Down-Regulated hsa-miR-190b-5p is Correlated With BCL11A Expression in Pediatric β-Thalassemia

Background: Transcription factor BCL11A is a key regulator of hemoglobin switching in adult β-thalassemia. Several microRNAs (miRNAs) involve in the pathology of β-thalassemia by regulations of BCL11A. However, the expressions and regulators of BCL11A in pediatric β-thalassemia were unclear. Methods: 18 pediatric β- thalassemia patients and 11 healthy controls were selected in this study. We applied reverse transcript quantitative real time PCR (RT-PCR) to analyze the expression levels of hsa-miR-190b-5p and γ-globin in pediatric β-thalassemia patients and luciferase activity assay to find out the direct regulations of BCL11A . Correlation between hsa-miR-190b-5p and biochemical indicators, BCL11A was assessed by the Pearson’s correlation test. Results: The expression levels of γ-globin in pediatric β-thalassemia patients were significantly increased. Moreover, the expression levels of hsa-miR-190b-5p were significantly down-regulated in pediatric β-thalassemia patients. Furthermore, hsa-miR-190b-5p was negatively correlated with BCL11A expression in pediatric β-thalassemia patients. Through luciferase activity assay, we found that hsa-miR-190b-5p was directly interacted with BCL11A 3’UTR 499-506 regions. Conclusion: Our results suggested that hsa-miR-190b-5p played key roles in regulating of BCL11A expression, which might provide novel therapies in pediatric patients with β-thalassemia .

TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

OsWRKY93 Dually Functions Between Leaf Senescence and in Response to Biotic Stress in Rice

Cross talking between natural senescence and cell death in response to pathogen attack is an interesting topic; however, its action mechanism is kept open. In this study, 33 OsWRKY genes were obtained by screening with leaf aging procedure through RNA-seq dataset, and 11 of them were confirmed a significant altered expression level in the flag leaves during aging by using the reverse transcript quantitative PCR (RT-qPCR). Among them, the OsWRKY2, OsWRKY14, OsWRKY26, OsWRKY69, and OsWRKY93 members exhibited short-term alteration in transcriptional levels in response to Magnaporthe grisea infection. The CRISPR/Cas9-edited mutants of five genes were developed and confirmed, and a significant sensitivity to M. oryzae infection was observed in CRISPR OsWRKY93-edited lines; on the other hand, a significant resistance to M. oryzae infection was shown in the enhanced expression OsWRKY93 plants compared to mock plants; however, enhanced expression of other four genes have no significant affection. Interestingly, ROS accumulation was also increased in OsWRKY93 enhanced plants after flg22 treatment, compared with the controls, suggesting that OsWRKY93 is involved in PAMP-triggered immune response in rice. It indicated that OsWRKY93 was involved in both flag leaf senescence and in response to fungi attack.

Development and Application of an RT-PCR to Differentiate the Prevalent NA-PRRSV Strains in China

Background: PRRSV features with genetic diversity and high mutation which leads to the emergence of a multiple of circulating virus strains with different virulence. North American (genotype 2) PRRSV (NA-PRRSV) can be divided into classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) according to their genomic characteristics and pathogenicity. So far, the above three subtypes of NA-PRRSV are now circulating in China. Objective and Method: In this study, a reverse transcript polymerase chain reaction (RT-PCR) was established to simultaneously differentiate three subtypes of NA-PRRSV. The established RT-PCR can be applied to PRRSV-infected samples originated from both supernatant of cell culture and pig tissues and showed specificity exclusively to PRRSV. The sensitivity of RT-PCR showed the minimum RNA detection was 0.04ng/µl. Result and Conclusion: The established RT-PCR was next used to differentiate the subtypes of 29 NA-PRRSV isolated in 2016 and the results showed that HP-PRRSV is still the dominant circulating virus strain in the presence of NADC30-like PRRSV in Henan province.

