Antibiotic Minocycline Prevents Respiratory Syncytial Virus Infection.

Treatment drugs, besides their specific activity, often have multiple effects on the body. The undesired effect of the drug may be repurposed as therapeutics, saving significant investigative time and effort. Minocycline has anti-cancer, anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Presently, minocycline is also known to show anti-viral activity against Influenza virus, Japanese encephalitis virus, Simian immunodeficiency virus, Human immunodeficiency virus and West Nile virus. Here, we investigate the effect of minocycline on Respiratory syncytial virus (RSV), a common respiratory virus that causes severe mortality and morbidity in infants, children, and older adult populations. Currently, there is no effective vaccine or treatment for RSV infection; hence, there is a critical need for alternative and effective drug choices. Our study shows that minocycline reduces the RSV-mediated cytopathic effect and prevents RSV infection. This is the first study demonstrating the anti-viral activity of minocycline against RSV.

A three-dimensional human skin model to evaluate the inhibition of Staphylococcus aureus by antimicrobial peptide-functionalized silver carbon nanotubes.

OBJECTIVE: To investigate the toxicity and antibacterial application of antimicrobial peptide-functionalized silver-coated carbon nanotubes against Staphylococcus infection using a full thickness human three-dimensional skin model. MATERIALS AND METHODS: The three-dimensional skin formation on the scaffolds was characterized by electron microscopy and investigation of several skin cell markers by real time-reverse transcriptase polymerase chain reaction. Functionalized silver-coated carbon nanotubes were prepared using carboxylated silver-coated carbon nanotubes with antimicrobial peptides such as TP359, TP226 and TP557. Following the characterization and toxicity evaluation, the antibacterial activity of functionalized silver-coated carbon nanotubes against Staphylococcus aureus was investigated using a bacterial enumeration assay and scanning electron microscopy. For this purpose, a scar on the human three-dimensional skin grown on Alvetex scaffold using keratinocytes and fibroblasts cells was created by taking precaution not to break the scaffold beneath, followed by incubation with 5 µg/mL of functionalized silver-coated carbon nanotubes re-suspended in minimum essential medium for 2 h. Post 2-h incubation, 200 µL of minimum essential medium containing 1 × 104 colony forming units of Staphylococcus aureus were incubated for 2 h. After incubation with bacteria, the colony forming unit/gram (cfu/g) of skin tissue were counted using the plate count assay and the samples were processed for scanning electron microscopy analysis. RESULTS: MTT assay revealed no toxicity of functionalized silver-coated carbon nanotubes to the skin cells such as keratinocytes and fibroblasts at 5 µg/mL with 98% cell viability. The bacterial count increased from 104 to 108 cfu/g in the non-treated skin model, whereas skin treated with functionalized silver-coated carbon nanotubes showed only a small increase from 104 to 105 cfu/g (1000-fold viable cfu difference). Scanning electron microscopy analysis showed the presence of Staphylococcus aureus on the non-treated skin as opposed to the treated skin. CONCLUSION: Thus, our results showed that functionalized silver-coated carbon nanotubes are not only non-toxic, but also help reduce the infection due to their antibacterial activity. These findings will aid in the development of novel antibacterial skin substitutes.

Evaluation of E. coli inhibition by plain and polymer-coated silver nanoparticles.

Escherichia coli causes various ailments such as septicemia, enteritis, foodborne illnesses, and urinary tract infections which are of concern in the public health field due to antibiotic resistance. Silver nanoparticles (AgNP) are known for their biocompatibility and antibacterial activity, and may prove to be an alternative method of treatment, especially as wound dressings. In this study, we compared the antibacterial efficacy of two polymer-coated silver nanoparticles either containing 10% Ag (Ag 10% + Polymer), or 99% Ag (AgPVP) in relation to plain uncoated silver nanoparticles (AgNP). Atomic force microscopy was used to characterize the nanoparticles, and their antibacterial efficacy was compared by the minimum inhibitory concentration (MIC) and bacterial growth curve assays, followed by molecular studies using scanning electron microscopy (SEM) and (qRT- PCR). AgNP inhibited the growth of E. coli only at 0.621 mg/mL, which was double the concentration required for both coated nanoparticles (0.312 mg/mL). Similarly, bacterial growth was impeded as early as 8 h at 0.156 mg/mL of both coated nanoparticles as compared to 0.312 mg/mL for plain AgNP. SEM data showed that nanoparticles damaged the cell membrane, resulting in bacterial cell lysis, expulsion of cellular contents, and complete disintegration of some cells. The expression of genes associated with the TCA cycle (aceF and frdB) and amino acid metabolism (gadB, metL, argC) were substantially downregulated in E. coli treated with nanoparticles. The reduction in the silver ion (Ag+) concentration of polymer-coated AgNP did not affect their antibacterial efficacy against E. coli.

