Engineering HEK293T cell line by lentivirus to produce miR34a-loaded exosomes.

BACKGROUND: RNA (ribonucleic acid) antisense is developing as a possible treatment option. As an RNA, miR-34a is involved in P53 function and cancer cell apoptosis. Although the therapeutic applications of miRNAs have several limitations, such as structural instability and susceptibility to nucleases. To resolve these issues, this study aims to apply exosomes as a delivery vehicle for miR-34a. AIMS: This study aims to create a cell factory to generate miR34a-enriched exosomes. The produced nanoparticles act as a delivery system and improve the structural stability of miR34a. METHODS: First exosome specific sequences were inserted into miR34a. The resulting miR34a oligonucleotide was transduced HEK293T cells genome with a lentiviral system. In the structure of miR34a oligonucleotide, six nucleotides were substituted to increase its packaging rate into exosomes. To maintain the secondary structure, stability, and expression of the miRNA gene, changes to the miR34a oligonucleotide were made using PCR (polymerase chain reaction) Extension. The forward-34a (5-TGGGGAGAGGCAGGACAGG-3) and Reverse-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA. RESULTS: The results confirmed that the changes in miR34a oligonucleotide do not affect its secondary structure. The energy level of the manipulated miR34a oligonucleotide was kept the same compared to the original one. Moreover, the loading of miR34a to the exosomes was increased. CONCLUSION: Our findings revealed that normal HEK293T did not express miR34a. However, lentiviral transduced miR34a oligonucleotide induced the loading of miR34a into the exosome. Moreover, replacing six nucleic acids in the 3' end of miR34a increased the loading of miR34a to exosome.

Aptamer-Based Magnetic Nanoprobe for Quantitative Measurement of Chloramphenicol in Milk through Portable Reader.

Background: We described here an aptamer-based magnetic nanoprobe for measuring the amount of chloramphenicol (CAP) in milk. Methods: The nanoprobe presented in this method consists of a magnetic nanoparticle conjugated to a specific CAP aptamer. If the target is detected in the sample, the nanoprobe binds to it, and the aptamer forms a G-quadruplex structure. This structure mimics the peroxidase activity in the presence of the hemin cofactor. If tetramethylbenzidine is added to the sample containing the nanoprobe, a blue color light is observed. After adding a stop reagent solution, the color produced is measured by a microplate reader and a portable meter. Results: This study proves a 99% positive linear relationship between the microplate reader's results and the portable meter results. Conclusion: Conjugation of the aptamer to magnetic nanoparticles and applying magnetic separation operations change the nanoprobe performance by 11% for both mentioned devices.

Fluorescent Detection of Methicillin Resistant Staphylococcus aureus by Loop-mediated Isothermal Amplification Assisted with Streptavidin-coated Quantum Dots.

Background: Methicillin Resistance Staphylococcus aureus (MRSA) could be considered as a major concern in medicine can cause nosocomial infection and bacteremia, especially in patients using catheter and household medical devices. Methods: Using molecular diagnostic methods are important for identification of MRSA from the Methicillin Sensitive Staphylococcus aureus (MSSA). Here we described a fluorescent assay using biotin-labelling Loop-mediated isothermal amplification (LAMP) method assisted with streptavidin-coated Quantum Dots (QDs) for detection of MRSA. For comparison, another fluorescent assay using LAMP assisted with Green Viewer (GV; a fluorescent dye) was applied for detection of MRSA. The mecA gene was selected as the target for amplification by LAMP and for biotin-labeling of the LAMP amplicons, biotin-11-dUTP was mixed with the dNTPs (deoxy Nucleotide Phosphates) in LAMP reaction. For determining the clinical performance of the developed assay, 30 blood samples with MRSA positive results were tested with QD-LAMP, the conventional LAMP, GV-LAMP, and Polymerase Chain Reaction (PCR). Results: Obtained results indicated that % sensitivity of QD-LAMP was 86.66% for detection of mecA positive MRSA samples; however, the Limit of Detection (LoD) of QD-LAMP was 1.5×104 Colony Forming Unit (CFU). Conclusion: The results suggested that the QD-LAMP assay was easy to operate and could be used for detection of MRSA in parallel to the blood culture with less sensitivity for detection of bacteremia and pediatric septicemia with low counts of MRSA.

