Background: The application of ribonucleic acids for molecular studies requires high integrity and quality of extracted total RNA samples.
In addition, the need to transfer RNA samples at room temperature without special treatments such as ice and liquid nitrogen storage according to international transport laws highlights the importance of low cost alternative methods such as RNA air-drying, lyophilisation and transportable agents.
In this study, the quality and quantity of air-dried RNA samples from leaf, petiole and bark tissues of different olive genotypes using several RNA extraction methods were compared with lyophilized ground leaves and RNAlater-stored tissue samples before precipitation.
The quality of RNA and prepared libraries were checked by several techniques including agarose and polyacrylamide gel electrophoresis, Agilent quality control, RT-PCR amplification of housekeeping and viral genes and high throughput sequencing.
Results: Although RNA value varied amongst cultivars, RNA extraction with TRIzol™ Reagent in fresh extractions and samples stored in RNAlater before RNA extraction resulted in 455.26 ng/µL and 63.46 ng/µL (mean value of cultivars) as the highest RNA concentration averages, respectively.
RNA samples extracted by TRIzol™ Reagents and stored for a short term at – 80 °C before air-drying showed the third-highest concentration (44.87 ng/µL). The synthesized cDNAs quality for PCR amplification of housekeeping genes (Rbc 1 and Nad 5) and partial genomes of Arabis mosaic virus and Cucumber mosaic virus showed satisfactory results in RNA samples extracted by TRIzol™ Reagents despite its variation amongst cultivars.
Conclusions: Considering the difficulties in the extraction of high quality and quantity RNA in olive for molecular analyses, this study demonstrated that RNA extraction method based on TRIzol™ Reagent can be considered for virobiome studies of both fresh and air-dried samples.
Keywords: Metavirome studies; Olive; RNA integrity; RNA quality.
A Variety of NucleicAcid Species Are Sensed by cGAS, Implications for Its Diverse Functions
Cyclic GMP-AMP synthase (cGAS) recognizes double-stranded DNA (dsDNA) derived from invading pathogens and induces an interferon response via activation of the key downstream adaptor protein stimulator of interferon genes (STING).
This is the most classic biological function of the cGAS-STING signaling pathway and is critical for preventing pathogenic microorganism invasion. In addition, cGAS can interact with various types of nucleic acids, including cDNA, DNA : RNA hybrids, and circular RNA, to contribute to a diverse set of biological functions. An increasing number of studies have revealed an important relationship between the cGAS-STING signaling pathway and autophagy, cellular senescence, antitumor immunity, inflammation, and autoimmune diseases.
This review details the mechanism of action of cGAS as it interacts with different types of nucleic acids, its rich biological functions, and the potential for targeting this pathway to treat various diseases.
Co-Delivery of p53 Restored and E7 Targeted NucleicAcids by Poly (Beta-Amino Ester) Complex Nanoparticles for the Treatment of HPV Related Cervical Lesions
The p53 gene has the highest mutation frequency in tumors, and its inactivation can lead to malignant transformation, such as cell cycle arrest and apoptotic inhibition.
Persistent high-risk human papillomavirus (HR-HPV) infection is the leading cause of cervical cancer. P53 was inactivated by HPV oncoprotein E6, promoting abnormal cell proliferation and carcinogenesis.
To study the treatment of cervical intraepithelial neoplasia (CIN) and cervical cancer by restoring p53 expression and inactivating HPV oncoprotein, and to verify the effectiveness of nano drugs based on nucleic acid delivery in cancer treatment, we developed poly (beta-amino ester)537, to form biocompatible and degradable nanoparticles with plasmids (expressing p53 and targeting E7).
In vitro and in vivo experiments show that nanoparticles have low toxicity and high transfection efficiency. Nanoparticles inhibited the growth of xenograft tumors and successfully reversed HPV transgenic mice’s cervical intraepithelial neoplasia.
Our work suggests that the restoration of p53 expression and the inactivation of HPV16 E7 are essential for blocking the development of cervical cancer. This study provides new insights into the precise treatment of HPV-related cervical lesions.
Treatment effect of DNA framework nucleicacids on diffuse microvascular endothelial cell injury after subarachnoid hemorrhage
Objectives: The purpose of this study was to investigate the treatment effect and molecular mechanism of tetrahedral framework nucleic acids (tFNAs), novel self-assembled nucleic acid nanomaterials, in diffuse BMEC injury after SAH.
