mRNA Ratio Research Articles

Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.

THE CORRELATION OF β/α MRNA RATIO WITH CLINICAL AND HEMATOLOGICAL PARAMETERS IN PATIENTS WITH β-THALASSEMIA SYNDROME

Background: Thalassemias are a group of genetically transmitted blood diseases characterized by defects in the production of α- or β-chains of hemoglobin called α-thalassemia and β-thalassemia, respectively. One of the common features of β-thalassemia is ineffective erythropoiesis because of the imbalance of globin chain production, which result in increased apoptosis during erythroblast maturation. Objective: To evaluate the ß/α globin mRNA ratio in patients with ß-thalassemia syndrome and to correlate the ß/α ratio with hematological and clinical condition of the patients. Methods: Thirty-five patient samples were collected from Thalassemia Centre of Ibn Al-Balady Hospital; 18 patients with β-thalassemia major and 17 patients with β-thalassemia intermedia. The patients were randomly selected regarding sex, whereas their age ranged from 3 to 17 years. Along with those samples, twenty control leftover samples that were the remaining of samples collected for laboratory investigation, were taken from Al-Kadhimiya Pediatric Hospital, and were age and sex matched with the patients group. The α and β globin chain ration were calculated by the real-time reverse transcription-polymerase chain reaction (qRT-PCR). The β/α -globin mRNA ratio of the samples was measured by the 2−ΔΔCT method. Results: Analysis of the β-globin/α-globin mRNA ratio showed that disease severity increased with a reduction of the ratio. There was a highly significant difference in α level, ß level and ß/α ratio among studied groups with a p value of (p < 0.001). The highest level was in control group, followed by that in β-thalassemia intermedia and the lowest was in β-thalassemia major. Conclusion: The severity of ß/α globin chain imbalance showed a significant and negative correlation with the mean corpuscular volume and there was no significant correlation between the ß/α ratio and markers of erythropoiesis in ß-thalassemia major and ß-thalassemia intermedia patients. The ß/α ratio was a good tool for diagnosis in ß-thalassemia major and ß-thalassemia intermedia as compared to control group with high sensitivity and specificity. A ß-chain gene expression was the lowest in ß-thalassemia major followed by ß-thalassemia intermedia as compared to control group. Keywords: Thalassemia, ß/α -globin mRNA ratio Citation: Yousif MH, Al-Mamoori HS. The Correlation of β/α mRNA Ratio with Clinical and Hematological Parameters in Patients with β-thalassemia Syndrome. Iraqi JMS. 2022; 20(1): 139-145. doi: 10.22578/IJMS.20.1.18

P1521: A SEVERE MOUSE MODEL OF ALPHA-THALASSEMIA SHOWS ABNORMAL IRON METABOLISM, ERYTHROPOIESIS AND COAGULATION, AND CAN BE RESCUED BY A NOVEL GENE THERAPY APPROACH

Background: Clinical presentation of deletional a-thal varies from an asymptomatic condition (one inactivated a-globin gene) to a complete knockout (Hb Bart’s Hydrops Fetalis). In patients with severe a-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation. Aims: The aims of this study are to develop a novel adult mouse model of a-thal and a gene therapy approach for this disease. Methods: We generated adult animals that do not produce a-globin chains (a-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. To generate a gene therapy tool, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human a-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human a-globin genes. Results: The a-KO animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only b-globin chains (HbH). RBC show aberrant morphology and aggregation of b-globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events, associated with neutrophil infiltration, fibrinogen staining, von Willebrand factor (vWF) released, platelet recruitment and activation. These animals also show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney. We identified ALS20a, a vector where a-globin is under control of the b-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Use of ALS20a resulted in correction of the disease phenotype in a dose-dependent manner in a-KO mice. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels. We tested ALS20a in CD34 cells isolated from four patients with both deletional and non-deletional HbH disease. We measured the change of b/a-globin mRNA ratio (b/aR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial b/aR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a b/aR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced a-thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2). Image:Summary/Conclusion: We generated an adult mouse model of lethal a-thal and, in preliminary experiments, we rescue it with ALS20a. Furthermore, ALS20a successfully improves a-globin levels in patient cells.