Cysteine/Histidine-Dependent Amidohydrolase/Peptidase (CHAP)-Displayed Nano Phages: Antimicrobial Function against Methicillin-Resistant Staphylococcus aureus (MRSA).

BACKGROUND: Emergence and prevalence of multi drug resistance strains such as Methicillin-Resistant Staphylococcus aureus (MRSA) call for new antibacterial option. Endolysins as a new option is suggested. The phage display technique is suggested for production of recombinant endolysins. The recombinant endolysins displayed nano phages specifically lysis bacteria, which penetrate to the depth of tissue and the effective dose is reduced. METHODS: CHAPK gene was ligated in T7Select vector arms in T7Select10-3b cloning kit. To produce recombinant nano phages, ligation reaction was added directly to the packaging extract. Recombinant nano phages were amplified by Double Layer Agar assay (DLA). The recombinant nano phages were characterized using TEM. Size of recombinant nano phages was determined using DLS. The spot test was performed to confirm CHAPk -displayed on the surface of nano phages. The turbidimetry was used to investigate lytic activity of recombinant nano phages against MRSA ATCC No. 33591. RESULTS: The results showed recombinant nano phages belonged to order Caudovirales and family Podoviridae with titer 2×107 PFU/ml. According to the results of DLS, size of recombinant nano phages was 71 nm. Formation inhibition zone confirmed the presence of CHAPk on the surface of nano phage phenotypically. The turbidimetry showed lytic activity recombinant nano phages against MRSA after 5 min. CONCLUSION: This study suggests that CHAPk -displayed nano phages can be effective in MRSA infections.

AS-LAMP: A New and Alternative Method for Genotyping.

In recent decades, different methods have been introduced for the genotyping of Single Nucleotide Polymorphisms (SNPs) and mutations in nucleic acid sequences. These methods have several applications ranging from agriculture to medicine. The Loop-mediated isothermal amplification (LAMP) method was first introduced by Notomi et al. Since then, different methods derived from LAMP have been extensively applied in detecting pathogens. The LAMP method is an isothermal technique that amplifies the target DNA segment using four different primers that have been uniquely designed for recognizing six distinct zones on the objective gene; the process of reaction continues at a constant temperature via a strand displacement reaction. Amplifying and detecting the targeted zone can be accomplished in one stage. Although the LAMP method is mostly used for pathogen detection, several studies have used this method for genotyping. The present article reviewed various studies that used the LAMP method for SNP detection. The outcomes indicated that the LAMP technique could be a reliable and alternative technique for genotyping. Further studies are recommended to use this approach for genotyping.

Ultra structural characteristics of methicillin resistant Staphylococcus aureus cell wall after affecting with lytic bacteriophages using atomic force microscopy.

OBJECTIVES: During the last years with increasing resistant bacteria to the most antibiotics bacteriophages are suggested as appropriate treatment option. To investigate lytic activity of bacteriophages there are indirect microbial procedures and direct methods. The present study to complement microbial procedures and investigate ultra-structural characteristics of infection bacterium-phage use atomic force microscopy technique. MATERIALS AND METHODS: The Siphoviridae bacteriophages were isolated from sewage at the Tertiary Pediatric Hospital. Bacteriophages (10×108 PFU/ml) were diluted and were mixed with 100 µl of methicillin resistant Staphylococcus aureus (MRSA) ATCC 33591 (1.5×108 CFU/ml). The tubes were incubated for 20 min at 37 °C, at intervals 10 min, 10 µl samples were removed and directly were investigated MRSA ATCC morphology, roughness parameter, 3D topography, cell height, and fast Fourier transform (FFT) by atomic force microscopy (AFM) technique. Concurrently turbidity assay were performed. RESULTS: Concentration of MRSA ATCC No. 33591 strain after 10 min in phage-treated MRSA S3 (1.5×106 CFU/ml), S4 (1.5×105 CFU/ml), S5 (1.5×104 CFU/ml), S6 (1.5×103 CFU/ml) decreased 2-log, 3-log, 4-log, and 5-log respectively. The results AFM micrographs shown the most changes in bacterial morphology and 3D topography, destruction of cell wall, decrease of cell height, and loss of their shape after 10 min at phage-treated MRSA S3 (1.5×106 CFU/ml), S4 (1.5×105 CFU/ml), S5 (1.5×104 CFU/ml), S6 (1.5×103 CFU/ml) respectively . CONCLUSION: In this study MRSA ATCC ultra-structural changes in phage-treated MRSA ATCC groups directly were detected using AFM technique.

