and changed the sensitivity of expressions to agonists of TLR1/2 (PGN), TLR4 (LPS), TLR3 (PIC), and TLR5 (FGN). Differences between low and high glucose-adapted cells were obtained for agonists of TLR1/2 (PPARα, PPARβ), TLR4 (PPAR β), TLR3 (PPARα). In the TLR4/p38/PPARβ signaling pathway, there was a stimulatory connection in normal glucose but an inhibitory connection in high glucose. TLR4/JNK/activated PPARβ, TLR4/JNK/inhibited PPARγ both in cells adapted to normal or high glucose, but PPARα expression was not affected. As PPARs in astrocytes are involved in inflammatory processes in the form of the recently published PPAR triad, the changes in expression revealed here are most likely resulting in implications of high glucose in inflammatory processes. Our data underline the complexity of multiple regulatory interactions between inflammatory responses and energy metabolism in astrocytes.

Molecular mechanism of chemoresistance and restoration in human gastric cancer cells.

e15544 Background: Gastric cancer is characterized by a high rate of relapse and failure of chemotherapy because of the emergence of drug resistant cells. Hence, resistance to chemotherapy is a major obstacle for the management of gastric cancer. It might be related with the development of cancer stem cells (CSCs). The aim of this study is to investigate the characteristics of the 5-fluorouracil (FU) resistant gastric cancer and to study how to restore the chemosensitivity. Methods: We used the AGS gastric cancer cell line (AGS) and transformed it into a 5-FU resistant cell line (AGS-R). AGS-R was established by continuous exposure of the cells to progressively increasing concentrations of 5-FU for about 1 year and modulating mRNA expression levels of four genes associated with thymidylate synthase (TS). The research methods used were MTT assay, flow cytometry analysis, luciferase reporter assay, western blotting, and siRNA transfection. Results: 5-FU-resistant gastric cancer cell, AGS-R was established by continuous exposure to 5-FU with gradual increase of its concentration for about 1 year. Comparing with AGS, thymidylate synthase (TS) expression in AGS-R was highly increase at transcriptional and translational level. And decreased transcriptional activity of β-catenin in AGS-R was confirmed by Western blotting and luciferase assay. Even though treatment with ICG-001, an inhibitor of β-catenin, showed growth inhibition of AGS in a dose- and time-dependent manner, but it could not in AGS-R. The expression of CD44, well-known CSCs marker, was significantly higher on AGS-R compared to AGS. And the Notch pathway was remarkably up-regulated. Flow cytometry analysis revealed that CD44 expression was reduced on AGS-R transfected with specific siRNA for notch intracellular domain (NICD). Conclusions: This study suggests that chemoresistance of gastric cancer against continuous exposure to 5-FU is associated with decrease of β-catenin activity and development of stemness via the activation of Notch pathway. Therefore, the inhibition of Notch pathway might be a potential therapeutic target in 5-FU-resistant gastric cancer.

Dynamic changes in rumen fermentation and bacterial community following rumen fluid transplantation in a sheep model of rumen acidosis: implications for rumen health in ruminants.

Over the years, rumen fluid transplantation (RT) has been successfully applied to treat acute rumen acidosis in ruminants, but how it functions in the ruminal microbial homeostasis and host function remains largely unknown. Here, we investigated the dynamic changes of rumen fermentation and bacterial communities following RT and its beneficial effects on rumen epithelial morphology and function in a sheep model of rumen acidosis. The results showed that RT resulted in dynamic changes in rumen fermentation and increased the concentrations of total volatile fatty acid, acetate, propionate, and butyrate, but it decreased the levels of lactate and LPS in the rumen. Illumina MiSeq Sequencing data showed that RT facilitated rapid rebuilt of ruminal bacterial homeostasis (8 d in control vs. 2 d in RT) from a markedly dysbiotic acidosis state to a healthy level (similar with those of donors). At the genus level, RT increased the relative abundance of unclassified Bacteroidales, unclassified Prevotellaceae, unclassified Ruminococcaceae, and Acetitomaculum. Additionally, RT also accelerated recovery of the predicted metagenomic function of ruminal bacteria. Rumen papillae morphology results showed that RT alleviated the damage of rumen epithelia induced by acute rumen acidosis and increased the length of rumen papillae. Furthermore, real-time PCR results showed that RT modulated mRNA expression of genes related to cytokines and tight junctions in the rumen epithelia. In summary, these results reveal that RT accelerates recovery of rumen fermentation and bacterial homeostasis and modulates rumen epithelial morphology and function for sheep suffering from rumen acidosis.-Liu, J., Li, H., Zhu, W., Mao, S. Dynamic changes in rumen fermentation and bacterial community following rumen fluid transplantation in a sheep model of rumen acidosis: implications for rumen health in ruminants.

