American Type Culture Collection Research Articles

In Vitro Evaluation of the Virucidal Activity of Different Povidone-Iodine Formulations Against Murine and Human Coronaviruses.

IntroductionPolyvinylpyrrolidone–iodine (PVP-I) demonstrates broad-spectrum anti-infective activity and is available in different formulations for oral rinse and topical use in medical and personal care settings. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need to supplement available preventive strategies.MethodsWe assessed virucidal activity of PVP-I formulations, including 0.5% (w/v) solution, 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution, versus placebos when challenged with coronaviruses in two in vitro studies. Murine coronavirus strain A59 (American Type Culture Collection [ATCC]® VR-764™), human coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF), human coronavirus strain NL63 (ZeptoMetrix Corp. #0810228CF), and human coronavirus strain 229E (ATCC® VR-740™) were used as surrogates for SARS-CoV-2. Both studies used the American Society for Testing and Materials in vitro time-kill method.ResultsAll active PVP-I formulations in study 1 demonstrated virucidal activity at 15 s, with mean log10 reduction of greater than 4.56 or greater than 99.99% inactivation; a cytotoxic effect against the National Collection of Type Cultures clone 1469 host cells was observed with 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution. Active PVP-I formulations in study 2 demonstrated effective virucidal activity against coronaviruses in less than 15 s; log10 reduction in viral titer for each coronavirus strain was consistently higher for 10.0% (w/v) solution and 0.5% (w/v) solution versus 7.5% (w/v) scrub.ConclusionBoth studies demonstrated in vitro virucidal activity of PVP-I formulations when challenged with SARS-CoV-2 surrogate coronaviruses. Although promising, further investigations are needed to evaluate SARS-CoV-2 inactivation.

Circ_0004018 suppresses cell proliferation and migration in hepatocellular carcinoma via miR-1197/PTEN/PI3K/AKT signaling pathway

ABSTRACT Hepatocellular carcinoma (HCC) is a common type of primary liver cancer. Circular RNAs (circRNAs) have been demonstrated to be a crucial player in multiple cancers. However, a large number of circRNAs remain to be explored. Our study focused on investigating hsa_circ_0004018 in HCC. Firstly, we conducted quantitative reverse transcription PCR (RT-qPCR) to find that circ_0004018 was down-regulated in HCC cells. Western blot analysis was performed to detect the protein levels of phosphatase and tensin homologue (PTEN) and related factors of PI3K/AKT signaling pathway. From the results of functional assays, we found that overexpression of circ_0004018 significantly inhibited the proliferative and migratory capacities of HCC cells. The regulatory mechanism of circ_0004018 in HCC was determined by RNA immunoprecipitation (RIP), RNA pull-down, and luciferase reporter assays, thereby we knew that circ_0004018 regulated PTEN by sequestering microRNA-1197 (miR-1197) to modulate PI3K/AKT signaling pathway. Finally, rescue assays verified that circ_0004018 participated in modulation of cell proliferation and migration in HCC via sponging miR-1197 and regulating PTEN. In conclusion, circ_0004018 suppresses the proliferation and migration of HCC cells via sponging miR-1197 to inactivate the PTEN/PI3K/AKT signaling pathway. Abbreviations: HCC: Hepatocellular carcinoma; circRNAs: Circular RNAs; PTEN: Phosphatase and tensin homologue; miR-1197: microRNA-1197; ceRNA: competitive endogenous RNA; ATCC: American Type Culture Collection; EMEM: Eagle’s Minimum Essential Medium; RT-qPCR: Quantitative real-time PCR; EdU: 5-ethynyl-20-deoxyuridine; FISH: Fluorescent in situ hybridization; RIP: RNA immunoprecipitation; 3ʹ-UTR: 3ʹ-untranslated region; Wt: wild-type; Mut; mutant type; gDNA: genomic DNA; Act D: Actinomycin D; PI3K: phosphatidylinositol-3-kinase; AKT: protein kinase; lncRNAs: long non-coding RNAs