Allele-Specific Loop-Mediated Isothermal Amplification for the Detection of IVSII-I G>A Mutation On ß-Globin Gene.

BACKGROUND: Thalassemia is one of the most common genetic health problems in the world. More than 200 different mutations have been identified in the beta-globin gene and among the 24 ß-globin gene mutations in ß-thalassemia carriers in the north of Iran IVSII-I G>A mutation has the highest frequency. Using fast, inexpensive, simple and reliable methods for the detection of the mutations in ß-thal carriers is very important in prenatal diagnosis, and introduction of alternative methods to the existing ones can help to simplify the detection of mutations. Since its introduction, different methods derived from LAMP have been widely used for SNPs detection. AIM: This study was aimed to design a new method for the detection of IVSII-I G>A mutation on ß-globin gene based on AS - LAMP technique. METHODS: Primer explorer V5 software was used for the design of LAMP primers. Three sets of primers were designed. In the first set, the BIP primers were exactly complementary to the normal and mutant alleles. In the second set, 1 nucleotide (T) was inserted at the 5' end of BIP primers, and in the last set, one nucleotide at the 5' end of BIP primer was changed. The other required primers for the LAMP reaction (FIP, B3, and F3) were the same for all 3 sets of primers. The LAMP reaction was applied on three DNA samples (Wild type, Heterozygote and Homozygote for IVSII-I G>A mutation) and synthetic DNA. RESULTS: The results of the present study showed that LAMP reaction using three sets of primers could not successfully detect the IVSII-I G > A mutation among subjects DNA sample and synthetic DNA. CONCLUSION: Although several studies have successfully used ARMS-LAMP method to detect the SNPs, and other studies use a variety of methods to identify IVSII-I G>A mutation, the present study was unable to differentiate between a normal allele and IVSII-I G>A mutation. Hence further studies are recommended to consider redesigning of primer set, DNA concentration and using commercial LAMP Master Mix to detect the mutation.

Isothermal Amplification Methods for the SNP Genotyping.

The demands for genotyping techniques with acceptable precision, accuracy, cost-effectiveness in high throughput formats made driving forces for continuous development of novel technologies. A wide range of mutation detection techniques based on polymerase chain reaction (PCR) have been introduced. The best alternatives were the isothermal amplification technologies that those did not require a thermal cycler. In this review, we aimed to describe the most known isothermal amplification techniques for SNP genotyping.

Ultrastructural Investigation of Leishmania major Promastigotes Treated with A New Potent Antileishmanial Azole Using Scanning Electron and Atomic Force Microscopes.

AIM AND BACKGROUND: Azoles as antifungal drugs have been used to treat leishmaniasis for many years. Several evidences suggesting that the primary target of azoles is the heme protein, which co-catalyzes cytochrome P-450-dependent 14α-demethylation of lanosterol. Little is known about the structural changes caused by azoles with atomic force microscopy (AFM) or scanning electron microscopy (SEM). In the current work, several patented antileishmanial agents reviewed (US8809555) (US 0269803 A1) (TW201802093 A). The present study aimed to determine ultrastructural damage in Leishmania major (L.major) induced by the newly synthesized azole. METHODS: In this study, we investigated the morphological alterations of the parasite treated with our new synthesized azole namely trans-2-(4-chlorophenyl)-2,3-dihydro-3-(1Himidazol- 1-yl)-4H-1-benzopyran-4-one (IF-2) against L.major promastigotes stage using two high-resolution microscopic techniques: atomic force microscopy and scanning electron microscopy. RESULTS: The results showed remarkable topographical and morphological alterations in the cell membrane at promastigote stage of L. major treated with the potent investigated azole (IF-2) ( IC50 values ≤8.9 µg/mL). Both techniques revealed membrane damage and also losing the flagellum in the observed cells. CONCLUSION: Our results strongly confirm the Leishmania cell wall as a potent target for the new synthesized azole (IF-2). Accordingly, focus on membrane integrity and glycoconjugates of Leishmania parasite to design new therapeutic agents is recommended.

