Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load.

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 µg/µL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 µg/µL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.

Antibacterial and antibiofilm effects of green synthesized selenium nanoparticles on clinical Klebsiella pneumoniae isolates.

Nanotechnology covers many disciplines, including the biological sciences. In this study, selenium nanoparticles (Se-NPs) were synthesized using Artemisia annua extract and investigated against clinical strains of klebsiella pneumoniae (K. pneumoniae) for their anti-biofilm effects. In this experimental study, from May 1998 to September 1998, 50 clinical samples of blood, urine, and sputum were collected, and K. pneumoniae strains were isolated using microbiological methods. Subsequently, the antibacterial effects of Se-NPs at concentrations of 12-25-50-100/5-6/3-25/125 µg/mL were studied. Finally, biofilm-producing strains were isolated, and the expression of mrkA biofilm gene was studied in real-time strains treated with Se-NPs using real-time polymerase chain reaction (PCR). Out of 50 clinical samples, 20 strains of K. pneumoniae were isolated. Minimum inhibitory concentration (MIC) results of Se-NPs showed that Se-NPs were capable of significant cell killing. Real-time PCR results also showed that mrkA gene expression was significantly reduced in strains treated with Se-NPs. According to this study, Se-NPs could reduce bacterial growth and biofilm formation, therefore, could be considered a candidate drug in the medical application for infections caused by K. pneumoniae.

Vancomycin-gingerol encapsulated niosomal formulation against carbapenem-resistantKlebsiella pneumoniae.

Alternatives to conventional antibiotics are critical in light of the increasing prevalence of antibiotic-resistant bacteria, posing a serious threat to humanity and imposing a financial burden on the community. The current study aimed to develop a Vancomycin (Van) and Gingerol (Gin) co-encapsulated in a niosomal (Nio-Gin/Van) formulation and to assess the optimized formulation as a potent antibacterial agent against carbapenem-resistantKlebsiella pneumoniae(CRKP) strains. The prepared Nio-Gin/Van was characterized via scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) and Fourier-transform infrared spectroscopy (FTIR) techniques. The F4 formulation was selected as the optimal formulation due to its low polydispersity index (PDI) (0.221 ± 0.023), small size (222.8 ± 6.35 nm), and suitable entrapment efficiency (EE%) (83.73 ± 1.12 for Gin and 66.25 ± 1.34 for Van). The Nio-Gin/Van had a sustained drug release up to 72 h and posed great stability to 60 d at 4 °C with low alterations in size, PDI and EE%, which introduced it as an appropriate candidate for medicinal utilization. The antibacterial activities of Nio-Gin/Van against CRKPs isolates were investigated using a MIC assay, which revealed MIC values of between 7.81/100-125/100 µg ml-1. Microtiter-plate assays and real-time polymerase chain reaction (PCR) were used to evaluate the antibiofilm properties of Nio-Gin/Van. A microtiter-plate assay indicated that approximately 53% of 15 CRKP isolates (n= 8) produced strong biofilms, while 26.6% (n= 4) produced moderate biofilms. Additionally, real-time PCR analysis revealed that Nio-Gin/Van significantly reduced the expression of thefimH, blaKPC, mrkD, andOmpk36genes in all CRKP isolates examined. It was concluded that encapsulating Gin-Van in niosome enhances their antibacterial and antibiofilm activity against CRKP strains and these preparations could be considered as a novel strategy for targeted drug delivery.

Synthesis of Gingerol-loaded Uio-66 nanoparticles and its anti-cancer effect against gastric cancer cell line (AGS).

BACKGROUND: Gastric cancer is the world's fifth most prevalent cancer and its treatments are associated with issues. In this investigation, a UIO-66 nanoparticle was loaded with Gingerol (UIO-66-Gin) as a great drug carrier vehicle for chemotherapy of the AGS cancer cell lines. METHODS AND RESULTS: UIO-66-Gin characterization was performed using SEM, DLS and FTIR tests. The release profile of Gin from UIO-66 was also assessed. The cytotoxicity of UIO-66-Gin against AGS cells was assessed using MTT assay. Caspase3, Caspase9, Bax, and Bcl2 genes expression was evaluated via Real-time PCR and apoptosis rate was performed using flow-cytometry assay. Size analysis indicated the spherical shape of nano-formulation with the mean size of 174.3 nm. Release analysis indicated that there was a 50% Gin release from the nanocarrier was reported in roughly 21 h, which revealed the regulated release of bioactive compound from the UIO-66 formulation in PBS medium. After 48 and 72 h, various concentration of both the Gin and UIO-66-Gin started to induce cytotoxicity in cancerous cells. However, the induction of cytotoxicity was higher in cells treated with UIO-66-Gin. UIO-66-Gin could induce the expression of pro-apoptotic (Bax, Caspase3, and Caspase9) genes and down-regulate the expression of Bcl2 as anti-apoptotic gene rather than other formulation. Flowcytometry results indicated that the elevation of apoptotic rate in cells treated with UIO-66-Gin was significantly higher than Gin treated cells. CONCLUSIONS: Our investigation revealed the potent anticancer effect and apoptotic induction ability of UIO-66-Gin against cancerous cells through altering the expression of genes involved in apoptosis.