P998: REGULATION OF BCL-X SPLICING BY SRC KINASES INHIBITORS IN MYELOPROLIFERATIVE NEOPLASMS

Background: Myeloproliferative neoplasms (MPN) are a group of incurable diseases that include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Both ET and PV are associated with prolonged clinical courses, with symptoms that affect their quality of life, and risk of progression to MF and acute leukemia. MF is the MPN subtype with the most adverse prognosis and is associated with a high risk of leukemic transformation. The FDA approved the use of ruxolitinib, a JAK1/2 kinase inhibitor, that proved to be effective in reducing symptoms and spleen volume, but not in inducing complete molecular remission. Although hematopoietic stem cell transplantation involves a high risk of mortality, it is nowadays the only curative treatment for MF. Therefore, it is relevant to identify new therapeutic strategies to improve the clinical outcome of MPN. Recently, there has been a growing interest in Bcl‐xL, the long isoform encoded by alternative splicing of the Bcl‐x gene, that acts as an anti‐apoptotic regulator. Bcl-xL appears to be up-regulated in MPN patients and we previously showed that its modulation correlates with the clinical severity of MPN subgroups. We also demonstrated that ABT-737, a specific Bcl-xL inhibitor, has a synergistic effect in combination with Ruxolitinib in HEL cell lines and MPN patients’ cells, suggesting that Bcl-xL inhibition could increase the power of treatment (Petiti et al. J Cell Mol Med. 2020). Unfortunately, ABT-737 is characterized by poor bioavailability, so a new approach to target Bcl-xL must be investigated. Paronetto et al. found that tyrosine phosphorylation of Sam68 in cancer cells may protect them from apoptosis by altering the Bcl-xS/L ratio in favor of Bcl-xL. Intriguingly, c-Src is one of the tyrosine kinases that phosphorylate Sam68 and several papers indicated its involvement in the MPN pathogenesis, without explaining the molecular mechanism. Aims: We aimed to molecular investigate the mechanism linking c-Src kinase activity with the deregulation of Bcl-x splicing in MPN, intending to consider c-Src kinase inhibitors, widely bioavailable, as indirect modulators of Bcl-xL to improve MPNs outcome. Methods: HEL cell lines, JAK2 V617F-mutated in homozygosis, has been incubated with different concentrations of ruxolitinib (JAK1/2 inhibitor) and dasatinib (SRC kinases inhibitor) at different concentrations and time points. Then, proliferation, and apoptosis were evaluated. Subsequently, the c-Src pathway, Sam68, and Bcl-xL/S ratio were investigated at both RNA and protein levels. Results: Performing in vitro study on HEL cell lines, we identified that dasatinib induced apoptosis and inhibition of proliferation in HEL cell lines, with a synergistic effect in combination with ruxolitinib. We also showed that dasatinib alone inhibited c-Src and Sam68 phosphorylation and down-modulated Bclx-L protein expression. Combination treatment with ruxolitinib and dasatinib increased both pro-apoptotic Cleaved Caspase 3 and BAX protein expression. Furthermore, we noted that treatment with ruxolitinib alone decreases the mRNA levels of Bclx-L, but also of the Bcl-xS isoform. On the contrary, combined drug therapy modulated the Bcl-xL/S mRNA ratio in favor of the pro-apoptotic Bcl-xS isoform. Summary/Conclusion: Although further studies will be necessary to better understand the c-Src role in Bcl-x splicing regulation in MPN, our preliminary data suggest that indirect downmodulation of Bcl-xL through SRC kinases inhibitors in combination with standard therapy could be useful in the future for the treatment of MPN patients.

Modulation of Vasomotor Function by Perivascular Adipose Tissue of Renal Artery Depends on Severity of Arterial Dysfunction to Nitric Oxide and Severity of Metabolic Parameters.

