Potential Target Sequences Research Articles

Investigating the role of aae-miR-34-5p in the regulation of juvenile hormone biosynthesis genes in the mosquito Aedes aegypti

Juvenile hormone (JH) controls the development and reproduction of insects. Therefore, a tight regulation of the expression of JH biosynthetic enzymes is critical. microRNAs (miRNAs) play significant roles in the post-transcriptional regulation of gene expression by interacting with complementary sequences in target genes. Previously, we reported that several miRNAs were differentially expressed during three developmental stages of Aedes aegypti mosquitoes with different JH levels (no JH, high JH, and low JH). One of these miRNAs was aae-miR-34-5p. In this study, we identified the presence of potential target sequences of aae-miR-34-5p in the transcripts of some genes encoding JH biosynthetic enzymes. We analysed the developmental expression patterns of aae-miR-34-5p and the predicted target genes involved in JH biogenesis. Increases in miRNA abundance were followed, with a delay, by decreases in transcript levels of target genes. Application of an inhibitor and a mimic of aae-miR-34-5p led respectively to increased and decreased levels of thiolase transcripts, which is one of the early genes of JH biosynthesis. Female adult mosquitoes injected with an aae-miR-34-5p inhibitor exhibited significantly increased transcript levels of three genes encoding JH biosynthetic enzymes, acetoacetyl-CoA thiolase (thiolase), farnesyl diphosphate phosphatase, and farnesal dehydrogenase. Overall, our results suggest a potential role of miRNAs in JH production by directly targeting genes involved in its biosynthesis.

The PBX1/miR-141-miR-200a/EGR2/SOCS3 Axis; Integrative Analysis of Interaction Networks to Discover the Possible Mechanism of MiR-141 and MiR-200a-Mediated Th17 Cell Differentiation.

Overexpression of miR-141 and miR-200a is known to be associated with the differentiation of T helper 17 (Th17) cells, which are key players in the pathophysiology of autoimmune disorders. However, the function and governing mechanism of these two microRNAs (miRNAs) in Th17 cell skewing are poorly defined. The aim of the present study was to identify the common upstream transcription factors and downstream target genes of miR-141 and miR-200a to obtain a better insight into the possible dysregulated molecular regulatory networks driving miR-141/miR-200a-mediated Th17 cell development. A consensus-based prediction strategy was applied for in-silico identification of potential transcription factors and putative gene targets of miR-141 and miR-200a. Thereafter, we analyzed the expression patterns of candidate transcription factors and target genes during human Th17 cell differentiation by quantitative real-time PCR and examined the direct interaction between both miRNAs and their potential target sequences using dual-luciferase reporter assays. According to our miRNA-based and gene-based interaction network analyses, pre-B cell leukemia homeobox (PBX1) and early growth response 2 (EGR2) were respectively taken into account as the potential upstream transcription factor and downstream target gene of miR-141 and miR-200a. There was a significant overexpression of the PBX1 gene during the Th17 cell induction period. Furthermore, both miRNAs could directly target EGR2 and inhibit its expression. As a downstream gene of EGR2, the suppressor of cytokine signaling 3 (SOCS3) was also downregulated during the differentiation process. These results indicate that activation of the PBX1/miR-141-miR-200a/EGR2/SOCS3 axis may promote Th17 cell development and, therefore, trigger or exacerbate Th17-mediated autoimmunity.

Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean.

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean (Glycinemax). The Streptococcuspyogenes Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5'-NGG-3' (SpCas9) or 5'-TTTN-3' (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from Staphylococcus aureus (SaCas9) recognizes the PAM sequence 5'-NNGRRT-3' (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5'-AAGGGT-3', 5'-GGGGAT-3', 5'-TTGAAT-3', and 5'-TAGGGT-3' in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.

Upregulation of Reg IV and Hgf mRNAs by Intermittent Hypoxia via Downregulation of microRNA-499 in Cardiomyocytes.

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and is a risk factor for cardiovascular disease (CVD) and insulin resistance/Type 2 diabetes. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to experimental IH or normoxia for 24 h to analyze the mRNA expression of several cardiomyokines. We found that the mRNA levels of regenerating gene IV (Reg IV) and hepatocyte growth factor (Hgf) in H9c2 and P19.CL6 cardiomyocytes were significantly increased by IH, whereas the promoter activities of the genes were not increased. A target mRNA search of microRNA (miR)s revealed that rat and mouse mRNAs have a potential target sequence for miR-499. The miR-499 level of IH-treated cells was significantly decreased compared to normoxia-treated cells. MiR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into P19.CL6 cells, and the IH-induced upregulation of the genes was abolished by introduction of the miR-499 mimic, but not by the miR-499 mimic NC. These results indicate that IH stress downregulates the miR-499 in cardiomyocytes, resulting in increased levels of Reg IV and Hgf mRNAs, leading to the protection of cardiomyocytes in SAS patients.