Expression Profile of miRNA-17-3p and miRNA-17-5p Genes in Gastric Cancer Patients with Helicobacter pylori Infection.

BACKGROUND: The most common chronic bacterial infection is Helicobacter pylori. The connection between chronic H. pylori infection and gastric cancer is recognized. The early detection of gastric cancer improves survival. miRNAs regulate gene expression in eukaryotes by inhibiting mRNA translocation or degradation. The objective of this study was to compare the expression of miRNA-17-3p and miRNA-17-5p genes in gastric cancer patients with Helicobacter pylori infection. METHODS: Herein, 30 isolates were identified as H. pylori based on urease test, and 30 and 12 cases were isolated from gastric cancer patients and non-Helicobacter pylori cases as control, respectively. A peripheral blood sample was collected from patients. Analysis of total mRNA extracts from peripheral blood samples, for gene expression changes (miRNA-17-3p and miRNA-17-5p) by quantitative real-time polymerase chain reaction (qRT-PCR), was done. RESULTS: As said by the results, p values showed that expression levels of miRNA-17-3p and miRNA-17-5p were significantly higher in H. pylori-positive GC patients and H. pylori-positive non-GC patients with comparing by healthy controls. So, there was no significant difference between expression levels of miRNA-17-3p and miRNA-17-5p in H. pylori-positive GC patients and H. pylori-positive non-GC patients. CONCLUSION: Considering our results, the high expression of miRNA-17-3p and miRNA-17-5p has a direct relationship with increased cell proliferation, inhibition of tumor cell apoptosis and tumor angiogenesis, in addition to miRNAs play an important role as biomarkers in helping for detection of the patient by H. pylori infection to become cancerous. Therefore, it can be used to make specific diagnostic kits and to treat patients.

Ecofriendly Biomolecule-Capped Bifidobacterium bifidum-Manufactured Silver Nanoparticles and Efflux Pump Genes Expression Alteration in Klebsiella pneumoniae.

Background: Klebsiella pneumoniae is currently considered as an immediate threat to human health due to its various multidrug efflux pumps. Microbially synthesized silver nanoparticles (AgNPs) are an attractive and eco-friendly approach to prevent antibiotic resistance in bacteria. In the present study, we compared the inhibitory effect of both commercial and green AgNPs by Bifidobacterium bifidum on OxqAB efflux pump genes in ciprofloxacin-resistant strains of K. pneumoniae. Materials and Methods: AgNPs were characterized by ultraviolet-visible spectrophotometer, Fourier transform infrared spectroscopy, X-ray diffraction, zeta potential, transmission electron microscopy, and scanning electron microscopy. Antibiogram was used to identify resistant isolates and the effect of the biosynthesized AgNPs against OxqAB efflux pump strains was assessed by the minimum inhibitory concentration (MIC) method. The expression levels of oxqAB genes were evaluated using real-time polymerase chain reaction (PCR) followed by exposure to subMICs of the AgNPs. Results: PCR results showed that 25 strains had OxqAB efflux pump and the MIC method indicated that AgNPs had an inhibitory effect on all resistant strains with OxqAB efflux pump. The efficacy of the synthetic nanoparticles was assessed by comparing the antiefflux pump activity with commercial AgNPs. In ciprofloxacin-resistant isolates, the oxqAB genes expression levels reduced in the subMIC of both AgNPs, whereas biosynthesized AgNPs had greater bactericidal effects compared with the commercial AgNPs. Conclusions: Efflux pumps could be an attractive target for our biosynthesized AgNPs. The oxqAB genes expression levels reduced in subMIC of both AgNPs, whereas biosynthesized AgNPs had greater bactericidal effects than the commercial AgNPs.

