scholarly journals Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients

2020 ◽  
Author(s):  
Tao Xu ◽  
Jingu Wang ◽  
Bingjie Hu ◽  
Guosi Zhang ◽  
Wu Zhou ◽  
...  
Keyword(s):  
Binding Site ◽  
Rna Stability ◽  
Viral Rna ◽  
N Gene ◽  
Pcr Analysis ◽  
Threshold Values ◽  
Anchor Primer ◽  
Rna Integrity ◽  
Rnase Treatment ◽  
Polymerase Chain

Abstract The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (−2.7 cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70–80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases (n = 252), 7% convalescent cases (n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.

Regulation of hepatitis C virus by microRNA-122

2008 ◽  
Vol 36 (6) ◽  
pp. 1220-1223 ◽  
Author(s):  
Catherine L. Jopling
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Binding Site ◽  
Rna Stability ◽  
Viral Rna ◽  
Hcv Rna ◽  
Mrna Targets ◽  
Virus Rna ◽  
Regulate Protein

Most metazoan miRNAs (microRNAs) bind to sites in the 3′-UTRs (untranslated regions) of mRNA targets and negatively regulate protein synthesis. The liver-specific miR-122, however, exerts a positive effect on HCV (hepatitis C virus) RNA levels by binding directly to a site in the 5′-UTR of the viral RNA. HCV translation and RNA stability are unaffected, and therefore miR-122 is likely to act at the level of viral replication. The miR-122-binding site in HCV RNA was examined to determine whether the nature of the site is responsible for the unusual mode of action for a miRNA. When the site was placed in the 3′-UTR of a reporter mRNA, miR-122 repressed translation, and therefore the location of the miR-122-binding site dictates its effect on gene expression. Additionally, a second binding site for miR-122 was identified in the HCV 5′-UTR, and miR-122 binding to both sites in the same viral RNA was found to be necessary for viral replication. The two sites are adjacent and are separated by a short spacer, which is largely conserved between HCV genotypes. The binding site requirements for miR-122 to positively regulate HCV replication provide an insight into this unusual mode of miRNA action.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Sensitivity of polymerase chain reaction (PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules

LWT ◽  
2015 ◽  
Vol 63 (1) ◽  
pp. 714-719 ◽  
Author(s):  
Sahilah Abd Mutalib ◽  
Nursheila Mustafa Muin ◽  
Aminah Abdullah ◽  
Osman Hassan ◽  
Wan Aida Wan Mustapha ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Southern Hybridization ◽  
Pcr Analysis ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Gelatin Capsules ◽  
Halal Authentication ◽  
Polymerase Chain

scholarly journals Effect of a Berry Polyphenolic Fraction on Biofilm Formation, Adherence Properties and Gene Expression of Streptococcus mutans and Its Biocompatibility with Oral Epithelial Cells

Antibiotics ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 46
Author(s):  
Mariem Souissi ◽  
Amel Ben Lagha ◽  
Kamel Chaieb ◽  
Daniel Grenier
Keyword(s):  
Epithelial Cells ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Cell Model ◽  
Pcr Analysis ◽  
Adhesion Properties ◽  
Oral Epithelial Cells ◽  
Polymerase Chain ◽  
Oral Epithelial ◽  
Wild Blueberry

The ability of Streptococcus mutans to adhere to oral surfaces and form biofilm is a key step in the tooth decay process. The aim of this study was to investigate a berry (wild blueberry, cranberry, and strawberry) polyphenolic fraction, commercialized as Orophenol®, for its antibacterial, anti-biofilm, and anti-adhesion properties on S. mutans. Moreover, the biocompatibility of the fraction with human oral epithelial cells was assessed. Phenolic acids, flavonoids (flavonols, anthocyanins, flavan-3-ols), and procyanidins made up 10.71%, 19.76%, and 5.29% of the berry polyphenolic fraction, respectively, as determined by chromatography and mass spectrometry. The berry polyphenolic preparation dose-dependently inhibited S. mutans biofilm formation while not reducing bacterial growth. At concentrations ranging from 250 to 1000 µg/mL, the fraction inhibited the adhesion of S. mutans to both saliva-coated hydroxyapatite and saliva-coated nickel–chrome alloy. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that incubating S. mutans with the berry polyphenolic fraction was associated with a reduced expression of luxS gene, which regulates quorum sensing in S. mutans. The berry fraction did not show any significant cytotoxicity in an oral epithelial cell model. In conclusion, Orophenol®, which is a mixture of polyphenols from wild blueberry, cranberry and strawberry, possesses interesting anti-caries properties while being compatible with oral epithelial cells.