In vitro biological activity of liposomal-containing antimony trioxide

Antimonials are used as chemotherapy for leishmaniasis, but have limited results due to their toxicity and broad resistance already acquired by the parasites. Nanotechnology offers an alternative to reduce these effects through the use of biocompatible nanocarriers, which can be vectorized to the target site. In addition, the redirection of molecules, already developed for the treatment of other pathologies, has the advantage of being already approved for therapy by regulatory agencies. The present study addresses the production of liposomal vesicles containing antimony trioxide (LC Sb2O3), as well as the evaluation of activity against tumor and bacterial cells. We produce liposomes in order of nanometric size, polydispersity index (PDI <0.3), pH value close to physiological (7.2), and zeta potential (anionic). Cytotoxicity was evaluated in 24 and 72 hours, in the HepG2, T98G, and U87MG tumor cell lines, by the method (3-4.5 dimethylthiazole-2.5 diphenyltetrazolium bromide) (MTT). The minimum inhibitory concentration (MIC) was tested on three bacterial strains (American Type Culture Collection – ATCC-Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) and mandatory (Staphylococcus aureus and Klebsiella pneumoniae). The liposomes were more cytotoxic than Sb2O3 in the free form, for all tested cell lines. This effect was stronger after 72 hours incubation. Antimony trioxide in both free and liposomal forms showed low antibacterial activity. Based on our results, we suggest that liposomes containing antimony trioxide have the potential for the repositioning of drugs addressing anticancer therapy.

Efficacy of porphyrin derivative, Chlorhexidine and PDT in the surface disinfection and roughness of Cobalt chromium alloy removable partial dentures

AimThe present study aimed to evaluate the effect of different cleansers and photodynamic therapy (PDT) on disinfection of Co/Cr removable partial denture colonized with C. albicans, S.aureus and S.mutans. Material and MethodsTwenty-five Co/Cr removable partial dentures were constructed. and inoculated by American Type Culture Collection (ATCC) of Candida albicans, S.aureus, and S.mutans in an in-vitro setup. Co/Cr removable partial dentures were randomly allocated into five groups and framework was immersed in 15 mL of contaminated culture medium broth. Based on the cleansing method, disinfected dentures in group 1 were cleansed with porphyrin derivative; group 2 was exposed to 0.12% chlorhexidine digluconate; samples in group 3 and group 4 were subjected to sodium perborate enzyme and citric acid respectively. Through a surface analyzer, the surface roughness of Co/Cr and methacrylate resin was measured in 3 different zones in micrometers before and after disinfection. Two-way ANOVA and Tukey's test was used to analyze the data. Levene test was used to assess the normal distribution of data. Significance level p< 0.05 ResultsThe results of metal roughness displayed that Co/Cr used as an alloy did not exhibit significant difference when exposed to different chemical disinfectants and photosensitizer (p > 0.05). Similarly, surface roughness (Ra) of heat polymerized acrylic resin using cleansers and PDT demonstrated no significant difference. Intergroup comparison showed that, hematoporphyrin derivative displayed comparable cleansing property against S. aureus, C.albicans, and S. mutans compared to to 0.12% CHX digluconate (p>0.05). The intragroup comparison showed hematoporphyrin derivative and CHX were equally effective against all bacteria (p>0.05). ConclusionCHX digluconate and hematoporphyrin derivative demonstrated high antimicrobial activity against S.aureus, S.mutans, and C.albicans with minimum Ra of an acrylic denture and Co/Cr metallic framework.

Estimation of limit of detection of Salmonella typhimurium in artificially contaminated chicken meat by cultured-based and polymerase chain reaction techniques

The objective of this study was to develop Polymerase Chain Reaction (PCR) procedure for detection of Salmonella Typhimurium in artificially contaminated chicken meat. The experiments were conducted with various dilutions of Salmonella Typhimurium reference the American Type Culture Collection ATCC (ATCC13311TM 4.4*107) High concentration 4.4*103 Colony Forming Units (CFU)/ml, low concentration 4.4*102 CFU/ml, very low concentration 4.4*101 CFU/ml inoculated in chicken meat, in order to determine limits of detection (LOD), optimum incubation times 18 to 20 hours of pre-enrichment in Buffered Peptone Water (BPW 1%). Hence, cultural methods and DNA extraction were performed according to kits instruction. The microbiological cultural test was capable to detect 1.76 CFU/mL, whereas PCR examination was able to detect 0.18 CFU/ml of initial dilution of Salmonella Typhimurium inoculated in chicken meat. Interestingly, the results were achieved in a less time period than that of classical culture. The PCR technique is beneficial in the methodology for detection of Salmonella in chicken meat.