Perivascular adipose tissue (PVAT) enhances vascular relaxation of mesenteric arteries in SHRSP.Z-Leprfa/IzmDmcr rats (SPZF), a metabolic syndrome model. We investigated and compared the effects of PVAT on the renal artery in SPZF with those on SHR/NDmcr-cp rats (CP). Renal arteries with and without PVAT were isolated from 23-week-old SPZF and CP. The effects of PVAT on acetylcholine- and nitroprusside-induced relaxation were examined using bioassays with phenylephrine-contracted arterial rings. Acetylcholine-induced relaxations without PVAT in SPZF and CP were 0.7- and 0.5-times lower in females than in males, respectively. In the presence of PVAT, acetylcholine-induced relaxations increased 1.4- and 2-times in male and female CP, respectively, but did not differ in SPZF. Nitroprusside-induced relaxation with and without PVAT was 0.7-times lower in female than in male SPZF but did not differ in CP. Angiotensin-II type-1 receptor (AT1R)/AT1R-associated protein mRNA ratios were lower in CP than in the SPZF and negatively correlated with the difference in arterial relaxation with and without PVAT. The effects of renal artery PVAT differed between the SPZF and CP groups. Higher levels of enhanced AT1R activity in SPZF PVAT may drive these differences by impairing the vascular smooth muscle responses to nitric oxide.

Development of transcriptomics-based growth rate indices in two model eukaryotes and relevance to metatranscriptomics datasets.

Growth rate estimation is important to understand the flow of energy and nutrient elements in an ecosystem, but it has remained challenging, especially on microscopic organisms. In this study, we propose four growth rate indices that use mRNA abundance ratios between nuclear and mitochondrial genes: (1) total nuclear and mitochondrial mRNA ratio (Nuc:Mito‐TmRNA); (2) nuclear and mitochondrial ribosomal protein mRNA ratio (Nuc:Mito‐RPmRNA); (3) gene ontology (GO) terms and total mitochondrial mRNA ratios; and (4) nuclear and mitochondrial specific gene mRNA ratio. We examine these proposed ratios using RNA‐Seq datasets of Daphnia magna, and Saccharomyces cerevisiae retrieved from the NCBI Short Read Archive. The results showed that both Nuc:Mito‐TmRNA and Nuc:Mito‐RPmRNA ratio indices showed significant correlations with the growth rate for both species. A large number of GO terms mRNA ratios showed significant correlations with the growth rate of S. cerevisiae. Lastly, we identified mRNA ratios of several specific nuclear and mitochondrial gene pairs that showed significant correlations. We foresee future implications for the proposed mRNA ratios used in metatranscriptome analyses to estimate the growth rate of communities and species.

Investigation of drugs for the prevention of doxorubicin-induced cardiac events using big data analysis

AimDoxorubicin, an anthracycline anti-tumour agent, is an essential chemotherapeutic drug; however, the adverse events associated with doxorubicin usage, including cardiotoxicity, prevent patients from continuing treatment. Here, we used databases to explore existing approved drugs with potential preventative effects against doxorubicin-induced cardiac events and examined their efficacy and mechanisms. MethodsThe Gene Expression Omnibus (GEO), Library of Integrated Network-based Cellular Signatures (LINCS), and Food and Drug Administration Adverse Events Reporting System (FAERS) databases were used to extract candidate prophylactic drugs. Mouse models of doxorubicin-induced cardiac events were generated by intraperitoneal administration of 20 mg/kg of doxorubicin on Day 1 and oral administration of prophylactic candidate drugs for 6 consecutive days beginning the day before doxorubicin administration. On Day 6, mouse hearts were extracted and examined for mRNA expression of apoptosis-related genes. ResultsGEO analysis showed that doxorubicin administration upregulated 490 genes and downregulated 862 genes, and LINCS data identified sirolimus, verapamil, minoxidil, prednisolone, guanabenz, and mosapride as drugs capable of counteracting these genetic alterations. Examination of the effects of these drugs on cardiac toxicity using FAERS identified sirolimus and mosapride as new prophylactic drug candidates. In model mice, mosapride and sirolimus suppressed the Bax/Bcl-2 mRNA ratio, which is elevated in doxorubicin-induced cardiotoxicity. These drugs also suppressed the expression of inflammatory cytokines Il1b and Il6 and markers associated with myocardial fibrosis, including Lgal3 and Timp1. ConclusionThese findings suggest that doxorubicin-induced cardiac events are suppressed by the administration of mosapride and sirolimus.

NKCC1 to KCC2 mRNA Ratio in Schizophrenia and Its Psychopathology: a Case-Control Study.