Preparation and modification of forcespun polypropylene nanofibers for adsorption of uranium (VI) from simulated seawater.

In this paper, polypropylene (PP) nanofibers were prepared using the melt forcespinning technology by a handmade device. Then, the surface of PP nanofibers was grafted through the high energy electron beams (EB) pre-irradiation method by acrylonitrile and methacrylic acid monomers with grafting percentage of 145.55%. The 92% of grafted cyano functional groups on nanofibers were converted to amidoxime groups, then modified by an alkaline solution. Characterization and surface morphology of nanofibers were investigated by Fourier Transform Infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The produced adsorbent was used to adsorb U(VI) ions from simulated seawater. The maximum adsorption was 83.24 mg/g in the optimal time of 60 min and optimal pH of 4. The optimum desorption efficiency was 80% in HCl 0.5 M. The kinetic data in optimum conditions showed that the adsorption followed an S-shaped kinetic model. The Adsorption equilibrium studies presented S-shape isotherm model that confirmed the adsorption occurs both on the adsorbent surface and in its pores The thermodynamic studies indicated spontaneous adsorption of uranyl ions and the higher efficiency adsorption at higher temperatures. The selectivity of adsorbent for metal ions followed the order V(V)>U(VI)>CO(II)>Ni(II)>Fe(II). These results shows that the prepared and modified nanofibers in this work can be considered as an effective and promising adsorbents for removal of uranium ions from seawater with high efficiency.

Antibiofilm effect of green engineered silver nanoparticles fabricated from Artemisia scoporia extract on the expression of icaA and icaR genes against multidrug-resistant Staphylococcus aureus.

Silver nanoparticles (AgNPs) are at the forefront of the swiftly developing scope of nanotechnology. In the current study, we investigated the green synthesis of AgNPs using Artemisia scoporia as a reducing and capping agent. The biosynthesized AgNPs were characterized using ultraviolet-visible spectroscopy, X-ray diffraction, Fourier-Transform infrared spectroscopy, dispersive absorption spectroscopy, scanning electron microscopy, and transmission electron microscopy. The efficacy of the nanoparticle synthesis was assessed by comparing the antibiofilm activity with commercial AgNPs. The effect of sub-minimum inhibitory concentrations (MICs) of AgNPs on biofilm formation was determined by microtiter plate assay. The expression level of the icaA and icaR genes was assessed by real-time polymerase chain reaction assay. The structural and functional aspects of AgNPs were confirmed. The expression levels of icaA and icaR in the isolates exposed to sub-MIC of both commercial and biosynthetic AgNPs were lower and higher than in the control group, respectively. Our results also indicated that greater reduction and induction in icaA and icaR gene expression were noticed with the sub-MIC doses of biosynthetic AgNP versus commercial AgNP, respectively. This study suggested the application of AgNPs as a significant therapeutic and clinical option in the future and usage for fabricating medical implants. Nevertheless, further investigation is required for examining the pharmaceutical and medicinal properties of AgNPs.

Antioxidant, antibacterial and anticancer properties of phyto-synthesised Artemisia quttensis Podlech extract mediated AgNPs.

The focus of this study is on a rapid and cost-effective approach for the synthesis of silver nanoparticles (AgNPs) using Artemisia quttensis Podlech aerial parts extract and assessment of their antioxidant, antibacterial and anticancer activities. The prepared AgNPs were determined by ultraviolet-visible spectroscopy, X-ray diffraction, Fourier transform infra-red spectroscopy, transmission electron microscopy, scanning electron microscopy, energy-dispersive spectroscopy, and dynamic light scattering and zeta-potential analysis. The AgNPs and A. quttensis extract were evaluated for their antiradical scavenging activity by 2, 2-diphenyl, 1-picryl hydrazyl assay and anticancer activity against colon cancer (human colorectal adenocarcinoma cell line 29) compared with normal human embryonic kidney (HEK293) cells. Also, the prepared AgNPs were studied for its antibacterial activity. The AgNPs revealed a higher antioxidant activity compared with A. quttensis extract alone. The phyto-synthesised AgNPs and A. quttensis extract showed a dose-response cytotoxicity effect against HT29 and HEK293 cells. As evidenced by Annexin V/propidium iodide staining, the number of apoptotic HT29 cells was significantly enhanced, following treatment with AgNPs as compared with untreated cells. Besides, the antibacterial property of the AgNPs indicated a significant effect against the selected pathogenic bacteria. These present obtained results show the potential applications of phyto-synthesised AgNPs using A. quttensis aerial parts extract.