Upregulation of REG IV gene in human intestinal epithelial cells by lipopolysaccharide via downregulation of microRNA-24.

The pathophysiology of inflammatory bowel diseases (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members (REG Iα, REG Iβ and REG IV) are expressed in Crohn's disease (CD) and ulcerative colitis (UC) and involved as proliferative mucosal factors in IBD. We revealed that REG Iα and REG Iβ were induced in cell culture system by IL‐6/IL‐22. Although REG IV was upregulated in IBD biopsy samples, the upregulation of REG IV was not at all induced in cell culture by autoimmune‐related cytokines such as IL‐6, IL‐22 and TNFα. Here, we analysed REG IV expression in LS‐174 T and HT‐29 human intestinal epithelial cells by real‐time RT–PCR and elisa. REG IV expression was induced by lipopolysaccharide (LPS). However, LPS did not activate REG IV promoter activity. As the LPS‐induced upregulation of REG IV was considered to be regulated post‐transcriptionally, we searched targeted microRNA (miR), which revealed that REG IV mRNA has a potential target sequence for miR‐24. We measured the miR‐24 level of LPS‐treated cells and found that the level was significantly lower. The LPS‐induced increase of REG IV mRNA was abolished by the introduction of miR‐24 mimic but not by non‐specific control RNA.

1317-P: Intermittent Hypoxia Increased the Expression of DBH and PNMT in Neuronal Cells via MicroRNA-Mediated Mechanism

Sleep apnea syndrome (SAS) , characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) , is a risk factor for hypertension and insulin resistance. We have reported correlation between IH and insulin resistance. However, why hypertension is induced by IH has been elusive. Here, we investigated the effect of IH on the expression of catecholamine metabolizing enzymes using in vitro IH system. Human and mouse neuronal cells, NB-1 and Neuro-2a, were exposed to IH (70 cycles of 5 min hypoxia [1% O2] and min normoxia [21% O2]) or normoxia for 24 hours. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in both NB-1 (P=0.0271 and P<0.0001, respectively) and Neuro-2a (P=0.0135 and P=0.0341, respectively) . Western blot showed that the expression of DBH and PNMT in the NB-1 cells significantly increased by IH (P=0.0296 and P=0.0007, respectively) . We next prepared reporter plasmids containing human DBH (-1018˜+10) and PNMT (-600˜+67) upstream of luciferase reporter gene and transfected them into NB-1 cells. The promoter activities of these genes were not increased by IH. Target mRNA search of microRNA (miR) s using MicroRNA.org program revealed that the mRNAs have a potential target sequence for miR-375. The miR-375 level of IH-treated cells was significantly decreased than that of normoxia-treated cells (P=0.0222) . To clarify the role of miR-375, miR-375 mimic and non-specific control RNA (miR-375 mimic NC) were introduced into NB-1 cells. The IH-induced up-regulation of mRNAs of DBH and PNMT was abolished by introduction of miR-375 mimic but not by miR-375 mimic NC. Theses results strongly suggest that IH stress down-regulates the miR-375 in neuronal cells, resulting in the increased levels of DBH and PNMT via the inhibition of the miR-375-mediated mRNA degradation, leading SAS patients to hypertension. Disclosure S.Takasawa: None. R.Shobatake: None. Y.Takeda: None. T.Uchiyama: None. A.Yamauchi: None. M.Makino: None. K.Asai: None. H.Ota: None.

Intermittent Hypoxia Upregulates the Renin and Cd38 mRNAs in Renin-Producing Cells via the Downregulation of miR-203.

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and it is a known risk factor for hypertension. The upregulation of the renin-angiotensin system has been reported in IH, and the correlation between renin and CD38 has been noted. We exposed human HEK293 and mouse As4.1 renal cells to experimental IH or normoxia for 24 h and then measured the mRNA levels using a real-time reverse transcription polymerase chain reaction. The mRNA levels of Renin (Ren) and Cd38 were significantly increased by IH, indicating that they could be involved in the CD38-cyclic ADP-ribose signaling pathway. We next investigated the promotor activities of both genes, which were not increased by IH. Yet, a target mRNA search of the microRNA (miRNA) revealed both mRNAs to have a potential target sequence for miR-203. The miR-203 level of the IH-treated cells was significantly decreased when compared with the normoxia-treated cells. The IH-induced upregulation of the genes was abolished by the introduction of the miR-203 mimic, but not the miR-203 mimic NC negative control. These results indicate that IH stress downregulates the miR-203 in renin-producing cells, thereby resulting in increased mRNA levels of Ren and Cd38, which leads to hypertension.

Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA.

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

378-P: Upregulation of Regenerating Gene IV and Hepatocyte Growth Factor in Cardiomyocytes by Intermittent Hypoxia and Its MicroRNA-Mediated Mechanism

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for cardiovascular disease (CVD) and insulin resistance. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes either to sustained hypoxia (1% O2 [SH]), 70 cycles/24 h of IH (5 min SH/10 min normoxia [21% O2]), mimicking SAS patients, or normoxia for 24 h, analyzed mRNA expression and found that IH significantly increased the mRNA levels of regenerating gene (Reg) IV and hepatocyte growth factor (Hgf). Reg IV and Hgf in IH-treated cell medium were also increased. In order to analyze the IH-induced expression mechanisms of Reg IV and Hgf genes, we prepared reporter plasmids containing Reg IV and Hgf upstream of luciferase reporter gene, and transfected them into P19.CL6 cardiomyocytes. The promoter activities of the genes were not increased by IH. Target mRNA search of microRNA (miR)s revealed that both the mRNAs have a potential target sequence for miR-499. The miR-499 level of IH-treated cells was significantly decreased than that of normoxia-treated cells. To clarify the role of miR-499, miR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into P19.CL6 cells, and the IH-induced up-regulation of Reg IV and Hgf was abolished by introduction of miR-499 mimic. We also measured apoptosis of IH/SH-stimulated P19.CL6 cells by TUNEL method and found that SH but not IH increased apoptosis and that Reg IV and Hgf in cultured medium significantly reduced. The addition of Reg IV and/or Hgf in the culture medium attenuated the SH-induced apoptosis. These results indicate that IH stress increased levels of Reg IV and Hgf mRNA via the inhibition of the miR-499-mediated mRNA degradation, leading to protect cardiomyocytes against hypoxia -induced apoptosis. Disclosure S. Takasawa: None. A. Itaya-hironaka: None. A. Yamauchi: None. M. Makino: None. S. Sakuramoto-tsuchida: None. T. Uchiyama: None. Y. Takeda: None. Y. Kyotani: None. H. Ota: None.

Nucleosome binding by the pioneer transcription factor OCT4

Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.

Hepatitis B virus X protein upregulates alpha-fetoprotein to promote hepatocellular carcinoma by targeting miR-1236 and miR-329.

Hepatitis B virus (HBV) infection is the most common cause of hepatocellular carcinoma (HCC) worldwide, wherein the expression of alpha-fetoprotein (AFP) is reactivated to promote tumorgenesis. Hepatitis B virus X protein (HBx) protein encoded by the HBV virus X gene has been considered to be oncogenic and implicated in hepatocarcinogenesis. However, the relationship between HBx and abnormal AFP expression in HCC is yet to be fully understood. To explore the potential regulation of HBx on AFP re-expression in HCC, 97 HCC samples of different etiologies were analyzed, and extremely higher serum AFP levels were found in patients with HBsAg+ . Analyses of HBV-related HCC specimens showed that the expression of AFP was negatively correlated with the levels of miR-1236 and miR-329. Further analyses indicated that HBx promotes the expression of AFP by orchestrating the levels of miR-1236 and miR-329 both in vitro and in vivo. Specifically, miR-1236 and miR-329 bind to the potential target sequences in AFP mRNA 3'-untranslated region to suppress its expression. HBx transfection resulted in the significant decrement of these microRNAs and increment of AFP expression. Moreover, AFP promotes the proliferation of hepatoma cells and attenuates the proapoptotic effect of chemotherapy agents. These findings revealed a novel regulatory mechanism of HBx on the abnormal AFP expression in HCC, which may provide a therapeutic approach for combating HBV-related HCC by targeting the regulation of AFP expression.