Schizophrenia (SCZ) is a debilitating, destructive, and chronic mental disorder and affects approximately one percent of the human population. Diagnosis in psychiatry is based on the patient's descriptions of his/her symptoms, interviewer's observations, history of disorder over time, and response to treatment. All of these data measure phenotype-based functions. But it appears that accurate diagnosis of such a complex disorder must be based on valid and reliable factors. In the present study, gene selection was based on the possible role of γ-aminobutyric acid (GABA) in psychopathology of SCZ and expression in blood. We evaluated the association of Na+-K+-Cl- co-transporter 1 (NKCC1) and K+-Cl- co-transporter 2 (KCC2) genes' messenger ribonucleic acid (mRNA) levels, and also the NKCC1/KCC2 ratio with positive and negative syndrome scale (PANSS) and brief psychiatric rating scale (BPRS) scores in an SCZ group. By using real-time PCR (RT-PCR), the present study is the first attempt to explore levels of NKCC1 and KCC2 expression at mRNA level and their relative expression in human peripheral blood of patients with SCZ. Our results showed that the NKCC1 to KCC2 mRNA ratio is significantly increased (but based on the delta cycle of threshold [∆Ct] is significantly lower) in the total sample of cases rather than controls (p = 0.045) and also higher in male sample cases rather than male controls (p = 0.016). In female samples, we found a trend toward a significant effect between the case and control participants (p = 0.075). We also found statistically significant association between mRNA of NKCC1 and KCC2 genes and NKCC1/KCC2 mRNA ratio with the positive and negative syndrome scale (PANSS) and brief psychiatric rating scale (BPRS) scores.

Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea.

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high rRNA:mRNA ratio (80–90%: ~10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA, and therefore, requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here, we show that after the discontinuation of the previous version of RiboZero (Illumina, San Diego, CA, USA) that was useful in partially or completely depleting rRNA from archaea, archaeal transcriptomics studies have experienced a slowdown. To overcome this limitation, here, we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper, we provide insights into the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.

Optimization of DOTAP/chol Cationic Lipid Nanoparticles for mRNA, pDNA, and Oligonucleotide Delivery.

Lipid nanoparticles (LNPs) can be used as delivery vehicles for nucleic acid biotherapeutics. In fact, LNPs are currently being used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. Cationic LNPs composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (chol) LNPs have been classified as one of the most efficient gene delivery systems and are being tested in numerous clinical trials. The objective of this study was to examine the effect of the molar ratio of DOTAP/chol, PEGylation, and lipid to mRNA ratio on mRNA transfection, and explore the applications of DOTAP/chol LNPs in pDNA and oligonucleotide transfection. Here we showed that PEGylation significantly decreased mRNA transfection efficiency of DOTAP/chol LNPs. Among non-PEGylated LNP formulations, 1:3 molar ratio of DOTAP/chol in DOTAP/chol LNPs showed the highest mRNA transfection efficiency. Furthermore, the optimal ratio of DOTAP/chol LNPs to mRNA was tested to be 62.5 µM lipid to 1 μg mRNA. More importantly, these mRNA-loaded nanoparticles were stable for 60 days at 4 °C storage without showing reduction in transfection efficacy. We further found that DOTAP/chol LNPs were able to transfect pDNA and oligonucleotides, demonstrating the ability of these LNPs to transport the cargo into the cell nucleus. The influence of various factors in the formulation of DOTAP/chol cationic LNPs is thus described and will help improve drug delivery of nucleic acid–based vaccines and therapies.Graphical abstract

Linking transcriptional dynamics of CH4-cycling grassland soil microbiomes to seasonal gas fluxes

Soil CH4 fluxes are driven by CH4-producing and -consuming microorganisms that determine whether soils are sources or sinks of this potent greenhouse gas. To date, a comprehensive understanding of underlying microbiome dynamics has rarely been obtained in situ. Using quantitative metatranscriptomics, we aimed to link CH4-cycling microbiomes to net surface CH4 fluxes throughout a year in two grassland soils. CH4 fluxes were highly dynamic: both soils were net CH4 sources in autumn and winter and sinks in spring and summer, respectively. Correspondingly, methanogen mRNA abundances per gram soil correlated well with CH4 fluxes. Methanotroph to methanogen mRNA ratios were higher in spring and summer, when the soils acted as net CH4 sinks. CH4 uptake was associated with an increased proportion of USCα and γ pmoA and pmoA2 transcripts. We assume that methanogen transcript abundance may be useful to approximate changes in net surface CH4 emissions from grassland soils. High methanotroph to methanogen ratios would indicate CH4 sink properties. Our study links for the first time the seasonal transcriptional dynamics of CH4-cycling soil microbiomes to gas fluxes in situ. It suggests mRNA transcript abundances as promising indicators of dynamic ecosystem-level processes.