Modulation of mRNA Expression in Peripheral Tissue in a Rodent E. coli Sepsis Model

The endogenous cannabinoid system (ECS) regulates cytokine production and mitigates pathophysiology in many pro‐inflammatory conditions, such as irritable bowel diseases, atherosclerosis and acute lung injury. Sepsis, a highly morbid condition, results from a dysregulated host response to infection. Despite extensive study the transcriptional regulation of cytokines and the ECS during sepsis remains to be established. We hypothesized that cannabinoid receptor (Cnr1 & 2) mRNA expression increases with systemic infection and these increases coincide with increases in pro‐inflammatory cytokines. We implanted a fibrin clot that contained either 25×106 or 0 E. coli in the peritoneal cavity of adult, male rats (n=8). Rats were euthanized at 3, 6, 12 or 24 h following the implant; lung, liver and serum were harvested. Total RNA was extracted using Trizol reagent, and cDNA was synthesized using reverse transcription PCR. Quantitative real‐time PCR was performed in duplicate for each sample using TaqMan Gene® Expression Assays specific for 18S ribosomal RNA (18S), Cnr1 & 2, interleukin (IL) IL‐4 & IL‐6 and tumor necrosis factor – alpha (TNF‐α). Each target gene was normalized to 18 S content, and relative quantitation of gene expression levels were performed using the comparative CT method (2−ΔΔCT method) with the 0 E. coli fibrin clot at each time point as the comparative control. The time course of cytokine expression depended on the cytokine and the tissue compartment. In liver, IL‐6 peaked at 3h (1086 ± 59% vs 0.36 ± 0.1%, p<0.01). In serum, IL‐4 peaked at 6h (168 ± 34% vs 86 ± 5%). In lung, both IL‐6 and TNF‐α expression levels peaked at 3h (IL‐6: 755 ± 33% vs 8 ± 0.5%, p<0.01; TNF‐a: 496 ± 230% vs 8 ± 0.2%); whereas, IL‐4 levels peaked at 24h (291 ± 31% vs 3 ± 2%, p<0.05). Interestingly, at 24h Cnr2 mRNA increased concomitantly with IL‐4 in the lung (360.0 ± 10% vs 30 ± 5%, p<0.01), whereas the expression levels of Cnr1 mRNA did not increase above sham surgical levels during the time course of this study. These preliminary data and analyses support our hypothesis that induction of ECS, as characterized by the increased transcription of Cnr2 mRNA, is associated with upregulation of mRNA of pro‐inflammatory cytokines.Support or Funding InformationNIH 5T32HL007913‐18, NIH U01 EB021960, VA Research Service I01BX004197This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Competitive Endogenous RNA (ceRNA) Regulation Network of lncRNA-miRNA-mRNA in Colorectal Carcinogenesis.

Competitive endogenous RNA (ceRNA) regulation suggested complex network of all transcript RNAs including long noncoding RNAs (lncRNAs), which can act as natural miRNA sponges to inhibit miRNA functions and modulate mRNA expression. Until now, the specific ceRNA regulatory mechanism of lncRNA-miRNA-mRNA in colorectal cancer (CRC) still remains unclear. RNA sequencing data of 478 colon adenocarcinoma cases and 41 controls as well as 166 rectum adenocarcinoma cases and 10 controls were obtained from The Cancer Genome Atlas (TCGA) to investigate the significant changes of lncRNAs, miRNAs and mRNAs in colorectal carcinogenesis. The target lncRNAs and mRNAs of miRNAs were predicted by miRWalk. Functional and enrichment analyses were conducted by DAVID database. The lncRNA-miRNA-mRNA interaction network was constructed using Cytoscape. We constructed ceRNA regulatory networks including 22 up-regulated lncRNAs, 12 down-regulated miRNAs and 122 up-regulated mRNAs, as well as 8 down-regulated lncRNAs, 43 up-regulated miRNAs and 139 down-regulated mRNAs. The GO enrichment showed that up-regulated genes mainly enriched in biological process including organic anion transport, collagen catabolic process, wound healing, Wnt receptor signalling and in pathways of tyrosine metabolism, taurine and hypotaurine metabolism, melanogenesis and phenylalanine metabolism. For down-regulated genes, significant enrichment was found in biological process of metal ion homeostasis, transmission of nerve impulse, cell-cell signalling, transmembrane transport and in pathways of ABC transporters, neuroactive ligand-receptor interaction, retinol metabolism, nitrogen metabolism and steroid hormone biosynthesis. We identified significantly altered lncRNAs, miRNAs and mRNAs in colorectal carcinogenesis, which might serve as potential biomarkers for tumorigenesis of CRC. In addition, the ceRNA regulatory network of lncRNA-miRNA-mRNA was constructed, which would elucidate novel molecular mechanisms involved in initiation and progression of CRC, thus providing promising clues for clinical diagnosis and therapy.