1771-P: Upregulation of RETN, TNFα, and CCL2 mRNAs in Adipocytes by Intermittent Hypoxia and Its MicroRNA-Mediated Mechanism

Sleep apnea syndrome (SAS), characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), is a risk factor for insulin resistance/type 2 diabetes. IH is considered to cause adipose tissue inflammation, leading to insulin resistance. In this study, we exposed mouse 3T3-L1 and human SW872 adipocytes to experimental IH (1% O2, 5% CO2 [5 min], 21% O2, 5% CO2 [10 min]) or normoxia (21% O2, 5% CO2) for 24 h, and analyzed mRNA expression of adipokines (leptin, adiponectin, resistin [RETN], tumor necrosis factor α [TNFα], IL-6, and C-C motif chemokine ligand 2 [CCL2]). We found that the mRNA levels of RETN, TNFα and CCL2 in SW872 cells (P=0.0257, P=0.0312, and P<0.0001, respectively) and 3T3-L1 adipocytes (P=0.0498, P=0.0237, and P=0.0002, respectively) were significantly increased by IH. RETN, TNFα, and CCL2 in IH-treated cell medium were also increased (P=0.0256, P=0.0215, and P<0.0001, respectively). We next prepared reporter plasmids containing human RETN (-979∼+20), TNFα (-966∼+19) and CCL2 (-3455∼+25) upstream of luciferase reporter gene, and transfected them into SW872 cells. The promoter activities of these genes were not increased by IH. Target mRNA search of microRNA (miR)s using MicroRNA.org program revealed that all the mRNAs have a potential target sequence for miR-452. The miR-452 level of IH-treated cells was significantly decreased than that of normoxia-treated cells (P=0.0458). To clarify the role of miR-452, miR-452 mimic and non-specific control RNA (miR-452 mimic NC) were introduced into SW872 cells, and the IH-induced up-regulation of RETN, TNFα, and CCL2 was abolished by introduction of miR-452 mimic but not by miR-452 mimic NC. These results indicate that IH stress down-regulates the miR-452 in adipocytes, resulting in the increased levels of RETN, TNFα, and CCL2 mRNA via the inhibition of the miR-452-mediated mRNA degradation, leading SAS patients to insulin resistance. Disclosure T. Uchiyama: None. A. Itaya-Hironaka: None. A. Yamauchi: None. M. Makino: None. S. Sakuramoto-Tsuchida: None. R. Shobatake: None. H. Ota: None. M. Takeda: None. C. Ohbayashi: None. S. Takasawa: None.

1959-P: Upregulation of PYY, GLP-1, and NTS by Intermittent Hypoxia in Enteroendocrine Cells

The patients with sleep apnea syndrome (SAS) are exposed to intermittent hypoxia (IH) during sleep. Although accumulating evidence indicates a strong relationship between SAS and obesity, we previously demonstrated that IH up-regulates the mRNA levels of anorexigenic peptides POMC and CART via GATA transcription factors in human neuronal cells (Int. J. Biochem. Cell Biol. 95, 100, 2018). Appetite is regulated not only by the central nervous system but also by gastrointestinal peptides. Here, we investigated the effect of IH on the appetite-inhibiting gut hormone expression using in vitro IH system. Human and mouse enteroendocrine cells, Caco-2 and STC-1, were exposed to IH [64 cycles of 5 min hypoxia (1% O2) and 10 min normoxia (21% O2)] or normoxia for 24 hours. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of PYY, GLP-1, and neurotensin (NTS) in both Caco-2 and STC-1. ELISA showed that the concentrations of PYY, GLP-1, and NTS in the culture medium were significantly increased by IH. To determine whether the IH-induced increases in PYY, GLP-1, and NTS in enteroendocrine cells were caused by transcriptional activation, reporter plasmids for PYY, GCG, which encodes a preprotein, part of which is cleaved into GLP-1, and NTS were transfected into Caco-2, and the luciferase activity was measured after the IH treatment. The promoter activities of PYY, GCG and NTS were not increased by IH. We then searched targeted miRNA and found that PYY, GLP-1 and NTS mRNAs have potential target sequences for miR-96, -527, and -2116. We measured the levels of the miRNAs and found that the levels of the miRNAs were not decreased by IH. Furthermore, the mRNA levels of PYY, GLP-1, and NTS were significantly up-regulated even in normoxia by trichostatin A and were significantly decreased even in IH by 5-azacytidine, suggesting that IH increases PYY, GLP-1, and NTS mRNAs via alterations in the chromatin structure in enteroendocrine cells. IH might have an anorexigenic influence on the enteric nervous system. Disclosure R. Shobatake: None. H. Ota: None. A. Itaya-Hironaka: None. A. Yamauchi: None. M. Makino: None. S. Sakuramoto-Tsuchida: None. T. Uchiyama: None. N. Takahashi: None. S. Ueno: None. K. Sugie: None. S. Takasawa: None.