Taxonomic status of the species Clostridium methoxybenzovorans Mechichi et al. 1999.

The taxonomic status of the species Clostridium methoxybenzovorans was assessed. The 16S rRNA gene sequence, whole-genome sequence and phenotypic characterizations suggested that the type strain deposited in the American Type Culture Collection (C. methoxybenzovorans ATCC 700855T) is a member of the species Eubacterium callanderi. Hence, C. methoxybenzovorans ATCC 700855T cannot be used as a reference for taxonomic study. The type strain deposited in the German Collection of Microorganism and Cell Cultures GmbH (DSM 12182T) is no longer listed in its online catalogue. Also, both the 16S rRNA gene and the whole-genome sequences of the original strain SR3T showed high sequence identity with those of Lacrimispora indolis (recently reclassified from Clostridium indolis) as the most closely related species. Analysis of the two genomes showed average nucleotide identity based on blast and digital DNA-DNA hybridization values of 98.3 and 87.9 %, respectively. Based on these results, C. methoxybenzovorans SR3T was considered to be a member of L. indolis.

Occurrence of Multidrug-Resistant Staphylococcus aureus among Humans, Rodents, Chickens, and Household Soils in Karatu, Northern Tanzania.

We conducted this study to investigate the isolation frequency and phenotypic antibiotic resistance pattern of Staphylococcus aureus isolated from rodents, chickens, humans, and household soils. Specimens were plated onto mannitol salt agar (Oxoid, Basingstoke, UK) and incubated aerobically at 37 °C for 24 h. Presumptive colonies of S. aureus were subjected to Gram staining, as well as catalase, deoxyribonuclease (DNAse), and coagulase tests for identification. Antibiotic susceptibility testing was performed by using the Kirby–Bauer disc diffusion method on Mueller–Hinton agar (Oxoid, Basingstoke, UK). The antibiotics tested were tetracycline (30 μg), erythromycin (15 μg), gentamicin (10 μg), ciprofloxacin (5 μg), clindamycin (2 μg), and amoxicillin-clavulanate (20 μg/10 μg). The S. aureus strain American Type Culture Collection (ATCC) 25,923 was used as the standard organism. We found that 483 out of 956 (50.2%) samples were positive for S. aureus. The isolation frequencies varied significantly between samples sources, being 52.1%, 66.5%, 74.3%, and 24.5%, respectively, in chickens, humans, rodents, and soil samples (p < 0.001). S. aureus isolates had high resistance against clindamycin (51.0%), erythromycin (50.9%), and tetracycline (62.5%). The overall prevalence of multidrug-resistant (MDR) S. aureus isolates was 30.2%, with 8.7% resistant to at least four different classes of antibiotics.

In vitro antimicrobial activity of cold atmospheric microwave plasma against bacteria causing canine skin and ear infections.

Cold atmospheric plasma (CAP) is a new generation medical therapeutic option for bacterial infections. CAP causes physical cell wall rupture and DNA damage, therefore making it highly useful in the treatment of various conditions such as skin infections. The antimicrobial activity of cold atmospheric microwave plasma (CAMP) against major strains in canine skin infections was tested and the difference in antimicrobial activity between the antibiotic-resistant and antibiotic-susceptible strains of Staphylococcus pseudintermedius was evaluated. American Type Culture Collection (ATCC) strains (Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli) and clinical isolates identified as methicillin-resistant S.pseudintermedius (n=27) and methicillin-susceptible S.pseudintermedius (n=13) were exposed to CAMP for 10s, 30s and 60s. Afterwards, the bacterial survival rate was confirmed. Gram-negative bacteria (P.aeruginosa and E.coli) were more susceptible than Gram-positive bacteria (S.aureus and S.pseudintermedius) for the same duration of CAMP exposure. Only the Gram-negative bacteria were completely killed after 60s exposure. In S.pseudintermedius isolates, CAMP exposure had similar antibacterial effects regardless of antibiotic resistance. CAMP has sufficient antimicrobial activity against major bacterial strains that cause pyoderma and otitis externa in dogs, and may be an alternative therapeutic option for S.pseudintermedius skin infections, for which antibiotics often are ineffective because of antimicrobial resistance in clinical veterinary medicine.

