Polymerase-endonuclease amplification reaction for large-scale enzymatic production of antisense oligonucleotide

Synthetic oligonucleotides are contaminated with highly homologous failure sequences. Oligonucleotide synthesis is difficult to scale up because it requires expensive equipments, hazardous chemicals, and tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves >100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation, so it has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs

Gene silencing in mammalian cells by PCR-based short hairpin RNA

AbstractRNA interference (RNAi) provides a powerful tool to silence genes in a sequence-specific manner in a variety of systems. However, not all sequences are effective in the RNAi-mediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)-based RNAi strategy for a quick screening of small interfering RNA (siRNA) efficiency. This method utilized a two-step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in specific depressions of exogenous (luciferase, green fluorescent protein and β-galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research

BACKGROUND: An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. RESULTS: In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. CONCLUSION: The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung

MicroRNA-206 regulates surfactant secretion by targeting VAMP-2

AbstractLung surfactant secretion is a highly regulated process. Our previous studies have shown that VAMP-2 is essential for surfactant secretion. In the present study we investigated the role of miR-206 in surfactant secretion through VAMP-2. VAMP-2 was confirmed to be a target of miR-206 by 3′-untranslational region (3′-UTR) luciferase assay. Mutations in the predicated miR-206 binding sites reduced the binding of miR-206 to the 3′-UTR of VAMP-2. miR-206 decreased the expression of VAMP-2 protein and decreased the lung surfactant secretion in alveolar type II cells. In conclusion, miR-206 regulates lung surfactant secretion by limiting the availability of VAMP-2 protein

MiR-20a regulates the PRKG1 gene by targeting its coding region in pulmonary arterial smooth muscle cells

AbstractChronic hypoxia triggers pulmonary vascular remodeling, which is associated with de-differentiation of pulmonary artery smooth muscle cells (PASMC). Here, we show that miR-20a expression is up-regulated in response to hypoxia in both mouse and human PASMC. We also observed that miR-20a represses the protein kinase, cGMP-dependent, type I (PRKG1) gene and we identified two crucial miR-20a binding sites within the coding region of PRKG1. Functional studies showed that miR-20a promotes the proliferation and migration of human PASMC, whereas it inhibits their differentiation. In summary, we provided a possible mechanism by which hypoxia results in decreased PRKG1 expression and in the phenotypic switching of PASMC

Methylation-mediated silencing of PTPRD induces pulmonary hypertension by promoting pulmonary arterial smooth muscle cell migration via the PDGFRB/PLCγ1 axis

OBJECTIVE: Pulmonary hypertension is a lethal disease characterized by pulmonary vascular remodeling and is mediated by abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Platelet-derived growth factor BB (PDGF-BB) is the most potent mitogen for PASMCs and is involved in vascular remodeling in pulmonary hypertension development. Therefore, the objective of our study is to identify novel mechanisms underlying vascular remodeling in pulmonary hypertension. METHODS: We explored the effects and mechanisms of PTPRD downregulation in PASMCs and PTPRD knockdown rats in pulmonary hypertension induced by hypoxia. RESULTS: We demonstrated that PTPRD is dramatically downregulated in PDGF-BB-treated PASMCs, pulmonary arteries from pulmonary hypertension rats, and blood and pulmonary arteries from lung specimens of patients with hypoxic pulmonary arterial hypertension (HPAH) and idiopathic PAH (iPAH). Subsequently, we found that PTPRD was downregulated by promoter methylation via DNMT1. Moreover, we found that PTPRD knockdown altered cell morphology and migration in PASMCs via modulating focal adhesion and cell cytoskeleton. We have demonstrated that the increase in cell migration is mediated by the PDGFRB/PLCγ1 pathway. Furthermore, under hypoxic condition, we observed significant pulmonary arterial remodeling and exacerbation of pulmonary hypertension in heterozygous PTPRD knock-out rats compared with the wild-type group. We also demonstrated that HET group treated with chronic hypoxia have higher expression and activity of PLCγ1 in the pulmonary arteries compared with wild-type group. CONCLUSION: We propose that PTPRD likely plays an important role in the process of pulmonary vascular remodeling and development of pulmonary hypertension in vivo

Cardiolipin externalization mediates prion protein (PrP) peptide 106–126-associated mitophagy and mitochondrial dysfunction

