ABSTRACT
Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development.
ABSTRACT
BACKGROUND: S. pyogenes, is a primary pathogen that leads to pharyngitis and can also trigger severe conditions like necrotizing fasciitis and streptococcal toxic shock syndrome (STSS), often resulting in high mortality rates. Therefore, prompt identification and appropriate treatment of S. pyogenes infections are crucial in preventing the worsening of symptoms and alleviating the disease's impact. RESULTS: In this study, a newly developed technique called multiple cross displacement amplification (MCDA) was employed to detect S. pyogenes,specifically targeting the speB gene, at a temperature of 63°C within 30 min. Then, an easily portable and user-friendly nanoparticles-based lateral flow biosensor (LFB) assay was introduced for the rapid analysis of MCDA products in just 2 min. The results indicated that the LFB offers greater objectivity compared to Malachite Green and is simpler than electrophoresis. The MCDA-LFB assay boasts a low detection limit of 200 fg and exhibits no cross-reaction with non-S. pyogenes strains. Among 230 clinical swab throat samples, the MCDA-LFB method identified 27 specimens as positive, demonstrating higher sensitivity compared to 23 samples detected positive by qPCR assay and 18 samples by culture. The only equipment needed for this assay is a portable dry block heater. Moreover, each MCDA-LFB test is cost-effective, priced at approximately $US 5.5. CONCLUSION: The MCDA-LFB assay emerges as a straightforward, specific, sensitive, portable, and user-friendly method for the rapid diagnosis of S. pyogenes in clinical samples.
Subject(s)
Biosensing Techniques , Nanoparticles , Streptococcus pyogenes/genetics , Nucleic Acid Amplification Techniques/methods , Biosensing Techniques/methods , Temperature , Sensitivity and SpecificityABSTRACT
Recently,plasma sterilization has attracted increasing attention in dental community for the atmospheric pressure non-equilibrium plasma jet (APNPs),which is driven by a kilohertz pulsed DC power,may be applied to the dental and oral diseases.However,it is still in doubt whether APNPs can effectively kill pathogenic bacteria in the oral cavity and produce no harmful effects on normal oral tissues,especially on normal mucosa.The aim of this study was to evaluate the bacterial-killing effect of APNPs in the biofilms containing a single breed of bacteria (Porphyromonas gingivalis,Pg.),and the pathological changes of the oral mucosa after treatment by APNPs.Pg.was incubated to form the biofilms in vitro,and the samples were divided into three groups randomly:group A (blank control);group B in which the biofilms were treated by APNPs (the setting of the equipment:10 kHz,1600 ns and 8 kV); group C in which the biofilms were exposed only to a gas jet without ignition of the plasma.Each group had three samples and each sample was processed for up to 5 min.The biofilms were then fluorescently stained,observed and photographed under a laser scanning confocal microscope.In the animal experiment,six male Japanese white rabbits were divided into two groups randomly (n=3 in each group) in terms of the different post-treatment time (1-day group and 5-day group).The buccal mucosa of the left side and the mucosa of the ventral surface of the tongue were treated by APNPs for 10 min in the same way as the bacterial biofilm experiment in each rabbit,and the corresponding mucosa of the other sides served as normal control.The clinical manifestations of the oral mucosa were observed and recorded every day.The rabbits were sacrificed one or five day(s) after APNPs treatment.The oral mucosa were harvested and prepared to haematoxylin and eosin-stained sections.Clinical observation and histopathological scores were used to assess mucosal changes.The results showed the obvious P.g.biofilms were formed at 10 days,and most of the bacteria in groups A and C were alive under a laser scanning confocal microscope,but the bacteria in the group B were almost all dead.In animal experiment,no ulcers,anabrosis and oral mucositis were found in both the 1-day and 5-day groups.The average mucous membrane irritation index was -0.83 and -0.67 in the 1-day and 5-day groups,respectively,suggesting that no intense mucosal membrane irritation responses occurred.It was concluded that APNPs could effectively kill P.g.in the biofilms and did not cause any pathological changes in the normal mucosa,suggesting that the plasma jet (APNPs) may be applied to oral diseases as a novel sterilization device in the future.