Antimicrobial susceptibilities and comparative whole genome analysis of two isolates of the probiotic bacterium Lactiplantibacillus plantarum, strain ATCC 202195

A synbiotic containing Lactiplantibacillus plantarum [American Type Culture Collection (ATCC) strain identifier 202195] and fructooligosaccharide was reported to reduce the risk of sepsis in young infants in rural India. Here, the whole genome of two isolates of L. plantarum ATCC 202195, which were deposited to the ATCC approximately 20 years apart, were sequenced and analyzed to verify their taxonomic and strain-level identities, identify potential antimicrobial resistant genes and virulence factors, and identify genetic characteristics that may explain the observed clinical effects of L. plantarum ATCC 202195. Minimum inhibitory concentrations for selected antimicrobial agents were determined using broth dilution and gradient strip diffusion techniques. The two L. plantarum ATCC 202195 isolates were genetically identical with only three high-quality single nucleotides polymorphisms identified, and with an average nucleotide identity of 99.99%. In contrast to previously published reports, this study determined that each isolate contained two putative plasmids. No concerning acquired or transferable antimicrobial resistance genes or virulence factors were identified. Both isolates were sensitive to several clinically important antibiotics including penicillin, ampicillin and gentamicin, but resistant to vancomycin. Genes involved in stress response, cellular adhesion, carbohydrate metabolism and vitamin biosynthesis are consistent with features of probiotic organisms.

Arbutin exerts anticancer activity against rat C6 glioma cells by inducing apoptosis and inhibiting the inflammatory markers and P13/Akt/mTOR cascade.

Gliomas are a type of brain cancer that occurs in the supporting glial cells of the brain. It is highly malignant and accountsfor 80% of brain tumors with high mortality and morbidity. Phytomedicines are potent alternatives for allopathic drugs which cause side effects. They have beenused from ancient times by traditional Chinese, Ayurveda, and Siddha medicine. Arubtin is a glycoside phytochemical extracted from plants and belongs to the family of Ericaceae. Arbutin possesses various pharmacological properties such as anti-inflammatory, antioxidant, antitumor, and so on. Hence in the present study, we analyzed the anticancer potency of arbutin against rat C6 glioma cells. Rat C6 glioma cells were procured from American Type Culture Collection and the cells were cultured in Roswell Park Memorial Institute-1640 medium. To assess the cytotoxicity effect of the arbutin against C6 glioma cells, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test was performed with different doses from 10 to 60 µM. Arbutin effectively induced apoptosis in the cells and the IC50 dose was obtained at 30 µM. For further studies, we selected the 30 µM IC50 dose and a higher dose of 40 µM. Reactive oxygen species (ROS) generated were analyzed with DCFDA/H2DCFDA stain and the destruction of mitochondrial membrane permeability which is the initiator of apoptosis was analyzed with a cationic stain Rhodamine 123. Dual staining with acridine orange and ethidium bromide was performed to assess the viable and dead cells. Cell adhesion propertiesof glioma cells were analyzed with Matrigel assay. The apoptotic, inflammatory, and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling molecules were analyzed with quantitative polymerase chain reaction (qPCR) analysis to confirm the anticancer effect of arbutin. Arbutin generated excessive ROS and disrupted the mitochondrial membrane, which induced apoptosis in cells, it also inhibited the cell adhesion property of C6 glioma cells. qPCR analysis clearly indicates arbutin increases the apoptotic genes and decreased the inflammatory and PI3K/mTOR signaling molecules. Overall, our results authentically confirm that arbutin can be a potent alternative for treating glioma.

Silver nanoparticles–chitosan composites activity against resistant bacteria: tolerance and biofilm inhibition

This study aimed to evaluate the effectiveness of silver nanoparticles–chitosan composites (AgNPs) with different morphologies and particle size distributions against resistant bacteria and biofilm formation. Four different samples were prepared by a two-step procedure using sodium borohydride and ascorbic acid as reducing agents and characterized by UV–Vis absorption spectra, scanning transmission electron microscopy. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the AgNPs were determined according to the Clinical and Laboratory Standards Institute (CLSI) against clinical isolates multidrug-resistant and strains of the American Type Culture Collection (ATCC). An assay was performed to determine the MICs during 20 successive bacteria exposures to AgNPs to investigate whether AgNPs induce tolerance in bacteria. The antibiofilm activities of AgNPs were also evaluated by determining the minimum biofilm inhibitory concentration (MBIC). The spherical AgNPs present diameters ranging from 9.3 to 62.4 nm, and some samples also have rod-, oval-, and triangle-shaped nanoparticles. The MIC and MBC values ranged from 0.8 to 25 μg/mL and 3.1 to 50 μg/mL, respectively. Smaller and spherical AgNPs exhibited the highest activity, but all the AgNPs developed in this study exhibit bactericidal activity. There was no significant MIC increase after 20 passages to the AgNPs. Regarding the antibiofilm activity, MBICs ranged from 12.5 to 50 μg/mL. Again, smaller and spherical nanoparticles presented the best results with phenotypic inhibition of production of slime or exopolysaccharide (EPS) matrix. Thus, it was concluded that AgNPs have a promising potential against resistant bacteria and bacteria that grow on biofilms without inducing tolerance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11051-021-05314-1.

