Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

A Long-Standing Giant Mandibular Ameloblastoma and its Management with Microvascular Free Fibular Graft: a Case Report.

Ameloblastoma is one of the most common benign epithelial odontogenic tumors of jaws. We report a case of long-standing slow-growing giant ameloblastoma involving almost all of mandibular bone. The solid multicystic lesion was excised, and the histopathological examination showed the follicular type of ameloblastoma. Furthermore, the defect was reconstructed with microvascular osteocutaneous free fibular graft.

A new post-synthetic polymerization strategy makes metal-organic frameworks more stable.

Metal-organic frameworks are of interest in a number of host-guest applications. However, their weak coordination bonding often leads to instability in aqueous environments, particularly at extreme pH, and hence, is a challenging topic in the field. In this work, a two-step, post-synthetic polymerization method is used to create a series of highly hydrophobic, stable MOF composites. The MOFs are first coated with thin layers of polydopamine from free-base dopamine under a mild oxygen atmosphere, which then undergoes a Michael addition to covalently graft hydrophobic molecules to the external MOF surface. This easy, mild post-synthetic modification is shown to significantly improve the stability of a number of structurally diverse MOFs including HKUST-1 (Cu), ZIF-67 (Co), ZIF-8 (Zn), UiO-66 (Zr), Cu-TDPAT (Cu), Mg-MOF-74 (Mg) and MIL-100 (Fe) in wet, caustic (acidic and basic) environments as determined by powder X-ray diffraction and surface area measurements.

Molecular epidemiology of the hemagglutinin gene of prevalent influenza virus A/H1N1/pdm09 among patient in Iran.

In 2015, the influenza virus A/H1N1/pdm09 strain outbreak became prevalent throughout the different provinces of Iran. There are relatively limited complete genetic sequences available for this virus from Asian countries. Diagnosis and virological surveillance of influenza is essential for detecting novel genetic variants causing epidemic potential. This study describes the genetic properties of HA genome of influenza A/H1N1 pdm09 viruses circulating in Iran during the 2015/2016 season. In order to investigate the genetic pattern of influenza A/H1N1 pdm09, a total of 1758 nasopharyngeal swabs were screened by real-time RT-PCR. Of those, 510 cases were found to be positive for A/H1N1/pdm09 virus. Evolution of the approximately 100 positive specimens with high virus load was conducted via genomic phylogeny. Phylogenetic analysis of the HA genes of the A/H1N1pdm09 viruses revealed the circulation of clade 6B1, characterized by amino acid substitutions S84N, S162N and I216T, where position 162 became glycosylated. The N-glycosylation of HA protein is post or co-translational modification that affect the evolution of influenza viruses. For influenza A(H1N1) pdm09 viruses, we found more mutations in the antigenic sites than in the stem region. The results of this study confirmed the necessity of constant regular antigenic and molecular surveillance of circulating seasonal influenza viruses.

Correction to: Blocking of opioid receptors in experimental formaline-inactivated respiratory syncytial virus (FI-RSV) immunopathogenesis: from beneficial to harmful impacts.

In the original publication, seventh author's name was incorrectly published as 'Maryam Golaram'.

Simultaneous formulation of influenza vaccine and chitosan nanoparticles within CpG oligodesoxi nucleotides leads to dose-sparing and protects against lethal challenge in the mouse model.

Lack of efficient delivery systems for transporting antigenic molecules to the cytosol of antigen-presenting cells presents a major obstacle for antigen uptake by immune cells. To this end, influenza whole inactivated virus vaccines were formulated with chitosan nanoparticles and CpG oligonucleotide as a biodegradable delivery system and a Th1-specific adjuvant, respectively. Intradermal injections of a single high dose and low dose of formulated candidate vaccines were carried out. Thirty days after injection, cell proliferation assay (MTT), IFN-gamma and IL-4 ELISpot assays were conducted. Sera samples were collected 21 days after immunization to measure IgG1 and IgG2a levels. In addition, the mice challenged with mouse-adopted virus were monitored for weight loss. The results show a significant stimulation of both humoral and cellular immunities; also, weight gain and a decrease in mortality in the mice receiving both dosages of inactivated influenza virus vaccines with CpG and Chitosan coating were observed. Based on the results, it can be concluded that formulation of inactivated influenza virus with CpG and its delivery by chitosan as low-dose can return the same results as with high-dose balanced between cellular and humeral immune responses. This formulation could potentially lead to a significant saving in vaccine production.

Blocking of opioid receptors in experimental formaline-inactivated respiratory syncytial virus (FI-RSV) immunopathogenesis: from beneficial to harmful impacts.