Overexpression of GRK6 alleviates chronic visceral hypersensitivity through downregulation of P2Y6 receptors in anterior cingulate cortex of rats with prenatal maternal stress

AimsVisceral hypersensitivity is a major clinic symptom in patients with irritable bowel syndrome (IBS). Anterior cingulate cortex (ACC) is involved in processing the information of pain. Both G protein‐coupled receptor kinase 6 (GRK6) and P2Y purinoceptor 6 (P2Y6) are associated with neuroinflammation and pathological pain. The aim of this study was to investigate the interaction between GRK6 and P2Y6 in ACC in the development of visceral hypersensitivity of adult offspring rats with prenatal maternal stress (PMS).MethodsVisceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). The expression and cellular distribution of GRK6 and P2Y6 were determined by Western blotting, qPCR, and fluorescence immunohistochemistry. Co‐immunoprecipitation was used to evaluate the interaction between GRK6 and P2Y6.ResultsThe mRNA and protein levels of GRK6 were significantly decreased in ACC of PMS rats. The injection of GRK6 overexpression virus significantly attenuated visceral hypersensitivity of PMS rats. P2Y6’s mRNA level, protein level, and ratio of membrane protein over total protein expression was markedly increased in PMS rats. P2Y6 antagonist MRS2578 microinjection reversed visceral hypersensitivity of PMS rats. GRK6 overexpression significantly reduced P2Y6’s expression in membrane proteins and P2Y6’s ratio of membrane protein over total protein expression.ConclusionsThese results indicate that decreased GRK6 leads to the accumulation of P2Y6 at neuron membrane in ACC, thereby contributing to visceral hypersensitivity of PMS rats.

METTL3 promotes proliferation and myogenic differentiation through m6A RNA methylation/YTHDF1/2 signaling axis in myoblasts

Skeletal muscle development has an important impact on muscle-related diseases and domestic animal meat production. The m6A RNA methylation is a common post-transcriptional modification, affecting the development and metabolism of various organs. However, the effect and regulatory mechanism of methyltransferase like 3 (METTL3) on myogenesis are still unclear. Here, we showed that the mRNA levels of METTL3 was greater in skeletal muscles including extensor digitorum longus (EDL), soleus (SOL), tibialis anterior (TA) and gastrocnemius (GAS). Moreover, METTL3 highly expressed in the early stage of myoblast proliferation at hour 0 and the late stage of myoblast differentiation at day 8, indicating it was involved in myogenesis. Interestingly, METTL3 knockdown inhibited myoblast proliferation and myogenic differentiation, whereas METTL3 overexpression promoted these processes. Mechanically, METTL3 overexpression increased the ratio of mRNA m6A/A and shortened the time of P21 and P27 mRNA half level, causing the mRNAs downregulation via reducing their stability. Meanwhile, the promotion of cell proliferation by METTL3 overexpression was attenuated by YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) knockdown. Furthermore, the promotion of myogenic differentiation by METTL3 overexpression was weakened by YTHDF1 knockdown through reducing the mRNA translation of MRFs including MyHC, MyoD and MyoG. Therefore, METTL3 facilitates myoblast proliferation and myogenic differentiation. Overall, these findings suggest that METTL3/m6A RNA methylation/YTHDF1/2 signaling axis is a novel strategy for the regulation of skeletal muscle development.

Methylmercury Induces Mitochondria- and Endoplasmic Reticulum Stress-Dependent Pancreatic β-Cell Apoptosis via an Oxidative Stress-Mediated JNK Signaling Pathway.