Isoflavones enhance the plasma cholesterol-lowering activity of 7S protein in hypercholesterolemic hamsters.

Previous studies have shown that 7S protein is the active ingredient responsible for the plasma cholesterol-lowering activity of soybean. It is hypothesized that isoflavones in soybean could enhance the blood cholesterol-lowering activity of 7S protein. Forty-eight hamsters were divided into six groups and fed a non-cholesterol diet or one of the five high-cholesterol diets containing 12.1% 7S protein with 0-15.62 mg g-1 isoflavones. The results showed that addition of isoflavones in diets dose-dependently enhanced the plasma total cholesterol-lowering activity of 7S protein. Addition of isoflavones in 7S protein-based diets significantly reduced hepatic cholesterol accumulation by 12.6-26.1%, compared with the high cholesterol control diet. Isoflavones could also facilitate excretion of neutral sterols in a dose-dependent manner. Supplementation of isoflavones in diets favourably modulated mRNA expression and the protein mass of HMG-CoA reductase. It was concluded that the enhancing effect of isoflavones on the blood cholesterol-lowering activity of 7S protein was mediated by inhibiting the cholesterol absorption and de novo cholesterol synthesis in hypercholesterolemic hamsters.

Enhanced expression of gene coding for β-endorphin in human monocytic cells exposed to pulsed radio frequency electric fields through thermal and non-thermal effects.

BackgroundThe enhanced expression of endogenous opioid peptides, including β-endorphin, has been implicated in the mechanism of action of pulsed radio frequency (PRF) application in pain modulation. Because thermal effects cannot be separated from the physical property of PRF application to biological tissues, we evaluated whether temperatures higher than that of the normal body temperature (37°C) modulate mRNA expression for the precursor of β-endorphin, proopiomelanocortin (POMC) in human monocytic cells THP-1. We also attempted to examine whether mechanisms other than thermal effects also modulate such gene expression.Methods and resultsThe mRNA for POMC in THP-1 cells increased by a 15-minutes incubation at 42°C, 45°C, or 70°C without PRF application as compared with that in cells incubated at 37°C. On the other hand, gene expression for POMC in cells incubated at 20°C as well as at 37°C with PRF application for 15 minutes increased as compared to that in cells incubated at 37°C without PRF application. Continuous radio frequency at 70°C but not PRF provoked apoptotic cell death at 1–2 hour, and necrotic cell death at 24 hours after the RF application.ConclusionA simple experimental system using human monocytic cells in culture demonstrated that a 15 minute elevation of temperature above 37°C enhanced gene expression for POMC in THP-1 cells, while a 15 minute application of PRF to these cells incubated at 37°C or lower, also enhanced gene expression, indicating that temperature-independent mechanisms as well as thermal effects may be involved in such gene expression.

Abstract 619: ALT-803 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NEO-201 against human carcinoma cells