Proper mitochondrial performance is imperative for the maintenance of normal neuronal function to prevent the development of neurodegenerative diseases. Persistent accumulation of damaged mitochondria plays a role in prion disease pathogenesis, which involves a chain of events that culminate in the generation of reactive oxygen species and neuronal death. Our previous studies have demonstrated that PINK1/Parkin-mediated mitophagy induced by PrP106−126 is defective and leads to an accumulation of damaged mitochondria after PrP106−126 treatment. Externalized cardiolipin (CL), a mitochondria-specific phospholipid, has been reported to play a role in mitophagy by directly interacting with LC3II at the outer mitochondrial membrane. The involvement of CL externalization in PrP106−126-induced mitophagy and its significance in other physiological processes of N2a cells treated with PrP106−126 remain unknown. We demonstrate that the PrP106−126 peptide caused a temporal course of mitophagy in N2a cells, which gradually increased and subsequently decreased. A similar trend in CL externalization to the mitochondrial surface was seen, resulting in a gradual decrease in CL content at the cellular level. Inhibition of CL externalization by knockdown of CL synthase, responsible for de novo synthesis of CL, or phospholipid scramblase-3 and NDPK-D, responsible for CL translocation to the mitochondrial surface, significantly decreased PrP106−126-induced mitophagy in N2a cells. Meanwhile, the inhibition of CL redistribution significantly decreased PINK1 and DRP1 recruitment in PrP106−126 treatment but had no significant decrease in Parkin recruitment. Furthermore, the inhibition of CL externalization resulted in impaired oxidative phosphorylation and severe oxidative stress, which led to mitochondrial dysfunction. Our results indicate that CL externalization induced by PrP106−126 on N2a cells plays a positive role in the initiation of mitophagy, leading to the stabilization of mitochondrial function

Gut Microbiota and Metabolome Changes in Three Pulmonary Hypertension Rat Models

Dysbiosis of the gut microbiota and metabolites is found in both pulmonary hypertension patients and pulmonary hypertension rodent models. However, the exact changes in gut microbiota during the development of pulmonary hypertension is unclear. The function of the gut microbiota is also ambiguous. Here, this study showed that the gut microbiota was disrupted in rats with hypoxia (Hyp)-, hypoxia/Sugen5416 (HySu)-, and monocrotaline (MCT)-induced pulmonary hypertension. The gut microbiota is dynamically changed during the development of Hyp-, HySu-, and MCT-induced rat pulmonary hypertension. The variation in the α diversity of the gut microbiota in Hyp-induced pulmonary hypertension rats was similar to that in rats with MCT-induced pulmonary hypertension and different from that in rats with HySu-induced pulmonary hypertension. In addition, six plasma biomarkers, His, Ala, Ser, ADMA, 2-hydroxybutyric acid, and cystathionine, were identified in Hyp-induced pulmonary hypertension rats. Furthermore, a disease-associated network connecting Streptococcus with Hyp-induced pulmonary hypertension-associated metabolites was described here, including trimethylamine N-oxide, Asp, Asn, Lys, His, Ser, Pro, and Ile

DNA^+Pro^: an Improved Progressive Multiple Sequence Alignment Algorithm for Evolutionary Analysis Using Combined DNA-Protein Sequences

Alignment of DNA and protein sequences is a basic tool in the study of evolutionary, structural and functional relationship among macromolecules. Present sequence alignment methods are somewhat error-prone, often producing systematic bias. Errors in sequence alignments sometimes lead to subsequent misinterpretation of evolutionary, structural and functional information in genes, proteins and genomes. In traditional sequence alignment algorithms, alignments of DNA and protein sequences are conducted separately. It has been long believed that the phylogenetic signal disappears more rapidly from DNA sequences than from encoded proteins. It is therefore generally preferable to align sequences at the amino acid level. Here we present a new method—DNA^+Pro^, which aggregates DNA and protein sequences into combined DNA-protein sequences and align them in a combined fashion. We demonstrate that combining sequences improve the quality of multiple sequence alignment and solve practical evolutionary problems in primate immunodeficiency virus proteins and bacterial restriction enzymes. In addition to increased theoretical information contents, the distance estimations are more biological significant in combined alignment than in protein only or DNA only alignments. By integrating information buried separately in DNA and protein sequences, DNA^+Pro^ improves the accuracy of multiple sequence alignment of closely-related proteins and prevents certain errors that may occur in phylogeny analysis using protein only approaches. The DNA^+Pro^ software and the supplementary data are downloadable free of charge from "our website, http://www.dnapluspro.com":http://www.dnapluspro.com.
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