Methylmercury (MeHg), a long-lasting organic pollutant, is known to induce cytotoxic effects in mammalian cells. Epidemiological studies have suggested that environmental exposure to MeHg is linked to the development of diabetes mellitus (DM). The exact molecular mechanism of MeHg-induced pancreatic β-cell cytotoxicity is still unclear. Here, we found that MeHg (1-4 μM) significantly decreased insulin secretion and cell viability in pancreatic β-cell-derived RIN-m5F cells. A concomitant elevation of mitochondrial-dependent apoptotic events was observed, including decreased mitochondrial membrane potential and increased proapoptotic (Bax, Bak, p53)/antiapoptotic (Bcl-2) mRNA ratio, cytochrome c release, annexin V-Cy3 binding, caspase-3 activity, and caspase-3/-7/-9 activation. Exposure of RIN-m5F cells to MeHg (2 μM) also induced protein expression of endoplasmic reticulum (ER) stress-related signaling molecules, including C/EBP homologous protein (CHOP), X-box binding protein (XBP-1), and caspase-12. Pretreatment with 4-phenylbutyric acid (4-PBA; an ER stress inhibitor) and specific siRNAs for CHOP and XBP-1 significantly inhibited their expression and caspase-3/-12 activation in MeHg-exposed RIN-mF cells. MeHg could also evoke c-Jun N-terminal kinase (JNK) activation and reactive oxygen species (ROS) generation. Antioxidant N-acetylcysteine (NAC; 1mM) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 100 μM) markedly prevented MeH-induced ROS generation and decreased cell viability in RIN-m5F cells. Furthermore, pretreatment of cells with SP600125 (JNK inhibitor; 10 μM) or NAC (1 mM) or transfection with JNK-specific siRNA obviously attenuated the MeHg-induced JNK phosphorylation, CHOP and XBP-1 protein expression, apoptotic events, and insulin secretion dysfunction. NAC significantly inhibited MeHg-activated JNK signaling, but SP600125 could not effectively reduce MeHg-induced ROS generation. Collectively, these findings demonstrate that the induction of ROS-activated JNK signaling is a crucial mechanism underlying MeHg-induced mitochondria- and ER stress-dependent apoptosis, ultimately leading to β-cell death.

4-Methoxydalbergione Inhibits Bladder Cancer Cell Growth via Inducing Autophagy and Inhibiting Akt/ERK Signaling Pathway.

Bladder cancer (BC) ranks the fourth in incidence in cancers of men and is a common malignant tumor in women. 4-Methoxydalbergione (4MOD), which is purified from Dalbergia sissoo Roxb, has been shown to have anticancer capacity for osteosarcoma and astroglioma. The role of 4MOD in bladder cancer has not been investigated. This study aims to evaluate the anticancer effect of 4MOD in BC cells and its possible mechanisms. The two human bladder cancer cell lines J82 and UMUC3 were used to evaluate the proliferation inhibitory effect of 4MOD by CCK8 and clonogenic assays. The migratory and invasive ability of tumor cells was examined by scratch test and transwell assay. Apoptosis was detected by flow cytometry and TUNEL assays. The autophagy-related molecules including Beclin-1 and LC3 were examined by Western blotting analysis. Furthermore, the RT-PCR was used to detect the mRNA expression of LC3. 4MOD repressed cell proliferation, migration, invasion and induced cell apoptosis in a concentration-dependent manner. The IC50 values of J82 and UMUC3 were 8.17 and 14.50 μM respectively. The mRNA and protein expression ratio of light chain 3-II (LC3-II)/LC3-I and the protein expression of Beclin-1 were increased when the BC cells were treated with 4MOD. The treatment of 4MOD attenuated the phosphorylation of Akt and ERK in the BC cells. We revealed that the 4MOD inhibits BC cells growth by inducing autophagy and inhibiting Akt/ERK signaling pathway. Our study provides new insights into the mechanism by which 4MOD weakens the proliferation of BC cells. This study demonstrates that 4MOD provided a lead compound for the development of novel compound with potent anticancer effect on BC cells.