Abstract Background: ALT-803 is a novel IL-15 superagonist complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α/IgG1 Fc fusion protein. It has been reported that ALT-803 exerted a significant anti-tumor activity in murine myeloma, breast, colon carcinomas, in rat bladder cancer, and in human bladder, ovarian carcinomas in vivo. ALT-803 was also found to enhance ADCC against wide range of human carcinoma cells in vitro. NEO-201 is a novel humanized IgG1 monoclonal antibody (mAb) which was found to react against a variety of cultured human carcinoma cell lines and tumor tissues. NEO-201 can mediate ADCC against human carcinoma cells. Methodology: We investigate the ability of ALT-803 to modulate the ADCC mediated by NE0-201, employing NK cells isolated from normal donors as effectors. Human NK cells were treated with ALT-803 at different concentrations for 48h prior to be used as effector cells, and human carcinoma cell lines expressing the NE0-201 antigen were utilized as targets in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and to modulate NK cells gene expression was evaluated by flow cytometry and by using the Nanostring analysis respectively. Results: The treatment with ALT-803 significantly enhanced the ADCC activity mediated by NEO-201 against NEO-201 positive carcinoma cells. The effect ALT-803 was dose-dependent and achieved statistical significance at all doses tested compared to vehicle control treatment. Treatment of NK cells with ALT-803 enhanced ADCC activity also from donors with minimal ADCC activity and lowered the effective dose of NEO-201 required to initiate the ADCC response compared to untreated NK cells. ADCC activity can be blocked by using anti-CD16 and anti-TIM3 blocking antibody. Phenotypic analysis of NK cells treated with 25 ng/ml of ALT-803 for 48h demonstrated that ALT-803 can enhance the expression of TIM3 and NKG2D and the mean fluorescence intensity (MFI) of granzyme B and CD107a in CD16/CD56 positive NK cells. Nanostring analysis of human NK cells treated with ALT-803 at different concentrations for 48h, showed that ALT-803 was able to modulate mRNA expression of 62 genes (1.6 log2 fold change compared to vehicle control was considered significant). ALT-803 up-regulated the mRNA expression of 43 genes, including NK activating receptors, factors involved in the NK cytotoxicity, cytokines and their receptors, and down-regulated the mRNA expression of 19 genes, including NK inhibiting receptors and factors involved in the activation of apoptosis. Conclusions: ALT-803 can enhance ADCC activity mediated by NEO-201 against human carcinoma cells. The enhancement of the ADCC activity may be in part due to the increase in the expression of TIM3, NKG2D, granzyme B, and CD107a positive NK cells, as well as to the modulation of transcripts that are involved in the NK activation and cytotoxicity. Citation Format: Massimo Fantini, Justin M. David, Hing C. Wong, Christina M. Annunziata, Philip M. Arlen, Kwong Yok Tsang. ALT-803 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NEO-201 against human carcinoma cells [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 619.

Repository corticotrophin injection exerts direct acute effects on human B cell gene expression distinct from the actions of glucocorticoids.

Repository corticotrophin injection (RCI, H.P Acthar® gel) has been approved for use in the management of multiple autoimmune and inflammatory diseases for more than a half-century, but its mechanism of action is not well understood. We used RNA-Seq methods to define RCI-regulated mRNAs in cultured human B cells under conditions of activation by interleukin (IL)-4 and CD40 ligand. Following IL-4/CD40L activation and RCI treatment we found up-regulation of 115 unique mRNA transcripts and down-regulation of 80 unique mRNAs. The effect on these RNA levels was dose-dependent for RCI and was distinct from changes in mRNA expression induced by treatment with a potent synthetic glucocorticoid. RCI down-regulated mRNAs were observed to include a significant over-representation of genes critical for B cell proliferation under activating conditions. These data confirm that RCI exerts direct effects on human B cells to modulate mRNA expression in specific pathways of importance to B cell function and that, at the molecular level, the effects of RCI are distinct from those exerted by glucocorticoids.

Roles of some antioxidants in modulation of cardiac myopathy induced by sodium nitrite via down-regulation of mRNA expression of NF-κB, Bax, and flt-1 and suppressing DNA damage

The underlying pathology of cardiac damage involves various molecular and signaling pathways. Therefore, this study aimed to explore the role of Quercetin (Querc), alone or in combination with Melatonin (Melat) against cardiac damage induced by sodium nitrite (Sod nit), as well as to elucidate different signaling pathways. Querc and Melat were injected intraperitoneally (i.p.), followed by induction of hypoxia in rats by using a single dose of Sod nit (60 mg/kg, s.c.). Treatment with Sod nit significantly decreased hemoglobin (Hb) levels in blood. Pretreatment of hypoxic rats with Querc and/or Melat elevated the declined Hb concentration. The forementioned antioxidants also successfully ameliorated the alteration of heat shock protein 70 (HSP-70) and markers of cardiac injury, including troponin T (Trop. T), creatine kinase-MB (CK-MB), tumor necrosis factor-α (TNF α), and C-reactive protein (CRP) in the rats serum. Furthermore, RT-PCR revealed that these antioxidants successfully modulated mRNA expression of NF-κB, Bax, Bcl-2, and flt-1. They also regulated vascular endothelial growth factor (VEGF), the apoptosis marker caspase 3, and oxidative DNA damage in cardiac tissue, compared to Sod nit-intoxicated rats. The present biochemical results are reinforced by histopathological examination. In Conclusion: The results reflected that treatment with Querc in combination with Melat was most effective in improving Sod nit-toxicity induced cardiac damage, thus confirming the promising role of this combination as an effective treatment for cardiac damage induced by other cardio-toxic agents.