Opioid system plays a significant role in pathophysiological processes, such as immune response and impacts on disease severity. Here, we investigated the effect of opioid system on the immunopathogenesis of respiratory syncytial virus (RSV) vaccine (FI-RSV)-mediated illness in a widely used mouse model. Female Balb/c mice were immunized at days 0 and 21 with FI-RSV (2 × 106 pfu, i.m.) and challenged with RSV-A2 (3 × 106 pfu, i.n.) at day 42. Nalmefene as a universal opioid receptors blocker administered at a dose of 1 mg/kg in combination with FI-RSV (FI-RSV + NL), and daily after live virus challenge (RSV + NL). Mice were sacrificed at day 5 after challenge and bronchoalveolar lavage (BAL) fluid and lungs were harvested to measure airway immune cells influx, T lymphocyte subtypes, cytokines/chemokines secretion, lung histopathology, and viral load. Administration of nalmefene in combination with FI-RSV (FI-RSV + NL-RSV) resulted in the reduction of the immune cells infiltration to the BAL fluid, the ratio of CD4/CD8 T lymphocyte, the level of IL-5, IL-10, MIP-1α, lung pathology, and restored weight loss after RSV infection. Blocking of opioid receptors during RSV infection in vaccinated mice (FI-RSV-RSV + NL) had no significant effects on RSV immunopathogenesis. Moreover, administration of nalmefene in combination with FI-RSV and blocking opioid receptors during RSV infection (FI-RSV + NL-RSV + NL) resulted in an increased influx of the immune cells to the BAL fluid, increases the level of IFN-γ, lung pathology, and weight loss in compared to control condition. Although nalmefene administration within FI-RSV vaccine decreases vaccine-enhanced infection during subsequent exposure to the virus, opioid receptor blocking during RSV infection aggravates the host inflammatory response to RSV infection. Thus, caution is required due to beneficial/harmful functions of opioid systems while targeting as potentially therapies.

IRES-based co-expression of influenza virus conserved genes can promote synergistic antiviral effects both in vitro and in vivo.

Vaccination is the most effective method for the prevention of influenza virus infection. Currently used influenza vaccines that target the highly polymorphic viral surface antigens can provide protection when well matched with circulating virus strains. Antigenic drift or cyclically occurring pandemics may hamper the efficacy of these vaccines, which are chosen prior to each flu season. Therefore, a universal vaccine, designed to induce broadly cross-protective immunity against the highly conserved internal antigens M1 and nucleoprotein could provide durable protection against various influenza virus subtypes, and it could also reduce the impact of pandemic influenza, which occurs less frequently. Here, we describe a new influenza vaccine candidate in which two highly conserved antigens, nucleoprotein (NP) and matrix (M1), are simultaneously expressed from a bicistronic vector termed pIRESM1/NP. Mice were immunized intradermally four times with the pIRESM1/NP construct. The protection efficacy of the gene-based vaccine was assessed by IFN-γ and Granzyme B ELISpot assays, follow-up observation of weight loss, and survival rates of the mice groups against lethal challenges with influenza A virus subtypes H1N1 and H5N1. The group that received pIRESM1/NP showed full protection against disease following lethal challenge with H1N1 and H5N1. This group also generated significantly higher host immune cellular responses than the other groups. These results demonstrate that a DNA vaccine strategy based on co-expression of the M1 and NP proteins could provide an effective way to control influenza virus infection.

Inhibiting influenza virus replication and inducing protection against lethal influenza virus challenge through chitosan nanoparticles loaded by siRNA.

Influenza virus causes a highly contagious viral respiratory tract infection with potentially fatal outcomes in humans and animals. There is now widespread influenza virus resistance to commercial drugs due to the genetic diversity of virus. Therefore, new therapeutic formulation needs to be developed. Chitosan/siRNA nanoparticles were generated as a new therapeutic approach against influenza virus infections both in vitro and in vivo. Designed siRNA against influenza nucleoprotein was formulated in chitosan polymer as siRNA/chitosan nanoparticle complex. Particle size and zeta potential of the nanoparticles were measured by dynamic light scattering. The uptake of labeled siRNA into Vero cells was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of enhanced green fluorescent protein (EGFP) was analyzed and quantified by flow cytometry in Vero cells. Results of the in vitro study showed that chitosan/siRNA nanoparticle was efficiently uptaken by Vero cells, leading to inhibition of influenza virus replication. Furthermore, nasal delivery of siRNA by chitosan nanoparticle complex has antiviral effects and significantly protected BALB/c mice from a lethal influenza challenge. These findings suggest that chitosan nanoparticle equipped with siRNA is a promising system for controlling influenza virus infection.

Autophagy induction regulates influenza virus replication in a time-dependent manner.