In-vitro evaluation of bactericidal activity of antiseptics and disinfectants commonly used in healthcare settings

Introduction: Healthcare-associated infections are common problems found in healthcare systems in most of the countries. Proper use of antiseptics and disinfectants is useful in reducing the magnitude of such infections. The aim of this study was to evaluate the bactericidal effect of different concentrations of selected antiseptics and disinfectants. Methods: Bactericidal activity of different concentrations of antiseptics and disinfectants; isopropyl alcohol, povidone iodine, chlorhexidine gluconate, sodium hypochlorite, hypertonic saline, peracetic acid and mixture of 2-aminoethanol, dodecyl dimethyl ammonium chloride, potassium carbonate &amp; bis(3-aminopropyl) dodecylamine were evaluated against nine strains of American Type Culture Collections and 11 strains isolated from the hospitals. Bacterial suspensions were prepared equal to 0.5 McFarland turbidity standards and 100 μl of each was mixed with 3 ml of antiseptic/ disinfectant solution. After desired contact time (1 minute, 15 minutes and 24 hours), 20 μl of reaction mixer was transferred into 4 ml of sterile physiological saline, mixed well and 20 μl of solution was evenly spread throughout Muller Hinton agar/ blood agar plates. Growth control was done using 3 ml of distilled water instead of 3 ml of antiseptic/ disinfectant solution. All plates incubated at 35oC ± 2oC for 18 – 24 hours. Colonies were counted and reduction percentage was calculated. Results: All concentrations of isopropyl alcohol, povidone iodine, chorhexidine, sodium hypochlorite, peracetic acid and the mixture of 2-aminoethanol, didecyldimethylammonium chloride, potassium carbonate and bis(3-aminopropyl)dodecylamine showed 100% bactericidal activities against all tested bacterial strains in1-minute contact time. Bactericidal activity of hypertonic saline was varied against tested bacterial strains with higher bactericidal activity against Gram positive organisms than Gram negative organisms. Conclusions: Currently using concentrations of antiseptics and disinfectants evaluated in this study have 100% bactericidal activity other than hypertonic saline.

Antibacterial Efficacy of Triple Antibiotic Medication With Macrogol (3Mix-MP), Traditional Triple Antibiotic Paste, Calcium Hydroxide, and Ethanol Extract of Propolis: An Intratubular Dentin Ex Vivo Confocal Laser Scanning Microscopic Study

IntroductionThis study aimed to assess the effectiveness of antibacterial activity of medications used in regenerative endodontic treatment. MethodsSixty-seven dentin cylinders of single-rooted teeth were contaminated with a culture of Enterococcus faecalis (ATCC 29212; American Type Culture Collection, Manassas, VA) for 5 days. Samples were divided into 1 control group and the following experimental groups according to the medication applied: traditional triple antibiotic paste (TAP), clindamycin-modified TAP (mTAP), triple antibiotic medication with macrogol (3Mix-MP), clindamycin-modified 3Mix-MP (m3Mix-MP), calcium hydroxide (CH), and ethanol extract of propolis (EEP). After 14 days, the medications were removed, and the samples were submitted to confocal laser scanning microscopic analysis to quantify the percentage of viable bacteria. The distribution of data was confirmed by the Shapiro-Wilk test. The Kruskal-Wallis and Dunn tests were used for intergroup comparisons, and the Wilcoxon test was used for comparison between superficial and deep antibacterial efficacy for the same medication. The level of significance was set at P < .05. Results3Mix-MP and m3Mix-MP presented significantly higher antibacterial efficacy compared with the other tested medications (P < .05), except for mTAP. mTAP was more effective than TAP (P < .05). The antibacterial efficacy of EEP and CH did not differ significantly from TAP and mTAP (P > .05). All medications showed effective antibacterial action compared with the control group (P < .05). Conclusions3Mix-MP and m3Mix-MP, which present extremely high concentrations of antibiotics (1500 mg/mL), were not more effective than mTAP at the concentration recommended by the American Association of Endodontists (5 mg/mL). Moreover, CH and EEP were as effective as TAP and mTAP.