Effects of bacilli, molasses, and reducing feeding rate on biofloc formation, growth, and gene expression in Litopenaeus vannamei cultured with zero water exchange

The effects of reducing feeding rate and molasses on water quality, growth, and gene expression of Litopenaeus vannamei were evaluated in a super intensive system with zero water exchange. A bioassay of 35 days (0,75 ± 0,22 g, 36 shrimp tank -1 , 450 shrimp m -3 ) with treatments in triplicate was performed: I) 100% commercial feed (control group) with water exchange; II) 100% commercial feed without water exchange; III) 100% commercial feed + Bacillus licheniformis BCR 4-3 + molasses without water exchange; IV) 90% commercial feed + B. licheniformis BCR 4-3 + molasses without water exchange, and V) 80% commercial feed + B. licheniformis BCR 4-3 + molasses without water exchange. Shrimp survival, weight, nitrogenous wastes, settleable solids (SS), and the relative expression (RT-qPCR) of stress and digestive genes were determined. Shrimp survival was 100% in all treatments. The reduced diet, bioflocs, and bacilli did not affect shrimp growth. The culture conditions reduced nitrogenous wastes and modulated mRNA expression of trypsin, chymotrypsin, and heat shock protein 70. Reduced feed ration can help to maintain water quality and lower costs in intensive shrimp cultures with bioflocs. Normal 0 21 false false false ES-MX JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif; mso-ansi-language:ES; mso-fareast-language:ES;}

Transcriptional profiling of antioxidant defense system and heat shock protein (Hsp) families in the cadmium- and copper-exposed marine ciliate Euplotes crassu.

To understand the transcriptional response of antioxidant defense system and heat shock protein (Hsp) families of the marine ciliate Euplotes crassus, we analyzed the transcriptome profile using RNA-seq technology after exposure to cadmium (Cd) and copper (Cu). De novo sequence assembly produced 61,240 unigenes with 21,330 BLAST hits and showed high sequence orthology with transcriptomes of other ciliates. Gene annotation and gene ontology (GO) comparison revealed that E. crassus expressed highly diversified but conserved stress-responsive gene families of the antioxidant defense system and Hsps. After waterborne exposure to 250μg/L of Cd and 25μg/L of Cu, transcriptional responses of the gene families were significantly modulated, suggesting that even the unicellular E. crassus has a conserved molecular defense mechanism, such as modulating mRNA expression, for homeostasis. These transcriptional responses make E. crassus a potential model for understanding the molecular response of single cell ciliates to heavy metal contamination.

Structural basis for the specific recognition of 18S rRNA by APUM23.

PUF (Pumilio/fem-3 mRNA binding factor) proteins, a conserved family of RNA-binding proteins, recognize specific single-strand RNA targets in a specific modular way. Although plants have a greater number of PUF protein members than do animal and fungal systems, they have been the subject of fewer structural and functional investigations. The aim of this study was to elucidate the involvement of APUM23, a nucleolar PUF protein in the plant Arabidopsis, in pre-rRNA processing. APUM23 is distinct from classical PUF family proteins, which are located in the cytoplasm and bind to 3′UTRs of mRNA to modulate mRNA expression and localization. We found that the complete RNA target sequence of APUM23 comprises 11 nt in 18S rRNA at positions 1141–1151. The complex structure shows that APUM23 has 10 PUF repeats; it assembles into a C-shape, with an insertion located within the inner concave surface. We found several different RNA recognition features. A notable structural feature of APUM23 is an insertion in the third PUF repeat that participates in nucleotide recognition and maintains the correct conformation of the target RNA. Our findings elucidate the mechanism for APUM23’s-specific recognition of 18S rRNA.