Intermittent Hypoxia Up-Regulates CCL2, RETN, and TNFα mRNAs in Adipocytes via Down-regulation of miR-452.

Sleep apnea syndrome (SAS), characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), is a risk factor for insulin resistance. Recently, IH is considered to independently cause adipose tissue inflammation/dysfunction, leading to worsening insulin resistance; however, the detailed mechanism remains unknown. We exposed mouse 3T3-L1 and human SW872 adipocytes to experimental IH or normoxia for 24 h, and analyzed mRNA expression of several adipokines. We found that the mRNA levels of RETN, TNFα, and CCL2 in SW872 and 3T3-L1 adipocytes were significantly increased by IH, whereas the promoter activities of these genes were not increased. A target mRNA search of microRNA (miR)s revealed that all human mRNAs have a potential target sequence for miR-452. The miR-452 level of IH-treated cells was significantly decreased compared to normoxia-treated cells. MiR-452 mimic and non-specific control RNA (miR-452 mimic NC) were introduced into SW872 cells, and the IH-induced up-regulation of the genes was abolished by introduction of the miR-452 mimic but not by the miR-452 mimic NC. These results indicate that IH stress down-regulates the miR-452 in adipocytes, resulting in increased levels of RETN, TNFα, and CCL2 mRNAs, leading to insulin resistance in SAS patients.

3′UTR variants of TNS3, PHLDB1, NTN4, and GNG2 genes are associated with IgA nephropathy risk in Chinese Han population

BackgroundImmunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis and is characterized by mesangial cell proliferation and agglomeration of the mesangial matrix. MethodsIn this study, we aimed to explore the role of TNS3, PHLDB1, NTN4, and GNG2 3′untranslated region (3′UTR) polymorphisms with the risk of IgAN in a Chinese Han cohort. A logistic recession model was used to calculate the effects of candidate single nucleotide polymorphism (SNP) on IgAN risk after adjusting age and gender difference. In silico prediction was conducted to identify potential functions of SNPs. ResultsThe analysis revealed a significant relationship between the homozygotic genotype for NTN4 rs1362970 A/A and higher risk of IgAN (p = 0.003). Statistically significant associations were found when the sample was stratified by gender and Lee's grade. As a result, NTN4 rs1362970 A/A and GNG2 rs3204008 G/G genotypes were associated with enhanced IgAN risk in males (p = 0.006, p = 0.023, respectively), and the association between the PHLDB1 rs7389 G/T genotype and higher IgAN risk was found in females (p = 0.008). In the Lee's grade III–V subgroup, the rs1369270 in NTN4 was significantly correlated with the risk of IgAN (p = 0.004). Bioinformatics prediction suggested that rs1362970 within NTN4 3′UTR was located in the potential target sequence of miR-483-5p. ConclusionsOur research confirmed that NTN4, GNG2, and PHLDB1 gene polymorphisms were implicated in IgAN susceptibility in Chinese Han population. Further research should be conducted to investigate and validate the mechanism by which the above-mentioned polymorphisms affect IgAN.

Genome-informed design of LAMP assays for specific detection of aster yellows phytoplasmas

Genome sequence data is becoming more available and represents a source of potential target sequences for specific molecular tests to verify the feasibility and efficiency of genome-inform test design the attempt to identify novel specific sequences in the publicly available genomes of aster yellows phytoplasmas and use them to design primers for a spcific LAMP assays. Coupling RUCS automated analysis with PrimerExplorer V5 primer designer and applying criteria for selection of assays at each stage of the process resulted in 13 promising LAMP assays targeting four newly identified regions specific to aster yellows phytoplasmas.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Bta-miR-2411 attenuates bovine viral diarrhea virus replication via directly suppressing Pelota protein in Madin-Darby bovine kidney cells