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

ANTIMICROBIAL ACTIVITY OF WATER-ETHANOLIC EXTRACTIONS FROM QUERCUS ROBUR L. LEAVES AND BUDS

The problem of finding new antimicrobial drugs based on medicinal plant raw materials in modern pharmaceutical practice, is still relevant. There are interesting plant objects that have an antimicrobial action due to the content of a complex of biologically active substances in them. Quercus robur L. is a promising plant object, medicinal plant raw materials of which can be used for the development of new antimicrobial drugs. The aim of the study is screening of the antimicrobial activity of water-ethanolic extractions from Quercus robur L leaves and buds. Materials and methods. The determination of the minimum inhibitory concentration was carried out by the method of double serial dilutions in Mueller-Hinton nutrient broth (Bio-Rad, USA). As test cultures, strains of microorganisms of the American Type Culture Collection (ATCC) were used: Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), as well as Candida albicans (a clinical strain). The incubation was carried out at the temperature of 35°C for 24 hours. Simultaneously, an experiment was carried out to establish a "negative" control. The results were assessed visually by the presence / absence of the growth of microorganisms in test tubes with the corresponding dilutions of the test samples. Results. In the course of the study, it was found out that water-ethanolic extractions of Quercus robur L. leaves have the greatest antimicrobial effect against strains of Staphylococcus aureus and Escherichia coli . The water-ethanolic extractions of Quercus robur L. buds exhibit a pronounced antimicrobial activity against Pseudomonas aeruginosa and Candida albicans strains. It was revealed that the preparation of Quercus robur L. leaves tincture in the raw material:extractant ratio of 1:5 has a pronounced antimicrobial effect on the strains of Pseudomonas aeruginosa , Staphylococcus aureus , and with a higher multiplicity of dilution - on the strains of Escherichia coli and Candida albicans . The drug tincture of Quercus robur L. buds in the raw material:extractant ratio of 1:5 has a pronounced antimicrobial effect on the strains of microorganisms P. aeruginosa, S. aureus, E. coli and C. albicans in an eight-fold dilution. With respect to P. aeruginosa strains, antimicrobial activity was observed in 16-fold dilutions. The most pronounced antimicrobial effect was recorded against the C. albicans strain in a 32-fold dilution. As a result of the study, it can be concluded that to obtain the antimicrobial drugs - tincture of Quercus robur L. leaves and buds - is advisable to use the optimal extractant – 70% alcohol in a raw material:extractant ratio of 1:5. With these parameters of extraction, the greatest antimicrobial effect is observed in relation to the studied strains of the microorganisms. 70% alcohol has also a better penetrating ability into the deep layers of the epidermis in comparison with higher concentrations. Conclusion. The results of the screening analysis of the antimicrobial activity will be used as a justification for the introduction of antimicrobial drugs based on the leaves and buds of the Quercus robur L. in a medical and pharmaceutical practice.

Layer-by-Layer Nanoparticles of Tamoxifen and Resveratrol for Dual Drug Delivery System and Potential Triple-Negative Breast Cancer Treatment.