The sensitive detection of IVSII-1(G˃A) mutation in beta globin gene using a Nano-based ligation genotyping system.

Beta-thalassemia (ß-thalassemia) is a globally genetic diseases, and is most prevalent in the Middle East, particularly in Iran. Carrier detection and prenatal diagnosis are the best ways to managing it, and to prevent new community cases from emerging. We report on a simple method for rapid detection of the worst ß-thalassemia point mutation in Iran (IVS-II-1 G>A), using a nano-based ligation assay, this was performed using probes with labeled magnetic nanoparticles and quantum dots. After optimizing the technique, 50 DNA samples were genotyped with this method. We found a frequency of 72% for IVSII-1 (G˃A) mutation (42% heterozygote, and 30% mutant homozygote) with a highly sensitive nano-based ligation genotyping system, offering excellent sensitivity and specificity for point mutation detection; it has been demonstrated to be inaccurate, sensitive, cost-effective, and rapid technique for single nucleotide polymorphism (SNP) genotyping.

Fabrication of DNA nanotubes with an array of exterior magnetic nanoparticles.

Described here a methodology for arraying of magnetic nanoparticles (MNPs) on the surface of DNA nanotubes (DNTs). Positioning of magnetic nanoparticles at exterior surface of DNTs were shaped after self-assembling of oligonucleotide staples within an M13mp18 DNA scaffold via an origami process. The staples were partially labeled with biotin to be arrayed at the surface of DNTs. Gel retardation assay of the DNTs carrying magnetic nanoparticles indicated a reversely behavioral electrophoretic movement in comparison to the nanotubes have been demonstrated previously. Also, high resolution transmission electron microscopy confirmed positioning magnetic nanoparticles at the exterior surface of DNTs, correctly. Ultrastructural characteristics of these DNA nanotubes using atomic force microscopy demonstrated topographic heights on their surfaces formed through positioning of magnetic nanoparticles outside the tubules. This nanoarchitecture would be potential for multiple arraying of nanoparticles that those be useful as functionalized chimeric nanocarriers for developing novel nanodrugs and nanobiosensors.

A multiplex restriction enzyme-PCR for unequivocal identification and differentiation of Trichostrongylus species in human samples.

Trichostrongylus species remain one of the major health challenges in the tropical and summer rainfall regions worldwide. Identification of strongylid species diagnostic methods is vital for obtaining a deep understanding of the epidemiology, population biology, anthelmintic treatment efficacy, and drug resistance in order to design effective parasite control strategies. We evaluated a multiplex RE-PCR for the diagnosis of key Trichostrongylus spp. Genomic DNA amplification of Trichostrongylus colubriformis, Trichostrongylus axei and Trichostrongylus vitrinus was achieved as standard sample using specific primers located in the second internal transcribed spacer (ITSII) of nuclear ribosomal DNA (rDNA). The mentioned method was based on isolation of Trichostrongylus ova from human fecal samples using Willis method, the extraction of ova genomic DNA samples, followed by rDNA ITSII PCR and one-step multiplex RE-PCR using three restriction enzymes of HinfI, DraI, and MseI. The multiplex RE-PCR technique provides a useful tool for discriminating all Trichostrongylus spp., being useful for diagnostic, epidemiological, ecological studies, and control programs. This method is rapid, especially when numerous restriction enzymes are required for species differentiation or identification.

The effect of green tea extract supplementation on sputum smear conversion and weight changes in pulmonary TB patients: A randomized controlled trial.