PURPOSE: Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research. METHODOLOGY: In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin-1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation.Results/Key findings. Beclin-1 overexpression in the cells infected by virus induced autophagy to 26 %. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50 % infection) of replicating virus were measured at 24 and 48 h post-infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post-infection (P≤0.01), but it was not significantly different from the control at 48 h post-infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post-infection. Additionally, we showed that inhibition of autophagy using 3-methyladenine reduced viral replication. CONCLUSION: This study revealed that the virus (H1N1) titre was controlled in a time-dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production.

Correction: Ghasemi, M., et al. Increase of Chamazulene and α-Bisabolol Contents of the Essential Oil of German Chamomile (Matricaria chamomilla L.) Using Salicylic Acid Treatments under Normal and Heat Stress Conditions Foods 2016, 5, 56.

The authors wish to make the following corrections to their paper [...].

Oil-in-water emulsion formulated with eucalyptus leaves extract inhibit influenza virus binding and replication in vitro.

Throughout human history, the human-beings have been used different types of plants as antimicrobial agents in fight against infectious diseases. Influenza virus is one of the most common causes of respiratory infection and transmitted through direct contact with flu infected individuals and contaminated substances or droplets. In the current study, both oil-in-water and water-in-oil emulsions with hydroalcoholic extract of eucalyptus leaves (OLHE) were developed and their antiviral efficiency was evaluated. To doing so, Madin-Darbey Canine Kidney (MDCK) cells were treated with effective minimal cytotoxic concentration of the formulated emulsions. The treated cells were then infected with 50% cell culture infectious dose (100 CCID50) of the A/H1N1 virus (the swine flu). The viral titers were measured by hemagglutination (HA) and cell culture infectious dose 50% (CCID50) assays. Also, to check the virus binding inhibition via the formulated extract, the viruses were incubated with the formulated extracts. Our study showed that the oil-in-water emulsions formulated with 2% eucalyptus leaves extract inhibited virus replication completely when the cells were infected by 100 CCID50 and decreased HA titer up to four fold. Therefore, this formulation, may hold promising application to prevent influenza virus transmission through direct contact among children and passengers.

Removal of Lead(II) ions from aqueous solutions using biocompatible polymeric nano-adsorbents: A comparative study.

Low-cost and biocompatible chitin nanofiber and chitosan nanoparticle as natural polymers synthesized successfully by means of a mechanical and a chemical procedure respectively. The surface properties and size of chitin nanofibers (CNFs) and chitosan nanoparticles (CNPs) were characterized using a scanning electron microscope (SEM). CNF and CNP were utilized for separation of lead(II) metal ions from aqueous solution, and the influence of effective factors were investigated. It was found that both adsorbents can adsorb lead ions from the solution effectively, but sorption efficiency for CNP at all the stages was greater than CNF. Furthermore, the results of corresponding equilibrium data with adsorption isotherms revealed that CNF and CNP are more compatible with Freundlich and Langmuir adsorption isotherms respectively. It shows that, for CNF, adsorption process occurs as a heterogeneous and multi-layer process, while for CNP, adsorption process is predicted to befall as a homogenous and single-layer process. At the end kinetics and thermodynamic studies were performed for both Nano-adsorbents.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

A novel chimeric influenza virosome containing Vesicular stomatitis G protein as a more efficient gene delivery system.

OBJECTIVES: To enhance the efficiency of influenza virosome-mediated gene delivery by engineering this virosome. RESULTS: A novel chimeric influenza virosome was constructed containing the glycoprotein of Vesicular stomatitis virus (VSV-G), along with its own hemagglutinin protein. To optimize the transfection efficiency of both chimeric and influenza cationic virosomes, HEK cells were transfected with plasmid DNA and virosomes and the transfection efficiency was assessed by FACS analysis. The chimeric virosome was significantly more efficient in mediating transfection for all amounts of DNA and virosomes compared to the influenza virosome. CONCLUSIONS: Chimeric influenza virosome, including VSV-G, is superior to the conventional influenza virosome for gene delivery.

Lanthanide metal-organic frameworks as selective microporous materials for adsorption of heavy metal ions.

Four microporous lanthanide metal-organic frameworks (MOFs), namely Ln(BTC)(H2O)(DMF)1.1 (Ln = Tb, Dy, Er and Yb, DMF = dimethylformamide, H3BTC = benzene-1,3,5-tricarboxylic acid), have been used for selective adsorption of Pb(ii) and Cu(ii). Among these MOFs, the Dy-based MOF shows better adsorption property and selectivity toward Pb(ii) and Cu(ii) ions. Adsorption isotherms indicate that sorption of Pb(ii) and Cu(ii) on MOFs is via monolayer coverage. Preconcentration is based on solid-phase extraction in which MOFs were rapidly injected into water samples and adsorption of metal ions was rapid because of good contact with analyte; then adsorbed Pb(ii) and Cu(ii) ions were analyzed by FAAS. The optimized methodology represents good linearity between 1 and 120 µg L(-1) and detection limit of 0.4 and 0.26 µg L(-1) for Pb(ii) and Cu(ii), respectively. Subsequently the method was evaluated for preconcentration of target metal ions in some environmental water samples.