Nanoparticle development demonstrates use in various physicochemical, biological, and functional properties for biomedical applications, including anti-cancer applications. In the current study, a cancer therapeutic conjugate was produced consisting of tamoxifen (TAM) and resveratrol (RES) by layer-by-layer (LbL) nanoparticles based on lipid-based drug delivery systems and liquid crystalline nanoparticles (LCNPs) coated with multiple layers of positively charged chitosan and negatively charged hyaluronic acid for the evaluation of biocompatibility and therapeutic properties against cancer cells. Multiple techniques characterized the synthesis of TAM/RES–LbL-LCNPs, such as Fourier-transform infrared spectroscopy (FTIR), X-ray crystallography (XRD), Zeta potential analysis, particle size analysis, Field Emission Scanning Electron Microscope (FESEM), and Transmission electron microscopy (TEM). The in vitro cytotoxic effects of TAM/RES–LbL-LCNPs were investigated against human breast cancer cell line, Michigan Cancer Foundation-7 (MCF-7), and human triple-negative breast cancer cell line, Centre Antoine Lacassagne-51 (CAL-51), using various parameters. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed that the treatment of cells with TAM/RES–LbL-LCNPs caused a reduction in cell proliferation, and no such inhibition was observed with human normal liver cell line: American Type Culture Collection Cell Line-48 (WRL-68 [ATCC CL-48]). Fluorescent microscopy examined the ability of Fluorescein isothiocyanate (FITC) to bind to TAM/RES–LbL-LCNPs along with their cellular uptake. Apoptosis determination was performed using hematoxylin–eosin and acridine orange–propidium iodide double staining. The expression of P53 and caspase-8 was analyzed by flow cytometry analysis. An in vivo study determined the toxicity of TAM/RES–LbL-LCNPs in mice and assessed the functional marker changes in the liver and kidneys. No significant statistical differences were found for the tested indicators. TAM/RES–LbL-LCNP treatment showed no apparent damages or histopathological abnormalities in the heart, lung, liver, spleen, and kidney histological images. The current findings observed for the first time propose that TAM/RES–LbL-LCNPs provide a new and safer method to use phytochemicals in combinatorial therapy and provide a novel treatment approach against breast cancers.

Comparing Effect of Annonaceae &amp; Emetine Nanoparticles on NFĸB p65 Pathway in Tongue Squamous Cell Carcinoma (scc-25)

Objective: The transformed cells in oral cancer secrete inflammatory mediators which generate an inflammatory microenvironment result in malignant transformation. Nuclear factor-κB (NF-κB) regulates a large number of genes which play a critical roles of the immune, inflammatory responses, cell proliferation, differentiation, and survival. Nanoparticles are 100 to 10,000 times smaller than human cells, could be designed to be highly selective for tumors and allow a slow release of active anticancer agents. We performed this study to investigate the effect of annonaceae nanoparticles and Emetine Emetine/Chitosan nanoparticles against scc-25 cell lines human tongue squamous cell carcinoma by studying their effect on NF-κB p65 expression. Methods: Cell Line cells were obtained from American Type Culture Collection, cells were cultured using DMEM (Invitrogen/Life Technologies). The effect of Annonaceae nanoparticles and EMETIN drug were investigated by Enzyme-Linked Immunosorbent Assay. The Signal is generated proportionally to the amount of bound NFĸB p65 and the intensity is measured by ROBONIK P2000 ELISA READER at 450 nm. Results: treatment of tongue scc-25 cells with both annonacea NP and Emetine/Chitosan NP revealed a significant increase in expression of NFĸB p65 in cancer cells by (227 & 261 pg/ml) respectively at incubation period 48 hours . While the control untreated cancer cells scc-25 revealed lower expression of NFĸB p65 at (147 pg/ml at incubation period 48 hour. Conclusion: Our findings demonstrate that annonaceae and Emetine nanoparticle might have regulatory effect on canonical NF-kb pathway by upregulation of NFĸB p65 expression signals.

The Potential of Exosomes Derived from Chronic Myelogenous Leukemia Cells As Biomarker Based on the BCR-ABL mRNA Transcript