BACKGROUND: Acceleration in sputum smear conversion helps faster improvement and decreased probability of the transfer of TB. In this study, we aimed to investigate the effect of green tea extract supplementation on sputum smear conversion and weight changes in smear positive pulmonary TB patients in Iran. METHODS: In this double blind clinical study, TB patients were divided into intervention, (n=43) receiving 500 mg green tea extract (GTE), and control groups (n=40) receiving placebo for two months, using balanced randomization. Random allocation and allocation concealment were observed. Height and weight were measured at the beginning, and two and six months post-treatment. Evaluations were performed on three slides, using the ZiehlNeelsen method. Independent and paired t test, McNemar's, Wilcoxon, Kaplan-Meier, Cox regression model and Log-Rank test were utilized. Statistical significance was set at p<0.05. This trial was registered under IRCT201212232602N11. RESULTS: The interventional changes and the interactive effect of intervention on weight were not significant (p>0.05). In terms of shortening the duration of conversion, the case to control proportion showed a significant difference (p=0.032). Based on the Cox regression model, the hazard ratio of the relative risk of delay in sputum smear conversion was 3.7 (p=0.002) in the higher microbial load group compared to the placebo group and 0.54 (95% CI: 0.31-0.94) in the intervention compared to the placebo group. CONCLUSION: GTE decreases the risk of delay in sputum smear conversion, but has no effect on weight gain. Moreover, it may be used as an adjuvant therapy for faster rehabilitation for pulmonary TB patients.

Non-invasive prenatal diagnosis of ß-thalassemia by detection of the cell-free fetal DNA in maternal circulation: a systematic review and meta-analysis.

The discovery of fetal DNA (f-DNA) opens the possibility of early non-invasive procedure for detection of paternally inherited mutation of beta-thalassemia. Since 2002, some studies have examined the sensitivity and specificity of this method for detection of paternally inherited mutation of thalassemia in pregnant women at risk of having affected babies. We conducted a systematic review of published articles that evaluated using this method for early detection of paternally inherited mutation in maternal plasma. A sensitive search of multiple databases was done in which nine studies met our inclusion criteria. The sensitivity and specificity was 99 and 99 %, respectively. The current study found that detection of paternally inherited mutation of thalassemia using analysis of cell-free fetal DNA is highly accurate. This method could replace conventional and invasive methods.

High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers.

OBJECTIVES: The high-resolution melting (HRM) technique is fast, effective and successful method for mutation detection. The aim of this study was to determine the sensitivity and specificity of the HRM method for detection of a paternally inherited mutation in a fetus as a noninvasive prenatal diagnosis of ß-thalassemia. METHODS: Genomic DNAs were prepared from 50 ß-thalassemia minor couples whose pregnancy was at risk for homozygous ß-thalassemia. Ten milliliters of the maternal blood from each pregnant woman were collected and after separating plasma stored at -80 °C until analysis. The extracted DNAs were analyzed by HRM real-time PCR for detection of IVS-II-I (G-A) as a paternally inherited mutation. The gold standard was the result of a chorionic villus sampling by a standard reverse dot blotting test. RESULTS: The sensitivity and specificity of HRM real-time PCR were 92.6% and 82.6%, respectively. Also, the positive and negative predictive values were 86.2% and 90.47%, respectively. CONCLUSIONS: HRM real-time PCR was a sensitive and specific method for determining the paternally inherited mutation in the fetus at risk with thalassemia major.

Ultrastructural characterizations of DNA nanotubes using scanning tunneling and atomic force microscopes.

The potential applications of scanning tunneling microscopy and atomic force microscopy for the characterizations of DNA nanotubes in nanoscale have been described here. The nanotubes were designed using the Cadnano software according to M13 mp18 DNA as a scaffold. DNA nanotubes were fabricated using the origami technique assisted with ligase treatment subsequently. Transmission electron microscopy confirmed the morphology of DNA nanotubes. For the topographic characterization of DNA nanotubes, an atomic force microscope was used in comparison to a scanning tunneling microscope. The scanning tunneling microscopy results revealed a high-resolution topography of DNA nanotubes in the constant-current mode; however, more details of the self-assembly in DNA strands in nanotubes were explored by atomic force microscopy with contact mode (or constant height). Our findings suggested that those two microscopes could be candidates for ultrastructural characterizations of DNA nanotubes for obtaining two- and three-dimensional micrographs.

Spectroscopic and microscopic analyses of rod-shaped gold nanoparticles interacting with single-stranded DNA oligonucleotides.