Development of Three-Stage Bioaerosol Sampler for Size-Selective Sampling

2022 ◽  
Author(s):  
Jun-Hyung Lim ◽  
Sang Hwan Nam ◽  
Jongwoo Kim ◽  
Nam Hoon Kim ◽  
Gun-Soo Park ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Size Range ◽  
Collection Efficiency ◽  
Commercial Product ◽  
Pcr Analysis ◽  
Cyclone Separator ◽  
Chain Reaction ◽  
Selective Sampling ◽  
Sampling Flow Rate ◽  
Polymerase Chain

Abstract In this study, a three-stage bioaerosol sampler with a sampling flow rate of 170 L/min was designed and fabricated for sampling the bioaerosols released during human breathing and coughing, and its performance was evaluated. The sampler was constructed using a cyclone separator with a cutoff size of 2.5 µm as a preseparator, a multi-nozzle virtual impactor with a cutoff size of 0.34 µm as an aerosol concentrator, and a BioSampler, which is a commercial product, for collecting bioaerosols in a collection fluid. The collection efficiency of the sampler was evaluated through simulations and experiments. Only particles with sizes of 0.1-4 µm were selectively collected in the collection fluid. Bacteriophage bioaerosols were sampled using the developed sampler and ACD-200 Bobcat sampler, which is a commercial product. The amounts of collected bacteriophages were compared using the polymerase chain reaction (PCR) technique. The sampling performance of the developed sampler was similar to that of the ACD-200 Bobcat sampler. Moreover, the developed sampler showed its ability to sample bioaerosols of a specific size-range and collect them directly in a collection fluid for the PCR analysis. Therefore, the developed sampler is expected to be useful for indoor environmental monitoring by effectively sampling the bioaerosols released indoors during human breathing and coughing.

TaffiX® nasal powder spray forms an effective barrier against infectious new variants of SARS-CoV-2 (COVID-19)

2021 ◽  
Author(s):  
Michal Mandelboim ◽  
Ella Mendelson ◽  
Yaron Drori ◽  
Nofar Atari ◽  
Tair Lapidot ◽  
...  
Keyword(s):  
South African ◽  
Rna Virus ◽  
Rna Extraction ◽  
Real Life ◽  
Preclinical Study ◽  
Viral Rna ◽  
Pcr Analysis ◽  
Effective Barrier ◽  
Gel Layer

Abstract Introduction: While vaccination efforts against SARS-CoV-2 around the world are ongoing -, new high-infectious variants of the virus are being detected. The protection of the available vaccines against some of the new variants is weaker, and experts are concerned that newer as yet undescribed variants of this mutated RNA virus will eventually prove stable against the current vaccines. Additional preventive measures will therefore be needed to protect the population until effective vaccinations are widely available.TaffiX® is a personal, anti-viral nasal powder spray comprised of low pH Hypromellose that upon insufflation into the nose creates a thin gel layer covering the nasal mucosa and forming a protective mechanical barrier that prevents viruses from engaging with nasal cells- the main portal of entry for viruses. Taffix is commercially available in many countries across Europe, Asia America and Africa. In a prior preclinical study, TaffiX® was found to be effective against SARS-CoV-2 Hong Kong/VM20001061/2020 in experimental in vitro conditions. A real-life clinical survey demonstrated that TaffiX® nasal spray significantly reduced the SARS-CoV-2 infection rate post mass-gathering event in a highly endemic community.Objective: The current study aimed to test the protective effect of Taffix against new pathogenic, highly infectious SARS-CoV-2 variants in vitro: the “British” B.1.1.7 (hCoV-19/Israel/CVL-46879-ngs/2020) and the “South African” B.1.351 (hCoV-19/Israel/CVL-2557-ngs/2020) variants.Study design: A TaffiX® gel was formed on a nylon filter, using an amount equivalent to a clinical dose of Taffix . Filters were then seeded with SARS-CoV-2 B.1.1.7 (“British”) and B.1.351 (“South African”) variants. After a 10 -minute incubation at room temperature, the bottom of each filter was washed, and the resulting flow-through was collected and seeded into 24 -well plates containing Vero-E6 cells. After 5 days of incubation, a 200 µl sample from each well was taken for viral RNA extraction followed by SARS-CoV 2 RT-PCR analysis.Results: The TaffiX® gel completely blocked SARS-CoV-2 highly infectious variants B.1.1.7 and B.1.351 in vitro, reducing the titer of recoverable infectious virus as well as viral RNA by 100%.Conclusions: Under in vitro conditions, TaffiX® formed an effective protective barrier against SARS-COV-2 variants (British variant and South African Variant). These results are consistent with prior findings demonstrating the in vitro high efficacy of Taffix gel in preventing viruses from reaching cells and infecting them. These results, added to clinical real-life studies performed with Taffix , support its use as an effective barrier against new variants of SARS-CoV-2 in conjunction with other protective measures.