PurposeExosomes are extracellular vesicles (EVs), 40-150 nm in diameter. They are released by all types of cells, and can potentially reflect the phenotypic state of the cell that produces them. Exosomes contain all the molecular constituents of a cell, including proteins, RNA, and DNA; they have been detected in many body fluids, including urine, cerebrospinal fluid, bile, ascites, and blood. Exosomes derived from chronic myelogenous leukemia (CML) cells may serve as novel targets for detecting the BCR-ABL mRNA transcript, and their contents may be used as a biomarker for the diagnosis and follow-up of patients with CML. However, no studies have been reported so far regarding this potential of exosomes. To explore the use of CML-derived exosomes as novel targets for detecting BCR-ABL mRNA transcripts, we isolated exosomes from two CML cell lines and from the sera of bone marrow (BM) blood of patients with CML, analyzed their characteristics, and performed PCR to measure the BCR-ABL mRNA transcript level.MethodsHuman CML cell lines (K562 and KU812) were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in an RPMI-1640 medium (Gibco, Carlsbad, CA, USA) with 10% exosome-depleted fetal bovine serum and 1% penicillin/streptomycin (Gibco). After culturing for 36-48 h, the supernatant was sequentially centrifuged at 5,000 × g for 30 min at 4 ºC, followed by another centrifugation at 10,000 × g for 30 min at 4 ºC to remove the detached cells. The exosomes were then isolated using a size-exclusion chromatography column packed with 10 mL of Sepharose CL-2B (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The exosomes from the serum of BM blood were isolated with ExoQuickTM Exosome Precipitation Solution (SBI, Mountain View, CA, USA). Total RNA from whole cells and exosomes was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Next, 25-100 ng of purified RNA was reverse-transcribed using the Prime Script RT-PCR kit (TaKaRa BIO, Shiga, Japan). Nested PCR was performed to detect the BCR-ABL mRNA transcripts.ResultsTransmission electron microscopy (TEM) was performed to study the morphology and size of the isolated exosomes. Exosomes derived from K562 and KU812 cells were found to be cup-shaped vesicles, 20-150 nm in diameter. Using dynamic light scattering, size distribution of the exosomes was found to be 30-190 nm in diameter. Exosome marker proteins (CD81 and CD9) were detected by western blotting; however, no endoplasmic reticulum marker protein (calnexin) was detected in the exosomes from either CML cell lines. This result confirmed that purified exosomes were obtained from the CML cell lines (Figure 1). Nested PCR was performed on these purified exosomes. A single band, corresponding to 250 bp, was detected in the exosomal RNA isolated from both the CML cell lines. RNA sequence analysis using NCBI/BLAST (National Center for Biotechnology Information, Bethesda, MD, USA) revealed 99% sequence homology between the detected 250-bp band and the partial mRNA for human BCR-ABL chimeric protein (Figure 2).Nested PCR was also performed on exosomes isolated from the serum of BM blood (one healthy control and three patients with CML). Bands of about 250 bp were detected in the exosomal RNA isolated from the sera of patients with CML. RNA sequence analysis revealed that exosomal RNA from patients with CML at the blast phase and accelerated phase showed 99% sequence homology with the partial mRNA for human BCR-ABL chimeric protein. Bands were observed in exosomal RNA from patients with CML at the chronic phase as well, although not to the extent that RNA sequence analysis could proceed (Figure 3).ConclusionsWe found that exosomes isolated from CML cells and the sera of BM blood contained the BCR-ABL mRNA transcript. This study revealed that CML-derived exosomes may act as novel targets for the detection of the BCR-ABL mRNA transcript, and can be used as a biomarker. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations.

Reverse transcription fluorescence resonance energy transfer‐polymerase chain reaction (FRET‐PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) (A23403G in the spike protein; C14408T in the RNA‐dependent RNA polymerase). Based on high‐resolution melting curve analysis, the reverse transcription (RT) FRET‐PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET‐PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.

Streptomyces venezuelae NRRL B-65442: genome sequence of a model strain used to study morphological differentiation in filamentous actinobacteria.

For over a decade, Streptomyces venezuelae has been used to study the molecular mechanisms that control morphological development in streptomycetes and is now a well-established model strain. Its rapid growth and ability to sporulate in a near-synchronised manner in liquid culture, unusual among streptomycetes, greatly facilitates the application of modern molecular techniques such as ChIP-seq and RNA-seq, as well as time-lapse fluorescence imaging of the complete Streptomyces life cycle. Here we describe a high-quality genome sequence of our isolate of the strain (Northern Regional Research Laboratory [NRRL] B-65442) consisting of an 8.2 Mb chromosome and a 158 kb plasmid, pSVJI1, which had not been reported previously. Surprisingly, while NRRL B-65442 yields green spores on MYM agar, the American Type Culture Collection (ATCC) type strain 10712 (from which NRRL B-65442 was derived) produces grey spores. While comparison of the genome sequences of the two isolates revealed almost total identity, it did reveal a single nucleotide substitution in a gene, vnz_33525, involved in spore pigment biosynthesis. Replacement of the vnz_33525 allele of ATCC 10712 with that of NRRL B-65442 resulted in green spores, explaining the discrepancy in spore pigmentation. We also applied CRISPR-Cas9 to delete the essential parB of pSVJI1 to cure the plasmid from the strain without obvious phenotypic consequences.