Induction of Anthocyanin Accumulation in Crabapple (Malus cv.) Leaves by Low Temperatures

Anthocyanins are protective pigments that accumulate in plant organs such as fruits and leaves, and are nutritionally valuable components of the human diet. There is thus considerable interest in the factors that regulate synthesis. Malus crabapple leaves are rich sources of these compounds, and in this study we analyzed leaf coloration, anthocyanin levels, and the expression levels of anthocyanin biosynthetic and regulatory genes in three crabapple cultivars (Royalty, Prairifire, and Flame) following various temperature treatments. We found that low temperatures (LTs) promoted anthocyanin accumulation in ‘Royalty’ and ‘Prairifire’, leading to red leaves, but not in ‘Flame’, which accumulated abundant colorless flavonols and retained green colored leaves. Quantitative reverse transcript PCR (RT-PCR) analyses indicated that the expression of several anthocyanin biosynthetic genes was induced by LTs, as were members of the R2R3-MYB, basic helix–loop–helix (bHLH) and WD40 transcription factor families that are thought to act in a complex. We propose that anthocyanin biosynthesis is differentially regulated in the three cultivars by LTs via the expression of members of this anthocyanin regulatory complex.

The CDK Inhibitor p21Cip1/WAF1 Is Induced by FcγR Activation and Restricts the Replication of Human Immunodeficiency Virus Type 1 and Related Primate Lentiviruses in Human Macrophages

ABSTRACT Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1). We have previously shown that aggregation of activating immunoglobulin G Fc receptors (FcγR) by immune complexes inhibits reverse transcript accumulation and integration of HIV-1 and related lentiviruses in monocyte-derived macrophages. Here, we show that FcγR-mediated restriction of HIV-1 is not due to enhanced degradation of incoming viral proteins or cDNA and is associated to the induction of the cyclin-dependent kinase inhibitor p21Cip1/WAF1 (p21). Small interfering RNA-mediated p21 knockdown rescued viral replication in FcγR-activated macrophages and enhanced HIV-1 infection in unstimulated macrophages by increasing reverse transcript and integrated DNA levels. p21 induction by other stimuli, such as phorbol myristate acetate and the histone deacetylase inhibitor MS-275, was also associated with preintegrative blocks of HIV-1 replication in macrophages. Binding of p21 to reverse transcription/preintegration complex-associated HIV-1 proteins was not detected in yeast two-hybrid, pulldown, or coimmunoprecipitation assays, suggesting that p21 may affect viral replication independently of a specific interaction with an HIV-1 component. Consistently, p21 silencing rescued viral replication from the FcγR-mediated restriction also in simian immunodeficiency virus SIVmac- and HIV-2-infected macrophages. Our results point to a role of p21 as an inhibitory factor of lentiviral infection in macrophages and to its implication in FcγR-mediated restriction.

Preparation of the Hydroxyapatite/Collagen Three-Dimensional Scaffold from its Membrane

Effect of osteogenic activities of MG63 on the HAp/Col membrane was examined at day 10 and 14 by reverse-transcript and real-time polymerase chain reactions. Osteogenic activities of MG63 were upregulated by culture them on the HAp/Col membrane in comparison to those on tissue culture polystyrene. The novel three-dimensional HAp/Col scaffold was prepared from the HAp/Col wavy membrane. The cylindrical HAp/Col scaffold was successfully prepared and indicated at least 2.5-times higher compressive strength and Young's modulus compared to the previous HAp/Col composites. The novel scaffold could be useful for regenerative medicine.

The Involvement of Cellular Recombination and Repair Genes in RNA-Mediated Recombination in Saccharomyces cerevisiae

We previously demonstrated that a reverse transcript of a cellular reporter gene (his3-AI) can serve as the donor for gene conversion of a chromosomal his3-ΔMscI target sequence, and that this process requires the yeast recombination gene RAD52. In this study, we examine the involvement of other recombination and repair genes in RNA-mediated recombination, and gain insight into the nature of the recombination intermediate. We find that mutation of the mitotic RecA homologs RAD51, RAD55, and RAD57 increases the rate of RNA-mediated recombination relative to the wild type, and that these gene functions are not required for RNA-mediated gene conversion. Interestingly, RAD1 is required for RNA-mediated gene conversion of chromosomal his3-ΔMscI sequences, suggesting that the cDNA intermediate has a region of nonhomology that must be removed during recombination with target sequences. The observation that both RAD1 and RAD52 are required for RNA-mediated gene conversion of chromosomal but not plasmid sequences indicates a clear difference between these two pathways of homologous RNA-mediated recombination.