Flowers Characteristics of Selected Species of Lime-Tree (Tilia spp.) in Terms of miRNA-Based Markers Activity, Mannose Expression and Biological Compounds Content

The significant healing effect of Tilia platyphyllos Scop. and Tilia cordata Mill. flowers are well known. However, the flowers of Tilia tomentosa Moench. are not suitable for harvest due to their toxic effects. To investigate the diverse background of this effect, we applied a functional miRNA-based marker, mannose expression analysis and determined the content of bioactive compounds. Out of nine tested markers, three (miR160, miR167 and miR408) provided reproducible miRNA-based loci and two of them (miR160 and miR398) enabled the acquisition of fingerprinting specific to flower and leaf samples of T. platyphyllos and T. tomentosa. The most pronounced profiling was specific for miR408 marker, the function of which is connected to plant defense and adaptation mechanisms. We confirmed the suitability of microRNA-based markers for polymorphism determination of flowers of selected species of lime-tree. The highest values of antioxidant activity, flavonoids, total polyphenols and phenolic acids content have been reached in silver linden flowers. When comparing the transcription activity of mannose in flowers, more than 30 times higher levels of mannose transcripts for the silver linden flowers was observed.

Development of a method for evaluating the mRNA transcription activity of influenza virus RNA-dependent RNA polymerase through real-time reverse transcription polymerase chain reaction

Background The development of an influenza RNA-dependent RNA polymerase (RdRp) inhibitor is required; therefore, a method for evaluating the activity of influenza RdRp needs to be developed. The current method uses an ultracentrifuge to separate viral particles and quantifies RdRp activity with radioisotope-labeled nucleosides, such as 32P-GTP. This method requires special equipment and radioisotope management, so it cannot be implemented in all institutions. We have developed a method to evaluate the mRNA transcription activity of RdRp without using ultracentrifugation and radioisotopes. Results RdRp was extracted from viral particles that were purified from the culture supernatant using anionic polymer-coated magnetic beads that can concentrate influenza virus particles from the culture supernatant in approximately 30 min. A strand-specific real-time reverse transcription polymerase chain reaction (RT-PCR) method was developed based on reverse transcription using tagged primers. RT primers were designed to bind to a sequence near the 3' end of mRNA containing a poly A tail for specific recognition of the mRNA, with an 18-nucleotide tag attached to the 5' end of the sequence. The RT reaction was performed with this tagged RT primer, and the amount of mRNA was analyzed using real-time qPCR. Real-time qPCR using the tag sequence as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the mRNA of segments 1, 4, and 5. The temperature, reaction time, and Mg2+ concentration were determined to select the optimum conditions for in vitro RNA synthesis by RdRp, and the amount of synthesized mRNAs of segments 1, 4, and 5 was determined with a detection sensitivity of 10 copies/reaction. In addition, mRNA synthesis was inhibited by ribavirin triphosphate, an RdRp inhibitor, thus indicating the usefulness of this evaluation method for screening RdRp inhibitors. Conclusion This method makes it possible to analyze the RdRp activity even in a laboratory where ultracentrifugation and radioisotopes cannot be used. This novel method for measuring influenza virus polymerase activity will further promote research to identify compounds that inhibit viral mRNA transcription activity of RdRp.

Breakthrough Science: Hypoxia-Inducible Factors, Oxygen Sensing, and Disorders of Hematopoiesis

Hypoxia-inducible factors (HIF) were discovered as activators of erythropoietin gene transcription in response to reduced O2 availability. O2-dependent hydroxylation of HIFs on proline and asparagine residues regulates protein stability and transcription activity, respectively. Mutations in genes encoding components of the oxygen sensing pathway cause familial erythrocytosis. Several small molecule inhibitors of HIF prolyl hydroxylases are currently in clinical trials as erythropoiesis stimulating agents. HIFs are overexpressed in bone marrow neoplasms, and the development of HIF inhibitors may improve outcome in these disorders.

The E3 Ligase PIAS1 Regulates p53 Sumoylation to Control Stress-Induced Apoptosis of Lens Epithelial Cells Through the Proapoptotic Regulator Bax