Phytochemical composition and biological activities of Artemisia quettensis Podlech ethanolic extract.

The present study aim to investigate the phytochemical composition, antibacterial, antioxidant and anticancer activities of the ethanolic extract from aerial parts of Artemisia quettensis Podlech. The aerial part of A. quettensis Podlech extract was used for Gas chromatography-mass spectrometry (GC-MS) analysis, antioxidant, antibacterial and anticancer activities. GC/MS analysis of extract from this plant showed 23 major components and the most dominant components were acetic acid, [4-(1-hydroxy-1-methylethyl) cyclohex-1-enyl] methyl ester (13.88%), trans-Phytol (10.06%) and 2,6-Dimethyl-2,6-octadiene-1,8-diol diacetate (6.8%). The extract had significant antibacterial and anticancer effects. The highest percentage of antioxidant activity was 78.46% at 2 mg/mL concentration of extract. Moreover, the highest antibacterial effects of extract were against to gram-positive bacteria and the IC50 cell cytotoxicity value on HT29 cell line in 24 h, 48 h and 72 h were 31.54, 6.08 and 2.96 mg/mL, respectively. From this study, A. quettensis Podlech could be considered as a promising source for novel drug compounds.

Chemical composition, antioxidant, antibacterial and cytotoxic effects of Artemisia marschalliana Sprengel extract.

The present study was to investigate the gas chromatography/mass spectrometry (GC/MS), in vitro antioxidant, antibacterial and anticancer activity of the ethanolic extract from aerial parts of Artemisia marschalliana Sprengel against human gastric carcinoma (AGS) and L929 cell lines. Phytochemical analysis of A. marschalliana Sprengel extract showed 22 major components and the most dominant compounds were trans-phytol (29.22%), α-Linolenic acid (13.47%) and n-Hexadecanoic acid (9.28%). In addition, the antioxidant and anticancer activity of A. marschalliana Sprengel extract were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, respectively. Antibacterial activity against selected pathogenic bacteria was also determined. According to the present obtained results, it seems that this plant has potential uses for pharmaceutical industries and further studies of pharmaceutical importance were suggested to be performed on A. marschalliana Sprengel.

Expression and Purification of the Uropathogenic Escherichia coli PapG Protein and its Surface Absorption on Lactobacillus reuteri: Implications for Surface Display System Vaccines.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is one of the most common bacteria that can cause urinary tract infections (UTIs). Unfortunately, no human vaccine against UTIs has been developed. Therefore, it is necessary to develop an efficient and safe vaccine that is able to induce mucosal and systemic immune responses. The use of lactic acid bacteria as a delivery system is a promising method to induce the immune system. OBJECTIVES: The aim of this study was to establish Lactobacillus reuteri harboring the E. coli PapG antigen on its surface. MATERIALS AND METHODS: In this study, the gene encoding PapG was fused to the AcmA gene (which encodes an anchor protein in Lactobacillus) and cloned into the pEX A vector. The PapG.AcmA fusion gene was digested with BamHI and NdeI and sub-cloned into the pET21a expression vector at the digestion sites. Subsequently, the recombinant plasmids (pET21a-PapG.AcmA and pET21a-PapG) were transformed into the E. coli Origami strain using the calcium chloride method and the fusion protein was expressed under 1 mM IPTG induction. The expression of the fusion protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Purification of the PapG and PapG.AcmA proteins was carried out using a Ni-NTA column, and surface adsorption was estimated on Lactobacillus. Finally, surface localization of the fusion protein was verified by an enzyme-linked immunosorbent assay (ELISA). RESULTS: The PapG.AcmA fusion was successfully sub-cloned in the pET21a expression vector. The expression of PapG and PapG.AcmA proteins in the E. coli Origami strain was indicated as protein bands in SDS-PAGE and confirmed by western blotting. In addition, the fusion protein was displayed on the surface of L. reuteri. CONCLUSIONS: In conclusion, we developed a method to express the PapG.AcmA protein on the surface of Lactobacillus. This is the first report on the successful application of lactic acid bacteria displaying the PapG.AcmA fusion protein. It will be interesting to determine the immune responses against the PapG protein in near future using this surface display strategy.