Heavy metals in vegetables sold in the local market in Jordan.

Heavy metals (As, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn) in various vegetables (cabbage, green onion, lettuce, parsley, rocket, spinach, carrot, onion, potato and cauliflower) from the market in Jordan were measured using inductively coupled plasma-mass spectrometry. As, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn ranged from 0.009-0.275 mg kg(-1) wet weight, 0.004-0.060 mg kg(-1), 0.003-0.401 mg kg(-1), 0.105-3.51 mg kg(-1), 0.15-1.15 mg kg(-1), 0.93-14.39 mg kg(-1), 0.044-0.702 mg kg(-1), 0.072-0.289 mg kg(-1) and 2.23-6.65 mg kg(-1), respectively. Parsley, followed by spinach, contained the highest concentration of heavy metals. Onion contained high levels of toxic heavy metals. The content of Cu in parsley and spinach and Pb in onion exceeded the Codex limits. However, the daily intake of heavy metals from the tested vegetables was lower than the maximum limits for allowable intake.

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production.

OBJECTIVES: To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection. RESULTS: Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively. CONCLUSIONS: MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

Increase of Chamazulene and α-Bisabolol Contents of the Essential Oil of German Chamomile (Matricaria chamomilla L.) Using Salicylic Acid Treatments under Normal and Heat Stress Conditions.

The chamazulene and α-(-)-bisabolol contents and quality of the chamomile oil are affected by genetic background and environmental conditions. Salicylic acid (SA), as a signaling molecule, plays a significant role in the plant physiological processes. The aim of this study was to evaluate the chemical profile, quantity, and improve the essential oil quality as a consequence of the increase of chamazulene and α-(-)-bisabol using salicylic acid under normal and heat stress conditions by the gas chromatography-mass spectrometry (GC-MS) technique. The factorial experiments were carried out during the 2011-2012 hot season using a randomized complete block design with three replications. The factors include four salicylic acid concentrations (0 (control), 10, 25 and 100 mg·L-1), and three chamomile cultivars (Bushehr, Bona, Bodegold) were sown on two different planting dates under field conditions. Fourteen compounds were identified from the extracted oil of the samples treated with salicylic acid under normal and heat stress conditions. The major identified oil compositions from chamomile cultivars treated with salicylic acid were chamazulene, α-(-)-bisabolol, bisabolone oxide, ß-farnesene, en-yn-dicycloether, and bisabolol oxide A and B. Analysis of variance showed that the simple effects (environmental conditions, cultivar and salicylic acid) and their interaction were significant on all identified compounds, but the environmental conditions had no significant effect on bisabolol oxide A. The greatest amount of chamazulene obtained was 6.66% at the concentration of 10 mg·L-1 SA for the Bona cultivar under heat stress conditions, whereas the highest α-(-)-bisabolol amount attained was 3.41% at the concentration of 100 mg·L-1 SA for the Bona cultivar under normal conditions. The results demonstrated that the application of exogenous salicylic acid increases the quantity and essential oil quality as a consequence of the increase of chamazulene and α-(-)-bisabolol under normal and heat stress conditions.

A Novel Cobalt-Sensitive Fluorescent Chemosensor Based on Ligand Capped CdS Quantum Dots.

In this work, a ligand capped CdS QDs was synthesized, characterized and its fluorescence behavior was studied. The surface of the CdS QDs was modified using N-(3-methyl-2-(thiophene-2-carboxamido) phenyl) thiophene-2-carboxamide. The immobilized ligand on the surface of the CdS QDs can interact by cationic species due to the existence of donating atoms in its structures. Thus, effect of some metal cations on the fluorescent intensity of the ligand capped CdS QDs were studied. It was found that fluorescence intensity of the modified CdS QDs quenched selectively by addition of Co(II) ion in comparison with other cations tested. The ligand capped CdS QDs can be used as a fluorescent bulk chemosensor for detection of Co(II) ions. The fluorescent quenching is linear in the range of 1.0 × 10(-5) to 1.5 × 10(-4) mol L(-1) of Co(II) ions. The limit of detection was obtained 8.3 × 10(-7) mol L(-1). The nanosensor exhibits high selectivity toward Co(II) ions in comparison with common metal ions.