B cell receptor signatures associated with strong and poor SARS-CoV-2 vaccine responses

ABSTRACT Breakthrough infection of SARS-CoV-2 is a serious challenge, as increased infections were documented in fully-vaccinated individuals. Recipients with poor antibody response are highly vulnerable to reinfection, whereas those with strong antibody responses achieve sterilizing immunity. Thus far, biomarkers associated with levels of vaccine-elicited antibody response are still lacking. Here, we studied the antibody response of age- and gender-controlled healthy cohort, who received inactivated SARS-CoV-2 vaccines and profiled the B cell receptor repertoires in longitudinally consecutive samples. Upon vaccination, all vaccinated individuals displayed a convergent antibody response with shared common antibody clones and public neutralizing antibodies. Strikingly, poor vaccine-responders are distinguishable from strong vaccine-responders by a biased V-usage before vaccination and IgG to IgM mRNA ratio. These findings reveal molecular signatures associated with the different levels of vaccine-induced antibody response, which could be further developed into biomarkers for the design of vaccination strategies.

Evaluation effects of ascorbic acid leads to activate and induce osteogenic protein marker expression: in silico and in-vitro study

Background: Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into several cell types including chondrocytes and osteocytes. The process of differentiation into such tissues is aided by transcription factors and collagen gene markers. Collagen biosynthesis is influenced by the presence of vascular endothelial growth factor (VEGF) and ascorbic acid cofactors of the prolyl hydroxylase 2 (PHD2) enzyme found in human cell receptors. The focus of this study is evaluation of how in silico methods can elucidate the effect of ascorbic acid on PHD2 and its expression. This study aimed to evaluate the role of ascorbic acid in its interaction with PHD2 (in silico) and the expression of type 1 collagen (COL1), type 2 collagen (COL2), and VEGF. Methods: The technique of in silico molecular docking consisted of three steps: preparation of PHD2 receptors using AutoDock tools, preparation of ligand ascorbic acid using AutoDock tools, and molecular docking using AutoDock Vina. The results of in silico molecular docking were visualized using pyMOL and BIOVIA discovery studio. Furthermore, in vitro MSCs from adipose tissue were isolated and characterized based on protein marker expression. The effect of ascorbic acid supplementation (50 and 100 mg/mL) on stem cell differentiation was evaluated based on the mRNA ratio of VEGF/COL1, VEGF/COL2, and COL2/COL1. Results: Molecular docking of ligand ascorbic acid with PHD2 resulted in nine poses. The third pose was chosen because it had an affinity of -5.7 kcal/mol and a root-mean-square deviation (RMSD) of 1.96, indicating a valid docking result. Meanwhile, the 2D conformer showed van der Waals bonds on Tyr303 and Try329 residues. MSCs were differentiated into adipocytes, chondrocytes, and osteocytes. Regarding the marker ratio of gene expression, at 50 mg/ml ascorbic acid there was an increase in the VEGF/COL1 and VEGF/COL2 ratios and a decrease in the COL2/COL1 ratio compared with the control; at 100 mg/ml ascorbic acid there was a decrease in both ratios (VEGF/COL1 and COL2/COL1), and the VEGF/COL2 ratio continued to increase. Conclusions: In silico molecular docking between PHD2 and ascorbic acid, which was studied via bioinformatics, resulted in a stable interaction with good ligand position. Further in vitro studies would support the influence of ascorbic acid on transcription factors, collagen gene markers, and the differentiation of MSCs.

Influence of N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine on the Expression of Neurotrophic Factors in Neuronal Differentiated Cultures of Human Induced Pluripotent Stem Cells under Conditions of Oxidative Stress.

Oxidative stress (OS) is implicated in the pathogenesis of several neurodegenerative diseases. We have previously shown that N-acyl dopamines (N-ADA and N-DDA) protect the neural cells of healthy donors and patients with Parkinson’s disease from OS. In this study, we assessed the effects of N-acyl dopamines on the expression of neurotrophic factors in human-induced pluripotent stem cell-derived neuronal cultures enriched with dopaminergic neurons under conditions of OS induced by hydrogen peroxide. We showed that hydrogen peroxide treatment increased BDNF but not GDNF mRNA levels, while it did not affect the secretion of corresponding proteins into the culture medium of these cells. Application of N-acyl dopamines promoted BDNF release into the culture medium. Under conditions of OS, N-DDA also increased TRKB, TRKC and RET mRNA levels. Furthermore, N-acyl dopamines prevented cell death 24 h after OS induction and promoted the expression of antioxidant enzymes GPX1, GPX7, SOD1, SOD2 and CAT, as well as reduced the BAX/BCL2 mRNA ratio. These findings indicate that stimulation of the expression of neurotrophic factors and their receptors may underlie the neuroprotective effects of N-acyl dopamines in human neurons.