Characterization of alpha-adrenoceptor gene expression in arterial and venous smooth muscle

1993 ◽  
Vol 265 (5) ◽  
pp. H1501-H1509 ◽  
Author(s):  
P. Ping ◽  
J. E. Faber
Keyword(s):  
Smooth Muscle ◽  
Messenger Rna ◽  
Vena Cava ◽  
Pcr Analysis ◽  
Relative Distribution ◽  
Alpha Adrenoceptor ◽  
Aortic Media ◽  
Unique Restriction ◽  
Polymerase Chain

Six genes coding for three unique alpha 1- (1A, 1B, 1C) and three unique alpha 2- (2A, 2B, 2C) adrenergic receptor (AR) subtypes have been cloned. Ligand binding and contractile studies have demonstrated that both alpha 1- and alpha 2-ARs can exist on vascular smooth muscle (VSM) cells, although less is known about the relative distribution and specific subtypes in different vascular segments. In the present study polymerase chain reaction (PCR) analysis was used to characterize the species of alpha-AR messenger RNA (mRNA) present in freshly isolated rat thoracic aortic media and vena cava and in cultured VSM cells (passage 2) derived from both sources. To prevent possible contamination of VSM mRNA, aortic media was separated from adventitia, and vessels were denuded of endothelial cells. Oligonucleotide primers specific for each of the six adrenergic genes were synthesized and used to probe for the presence of alpha-AR mRNA species after reverse transcription of total cellular RNA to cDNA. PCR-amplified AR transcripts were distinguished by the size of amplified DNA fragments and unique restriction endonuclease cleavage. Expression of alpha 1C- or alpha 2C-mRNA was not detected in vascular tissues or cultured VSM cells, although the alpha 2C-primers detected the expected alpha 2C expression in cerebral cortex. Only alpha 1A-mRNA was detected in aortic adventitia. VSM from aorta expressed alpha 1A-, alpha 1B-, and alpha 2A-mRNA, and this pattern was preserved in cultured aortic VSM. Vena cava also expressed both alpha 1A and alpha 1B; however only alpha 2B-mRNA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Aberrant Long Noncoding RNAs Expression Profiles Affect Cisplatin Resistance in Lung Adenocarcinoma

2017 ◽  
Vol 2017 ◽  
pp. 1-14 ◽  
Author(s):  
Lijuan Hu ◽  
Jian Chen ◽  
Fan Zhang ◽  
Junjun Wang ◽  
Jingye Pan ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
A549 Cell ◽  
Cisplatin Resistance ◽  
Expression Profiles ◽  
Noncoding Rnas ◽  
Long Noncoding Rnas ◽  
Pcr Analysis ◽  
Sensitive Cell ◽  
Chain Reaction ◽  
Polymerase Chain

Background. Long noncoding RNAs (lncRNAs) have been shown to be involved in the mechanism of cisplatin resistance in lung adenocarcinoma (LAD). However, the roles of lncRNAs in cisplatin resistance in LAD are not well understood. Methods. We used a high-throughput microarray to compare the lncRNA and mRNA expression profiles in cisplatin resistance cell A549/DDP and cisplatin sensitive cell A549. Several candidate cisplatin resistance-associated lncRNAs were verified by real-time quantitative reverse transcription polymerase chain reaction (PCR) analysis. Results. We found that 1,543 lncRNAs and 1,713 mRNAs were differentially expressed in A549/DDP cell and A549 cell, hinting that many lncRNAs were irregular from cisplatin resistance in LAD. We also obtain the fact that 12 lncRNAs were aberrantly expressed in A549/DDP cell compared with A549 cell by quantitative PCR. Among these, UCA1 was the aberrantly expressed lncRNA and can significantly reduce the IC50 of cisplatin in A549/DDP cell after knockdown, while it can increase the IC50 of cisplatin after UCA1 was overexpressed in NCI-H1299. Conclusions. We obtained patterns of irregular lncRNAs and they may play a key role in cisplatin resistance of LAD.

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