Materials and methods: tFNAs were synthesized from four ssDNAs. The effects of tFNAs on SAH-induced diffuse BMEC injury were explored by a cytotoxicity model induced by hemin, a breakdown product of hemoglobin, in vitro and a mouse model of SAH via internal carotid artery puncture in vivo.
Cell viability assays, wound healing assays, transwell assays, and tube formation assays were performed to explore cellular function like angiogenesis.
Results: In vitro cellular function assays demonstrated that tFNAs could alleviate hemin-induced injury, promote angiogenesis, and inhibit apoptosis in the hemin cytotoxicity model. In vivo study using H&E and TEM results jointly indicated that the tFNAs attenuate the damage caused by SAH in situ, showing restored number of BMECs in the endothelium layer and more tight intercellular connectivity.
Histological examination of SAH model animals confirmed the results of the in vitro study, as tFNAs exhibited treatment effects against diffuse BMEC injury in the cerebral microvascular bed.
Conclusions: Our study suggests the potential of tFNAs in ameliorating diffuse injury to BMECs after SAH, which laid theoretical foundation for the further study and use of these nucleic acid nanomaterials for tissue engineering vascularization.
DNA Hairpins and Dumbbell-Wheel Transitions Amplified Walking Nanomachine for Ultrasensitive Nucleic Acid Detection
Nucleic acids, including circulating tumor DNA (ctDNA), microRNA, and virus DNA/RNA, have been widely applied as potential disease biomarkers for early clinical diagnosis.
In this study, we present a concept of DNA nanostructures transitions for the construction of DNA bipedal walking nanomachine, which integrates dual signal amplification for direct nucleic acid assay.
DNA hairpins transition is developed to facilitate the generation of multiple target sequences; meanwhile, the subsequent DNA dumbbell-wheel transition is controlled to achieve the bipedal walker, which cleaves multiple tracks around electrode surface.
Through combination of strand displacement reaction and digestion cycles, DNA monolayer at the electrode interface could be engineered and target-induced signal variation is realized. In addition, pH-assisted detachable intermolecular DNA triplex design is utilized for the regeneration of electrochemical biosensors.
The high consistency between this work and a standard quantitative polymerase chain reaction is validated. Moreover, the feasibilities of this biosensor to detect ctDNA and SARS-CoV-2 RNA in clinical samples are demonstrated with satisfactory accuracy and reliability. Therefore, the proposed approach has great potential applications for nucleic acid-based clinical diagnostics.
Time distribution of positive nucleic acid detection in imported cases infected with SARS-CoV-2 in China
Objective: To analyze the time distribution of the first positive nucleic acid detection in imported cases infected with SARS-CoV-2 reported nationwide in China and provide references for further improvement of the prevention and control of COVID-19 in international travelers.
Methods: The data of imported cases infected with SARS-CoV-2 reported by provinces from 24 July 2020 and 23 July 2021 were collected for the analysis on the time distribution of the first positive nucleic acid detection after entering China.
Results: A total of 7 199 imported cases infected with SARS-CoV-2 were reported in 28 provinces from 24 July 2020 to 23 July 2021. The median interval (Q1, Q3) from the entry to the first positive nucleic acid detection of SARS-CoV-2 was 1 (0, 5) day.
The imported cases who had the first positive nucleic acid detections within 14 days and 14 days later after the entry accounted for 95.15% (6 850/7 199) and 4.85% (349/7 199) respectively. Among these cases, 3.65% (263/7 199), 0.88% (63/7 199) and 0.32% (23/7 199) had the first positive nucleic acid detections within 15-21 days, 22-28 days and 28 days later after the entry respectively.
Nucleic Acid |
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Locked nucleic acid 1 |
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Locked nucleic acid 1 |
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Locked nucleic acid 1 |
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Locked nucleic acid 1 |
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Locked nucleic acid 1 |
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Ecl Direct Nucleic Acid |
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The proportion of asymptomatic infections were 47.24% (3 236/6 850) and 63.61% (222/349) among the cases who had the first positive nucleic acid detections within 14 days and 14 days later after the entry respectively. A total of 39.54% (138/349) of cases infected with SARS-CoV-2 with the first positive nucleic acid detections 14 days later after the entry had inter-provincial travel after the discharge of entry point isolation.
Conclusions: About 5% of the imported cases infected with SARS-CoV-2 were first positive 14 days later after the entry. In order to effectively reduce the risk of domestic COVID-19 secondary outbreaks caused by imported cases, it is suggested to add a nucleic acid test on 8th -13th day after the entry.