MicroRNAs (miRNAs) are endogenous ∼22 nt noncoding RNAs that control the translation initiation and stability of target genes in a sequence-specific manner and, thus, play important regulatory roles in animals and plants. Homologs of Dom34, called Pelota or PELO, are broadly conserved in eukaryotes and archaea. Biochemical and genetic studies indicate that eukaryotic Dom34/Pelota plays an important role in cell division, differentiation of germline stem cells, and stem cell self-renewal by controlling the expression of specific genes at the translational level. Additionally, it is reported that Pelota is specifically required for high efficiency synthesis of proteins in numerous viruses. In earlier studies, we found the Bos taurus bta-miR-2411 (shortly miR-2411 herein) was significantly upregulated by more than 2.1 times in bovine viral diarrhea virus (BVDV) strain NADL-infected Madin-Darby bovine kidney (MDBK) cells after 8 h post-infection (pi) compared to normal MDBK cells without BVDV infection. Moreover, miR-2411 overexpression significantly reduced the BVDV E1 mRNA level and viral titer. Nevertheless, the mechanisms of miR-2411 attenuating on viral replication remain unclear. Here, we report that miR-2411 as a novel microRNA regulates BVDV NADL replication via directly targeting the Pelota gene in MDBK cells. We investigated whether the potential target sequences of miR-2411, located in the Pelota 3′UTR, and miR-2411 agomir transfection attenuated Pelota mRNA and protein levels. Indeed, upon miR-2411 overexpression, BVDV NADL replication was prevented. Importantly, BVDV NADL replication levels were reversed to normal levels as a result of the Pelota rescuing experiment even though miR-2411 was existent. Overall, we profiled the unique role of miR-2411 in regulating BVDV NADL replication and provided a novel strategy for generalized inhibition of viral infection.

Up-regulation of selenoprotein P and HIP/PAP mRNAs in hepatocytes by intermittent hypoxia via down-regulation of miR-203

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human hepatocytes (JHH5, JHH7, and HepG2) to experimental IH or normoxia for 24h, measured mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR), and found that IH significantly increased the mRNA levels of selenoprotein P (SELENOP) — a hepatokine — and hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) — one of REG (Regenerating gene) family. We next investigated promoter activities of both genes and discovered that they were not increased by IH. On the other hand, a target mRNA search of micro RNA (miRNA) revealed that both mRNAs have a potential target sequence for miR-203. The miR-203 level of IH-treated cells was significantly lower than that of normoxia-treated cells. Thus, we introduced miR-203 inhibitor and a non-specific control RNA (miR-203 inhibitor NC) into HepG2 cells and measured the mRNA levels of SELENOP and HIP/PAP. The IH-induced expression of SELENOP and HIP/PAP was abolished by the introduction of miR-203 inhibitor but not by miR-203 inhibitor NC. These results demonstrate that IH stress up-regulates the levels of SELENOP in human hepatocytes to accelerate insulin resistance and up-regulates the levels of HIP/PAP mRNAs to proliferate such hepatocytes, via the miR-203 mediated mechanism.

VSEARCH: a versatile open source tool for metagenomics.

BackgroundVSEARCH is an open source and free of charge multithreaded 64-bit tool for processing and preparing metagenomics, genomics and population genomics nucleotide sequence data. It is designed as an alternative to the widely used USEARCH tool (Edgar, 2010) for which the source code is not publicly available, algorithm details are only rudimentarily described, and only a memory-confined 32-bit version is freely available for academic use.MethodsWhen searching nucleotide sequences, VSEARCH uses a fast heuristic based on words shared by the query and target sequences in order to quickly identify similar sequences, a similar strategy is probably used in USEARCH. VSEARCH then performs optimal global sequence alignment of the query against potential target sequences, using full dynamic programming instead of the seed-and-extend heuristic used by USEARCH. Pairwise alignments are computed in parallel using vectorisation and multiple threads.ResultsVSEARCH includes most commands for analysing nucleotide sequences available in USEARCH version 7 and several of those available in USEARCH version 8, including searching (exact or based on global alignment), clustering by similarity (using length pre-sorting, abundance pre-sorting or a user-defined order), chimera detection (reference-based or de novo), dereplication (full length or prefix), pairwise alignment, reverse complementation, sorting, and subsampling. VSEARCH also includes commands for FASTQ file processing, i.e., format detection, filtering, read quality statistics, and merging of paired reads. Furthermore, VSEARCH extends functionality with several new commands and improvements, including shuffling, rereplication, masking of low-complexity sequences with the well-known DUST algorithm, a choice among different similarity definitions, and FASTQ file format conversion. VSEARCH is here shown to be more accurate than USEARCH when performing searching, clustering, chimera detection and subsampling, while on a par with USEARCH for paired-ends read merging. VSEARCH is slower than USEARCH when performing clustering and chimera detection, but significantly faster when performing paired-end reads merging and dereplication. VSEARCH is available at https://github.com/torognes/vsearch under either the BSD 2-clause license or the GNU General Public License version 3.0.DiscussionVSEARCH has been shown to be a fast, accurate and full-fledged alternative to USEARCH. A free and open-source versatile tool for sequence analysis is now available to the metagenomics community.