Protein sumoylation is one of the most important post-translational modifications regulating many biological processes (Flotho A & Melchior F. 2013. Ann Rev. Biochem. 82:357–85). Our previous studies have shown that sumoylation plays a fundamental role in regulating lens differentiation (Yan et al., 2010. PNAS, 107(49):21034-9.; Gong et al., 2014. PNAS. 111(15):5574–9). Whether sumoylation is implicated in lens pathogenesis remains elusive. Here, we present evidence to show that the protein inhibitor of activated STAT-1 (PIAS1), a E3 ligase for sumoylation, is implicated in regulating stress-induced lens pathogenesis. During oxidative stress-induced cataractogenesis, expression of PIAS1 is significantly altered at both mRNA and protein levels. Upregulation and overexpression of exogenous PIAS1 significantly enhances stress-induced apoptosis. In contrast, silence of PIAS1 with CRISPR/Cas9 technology attenuates stress-induced apoptosis. Mechanistically, different from other cells, PIAS1 has little effect to activate JNK but upregulates Bax, a major proapoptotic regulator. Moreover, Bax upregulation is derived from the enhanced transcription activity of the upstream transcription factor, p53. As revealed previously in other cells by different laboratories, our data also demonstrate that PIAS1 promotes SUMO1 conjugation of p53 at K386 residue in lens epithelial cells and thus enhances p53 transcription activity to promote Bax upregulation. Silence of Bax expression largely abrogates PIAS1-mediated enhancement of stress-induced apoptosis. Thus, our results demonstrated that PIAS1 promotes oxidative stress-induced apoptosis through positive control of p53, which specifically upregulates expression of the downstream proapoptotic regulator Bax. As a result, PIAS1-promoted apoptosis induced by oxidative stress is implicated in lens pathogenesis.

Genetic Findings in a Cohort of Patients with Androgen Insensitivity Syndrome

Background: Androgen insensitivity syndrome (AIS) is a rare X-linked recessive inherited disorder caused by mutations in AR, a gene encoding androgen receptor. The aim of this study was to expand genetic spectrum of AIS.Methods: We performed a retrospective study on consecutive patients diagnosed as AIS from 2010 to 2020 in a single tertiary center. Variant analysis of AR gene was performed by PCR-Sanger sequencing. The pathogenicity of novel variants was evaluated by dual-luciferase reporter assay and immunofluorescence of AR protein in vitro. Results: A total of 19 unrelated 46,XY patients were enrolled, 14 with complete insensitivity syndrome (CAIS) and 5 with partial insensitivity syndrome (PAIS). We identified 19 AR variants: 12 (63.2%) were missense variants and 7 variants (36.8%) resulted in premature stop codon. Eight AR variants were novel, including P15Afs*69, S258Efs*47, W435Gfs*44, C560F, C577W, C580Afs*46, K718X and V819G. Dual-luciferase reporter assay found residual transcription activity of approximately 1% in six novel variants, which may explain the CAIS phenotype. The AR mutant protein (V819G) showed transcription activity of 59%, consistent with mild clinical features in one PAIS patient. Interestingly, another AR mutant protein (K718X) related to CAIS showed increased transcription activity but impaired nuclear localization. Conclusions: We identified eight novel AR variants related to AIS. Both residual transcription activity of AR and nuclear localization of AR protein were associated with the severity of AIS.

Identification of Potential miRNA-mRNA Regulatory Network in Tetralogy of Fallot: A Network Biology Approach

BackgroundTetralogy of Fallot (ToF) is one of the most prevalent and fatal birth defects. Its pathogenesis remains unknown and it’s not easily diagnosed at early stage. Therefore, it’s necessary to explore the critical regulators and molecular mechanism in ToF to find out novel molecular markers for early diagnosis.MethodsThree ToF datasets (GSE35490, GSE40128, GSE36761) were downloaded from the GEO database. The differentially expressed microRNAs (DEMs) and mRNAs (DEGs) were identified between ToF infants and normal infants after data preprocessing, followed by GO and KEGG analysis of DEGs. Then, PPI network and modular analysis were performed to identify the hub genes. Ultimately, potential miRNA-mRNA pathways in ToF were constructed based on the integrated data.Results22 overlapping DEMs were found in the GES35490 and GSE40128. Simultaneous, 181 intersected genes were found and identified as the significant DEGs including 84 down-regulated DEGs and 97 up-regulated DEGs. Further, 20 hub genes ranked by top 10% with high connectivity, including STAT3, FBN1, RUNX2, were found by PPT network and modular analysis. Furthermore, GO and KEGG analysis showed these DEGs were linked to cell cycle, apoptotic process, transcription activity and FOX signaling pathway. Additionally, we establish three potential miRNA-mRNA pathway, including miR-22-STAT3, miR-336/miR486-FBN1, miR-222-RUNX2, which can participate in the formation of ToF by cell cycle and transcription activity.ConclusionsThree miRNA-mRNA pathways were established and indicated cell cycle and transcription activity may be involved in the pathogenesis of ToF. Our study provided a novel bio-marker for early diagnosis of ToF.

Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions

ABSTRACTDNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 °C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.