The application of rod-shaped gold nanoparticles as probes and carriers in biological systems have recently attracted great interest. UV-vis spectroscopy, circular dichroism, Fourier transform infrared spectroscopy, scanning electron microscopy and atomic force microscopy were used to study optical and structural properties of rod-shaped gold nanoparticles when interacting with DNA oligomers in phosphate sodium salt buffer. The morphological transformation process of rod-shaped gold nanoparticles resulting from the interaction with single-stranded DNA (ssDNA) showed the role of hexadecyltrimethylammonium bromide (CTAB) in nanostructures as the main interacting agent. The obtained results confirmed that the CTAB coat of rod-shaped gold nanoparticles have powerful positive charges for conjugations with surface negative charges of phosphate groups on ssDNA oligomers. The CTAB also inhibit the formation of covalent sulphide bonds between the gold core of rod-shaped nanoparticles and alkanethiol oligonucleotides. The authors found that when the nanorods were exposed to ssDNA oligonucleotides, the gold nanorods changed their shapes and sizes, and exposed some microscopic malformations which could be used in the development of colorimetric assays of nucleic acids.

A nanodiagnostic colorimetric assay for 18S rRNA of Leishmania pathogens using nucleic acid sequence-based amplification and gold nanorods.

BACKGROUND AND OBJECTIVE: We describe here a nanodiagnostic colorimetric assay for 18S rRNA of Leishmania pathogens that uses nucleic acid sequence-based amplification (NASBA) and gold nanorods (GNRs). METHODS: NASBA primers targeting 18S rRNA were used for amplification of RNA in an isothermal process. The electrostatic interactions between the phosphate groups of the RNA amplicons and the cetyl trimethylammonium bromide layer on the GNRs resulting in their aggregation. This phenomenon changes the color of the GNR solution from red to purple. RESULTS: Our data showed 100% sensitivity and 80% specificity with the colorimetric assay compared with results using reverse transcription polymerase chain reaction. CONCLUSION: The nanodiagnostic method we describe simplifies the detection of NASBA amplicons without the need for gel electrophoresis.

Biomolecular and structural analyses of cauliflower-like DNAs by ultraviolet, circular dichroism, and fluorescence spectroscopies in comparison with natural DNA.

Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template, and a Bst DNA polymerase in 75- and 90-min periods. These times led to manufacturing two types of cauliflower-like DNAs with different contents of inverted repetitions and stem-loops, which were clearly indicated by two comparable electrophoresis patterns in agarose gel. LAMP-fabricated DNAs and natural dsB-DNA (salmon genomic DNA) were dialyzed in Gomori phosphate buffer (10 mM, pH 7.4) to be isolated from salts, nucleotides, and primers. Dialyzed DNAs were studied using UV spectroscopy, circular dichroism spectropolarimetry, and fluorescence spectrophotometry. Structural analyses indicated reduction of the molecular ellipticity and extinction coefficients in comparison with B-DNA. Also, cauliflower-like DNAs demonstrated less intrinsic and more extrinsic fluorescence in comparison with natural DNA. The overwinding and lengthening of the cauliflower-like configurations of LAMP DNAs led to changes in physical parameters of this type of DNA in comparison with natural DNA. The results obtained introduced new biomolecular characteristics of DNA macromolecules fabricated within a LAMP process and show the effects of more inverted repeats and stem-loops, which are manufactured by lengthening the process.

Effect of ionic liquids on the structure, stability and activity of two related α-amylases.

Ionic liquids are recognized as green solvents for carbohydrates dissolution. However, only a limited number of studies have been carried out to investigate their effect on carbohydrate hydrolyzing enzymes. We have investigated the influence of two water miscible ionic liquids on the activity, stability and structure of two related α-amylases from Bacillus amyloliquefaciens and Bacillus lichiniformis. Upon changes in ionic liquids concentrations, both enzymes activity and stability were reduced. Associated thermodynamic and conformational changes were observed using differential scanning calorimetry and fluorescence techniques. Thermal denaturation was accompanied by aggregation in both aqueous buffer and [BMIm][Cl] but [HMIm][Cl] significantly suppressed aggregation.