P104: BASE EDITING REPAIRS THE HBE MUTATION RESTORING THE PRODUCTION OF NORMAL GLOBIN CHAINS IN SEVERE HBE/β-THALASSEMIA PATIENT HEMATOPOIETIC STEM AND ERYTHROID CELLS

Aims: HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide1. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any β0-thalassemia mutation on the other. There is a reduction in β-globin production, resulting in a relative excess in α-globin chains that leads to ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype shared by 1.5% of the world’s population2. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies rely on non-homologous end joining targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Methods: Adenine base editors (ABEs) circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C transitions. Conversion of the HbE codon to WT through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions. ABEs are able to convert the HbE codon (AAG) to wild-type (GAG), but also to GGG or AGG (Fig A). GGG at codon 26 is found in a naturally occurring haemoglobin, Hb Aubenas3. Heterozygotes have normal red cell indices and are phenotypically normal. We electroporated the latest generation of ABE8 editors4 as mRNA into 3 different severe HbE/β-thalassemia donor HSPCs with sgRNAs targeting the HbE codon. Results: The mean conversion from the HbE codon to a normal or normal variant in unselected cells was 86.2 (SD±8.1%, Fig B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure C), indicating a reduction in the excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Fig D & E). In serial NSG-murine xenotransplantation experiments, base edited cells were found to persist in secondary transplants, showing editing is possible in long-term HSCs (mean editing efficiency 34.5%, Fig F). Potential off-target effects were assessed in-vitro by CIRCLE-seq5; most candidate sites were in intergenic and intronic regions (Fig G & H). The top 250 sites were sequenced using deep targeted NGS. Only 5 sites showed OT deaminations at low levels in patient cells (mean 1.5%). We developed a machine learning model to assess potential OT-effects on chromatin accessibility, at all candidate sites in 49 different blood cell types6. Only 17 potential edits were predicted to result in a significant change in chromatin state (Fig I). 3 of these were in the top 250 sites sequenced previously, and none showed deamination in-vivo. Conclusion: Together these data provide robust evidence for base editing being used as an effective and safe therapeutic strategy for HbE/β-thalassemia.

Using Deep Learning Algorithm: The Study of Sperm Head Vacuoles and Its Correlation with Protamine mRNA Ratio.

Objective It is necessary to evaluate fertility effective agents to predict assisted reproduction outcomes. This study was designed to examine sperm vacuole characteristics, and its association with sperm chromatin status and protamine-1 (PRM1) to protamine-2 (PRM2) ratio, to predict assisted pregnancy outcomes. Materials and Methods In this experimental study, ninety eight semen samples from infertile men were classified based on Vanderzwalmen’s criteria as follows: grade I: no vacuoles; grade II: <2 small vacuoles; grade III: <1 large vacuole and grade IV: large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscopy (SEM). The chromatin integrity, condensation, viability and acrosome integrity, and protamination status were evaluated for vacuolated samples by toluidine blue (TB) staining, aniline blue, triple staining, and CMA3 staining, respectively. Also, Protamine-1 and protamine-2 genes expression was analysed by reverse transcription-quantitative polymerase chain reaction (PCR). The assisted reproduction outcomes were also followed for each cycle. Results The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (P=0.03 and P=0.02, respectively), and also, protamine-deficient (P=0.04 and P=0.03, respectively). The percentage of reacting acrosomes was significantly higher in the grades III and IV spermatozoa in comparison with normal group. The vacuolated spermatozoa with grade IV showed a high protamine mRNA ratio (PRM-2 was underexpressed, P=0.01). In the IVF cycles, we observed a negative association between sperm head vacuole and fertilization rate (P=0.01). This negative association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P=0.04 and P=0.03, respectively). ConclusionThe results of our study highlight the importance sperm parameters such as sperm head vacuole characteristics, particularly those parameters with the potency of reflecting protamine-deficiency and in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) outcomes predicting.