Proteomic analysis of antimicrobial effects of pegylated silver coated carbon nanotubes in Salmonella enterica serovar Typhimurium.

BACKGROUND: Synthesis of silver nano-compounds with enhanced antimicrobial effects is of great interest for the development of new antibacterial agents. Previous studies have reported the antibacterial properties of pegylated silver-coated carbon nanotubes (pSWCNT-Ag) showing less toxicity in human cell lines. However, the mechanism underlining the pSWCNT-Ag as a bactericidal agent remained unfolded. Here we assessed the pSWCNT-Ag effects against foodborne pathogenic bacteria growth and proteome profile changes. RESULTS: Measurements of bioluminescent imaging, optical density, and bacteria colony forming units revealed dose-dependent and stronger bactericidal activity of pSWCNT-Ag than their non-pegylated counterparts (SWCNT-Ag). In ovo administration of pSWCNT-Ag or phosphate-buffered saline resulted in comparable chicken embryo development and growth. The proteomic analysis, using two-dimensional electrophoresis combined with matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry, was performed on control and surviving Salmonella enterica serovar Typhimurium to pSWCNT-Ag. A total of 15 proteins (ten up-regulated and five down-regulated) differentially expressed proteins were identified. Functional analyses showed significant reduction of proteins associated with biofilm formation, nutrient and energy metabolism, quorum sensing and maintenance of cell structure and cell motility in surviving S. Typhimurium. In contrast, proteins associated with oxygen stress, DNA protection, starvation, membrane rebuilding, and alternative nutrient formation were induced as the compensatory reaction. CONCLUSIONS: This study provides further evidence of the antibacterial effects of pSWCNT-Ag nanocomposites and knowledge of their mechanism of action through various protein changes. The findings may lead to the development of more effective and safe antimicrobial agents.

Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin.

The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolerance induction and outlines in detail current available strategies and alternatives that may allow achieving full-thickness skin replacement and repair.

The anti-microbial peptide TP359 attenuates inflammation in human lung cells infected with Pseudomonas aeruginosa via TLR5 and MAPK pathways.

Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial cells, which do not necessarily eradicate the pathogen. Nonetheless, it reduces lung function due to significant airway damage, most importantly in cystic fibrosis patients. Recently, we published that TP359, a proprietary cationic peptide had potent bactericidal effects against P. aeruginosa, which were mediated by down-regulating its outer membrane biogenesis genes. Herein, we hypothesized that TP359 bactericidal effects could also serve to regulate P. aeruginosa-induced lung inflammation. We explored this hypothesis by infecting human A549 lung cells with live P. aeruginosa non-isogenic, mucoid and non-mucoid strains and assessed the capacity of TP359 to regulate the levels of elicited TNFα, IL-6 and IL-8 inflammatory cytokines. In all instances, the mucoid strain elicited higher concentrations of cytokines in comparison to the non-mucoid strain, and TP359 dose-dependently down-regulated their respective levels, suggesting its regulation of lung inflammation. Surprisingly, P. aeruginosa flagellin, and not its lipopolysaccharide moiety, was the primary inducer of inflammatory cytokines in lung cells, which were similarly down-regulated by TP359. Blocking of TLR5, the putative flagellin receptor, completely abrogated the capacity of infected lung cells to secrete cytokines, underscoring that TP359 regulates inflammation via the TLR5-dependent signaling pathway. Downstream pathway-specific inhibition studies further revealed that the MAPK pathway, essentially p38 and JNK are necessary for induction of P. aeruginosa elicited inflammatory cytokines and their down-regulation by TP359. Collectively, our data provides evidence to support exploring the relevancy of TP359 as an anti-microbial and anti-inflammatory agent against P. aeruginosa for clinical applications.

Advances in Skin Regeneration Using Tissue Engineering.

Tissue engineered skin substitutes for wound healing have evolved tremendously over the last couple of years. New advances have been made toward developing skin substitutes made up of artificial and natural materials. Engineered skin substitutes are developed from acellular materials or can be synthesized from autologous, allograft, xenogenic, or synthetic sources. Each of these engineered skin substitutes has their advantages and disadvantages. However, to this date, a complete functional skin substitute is not available, and research is continuing to develop a competent full thickness skin substitute product that can vascularize rapidly. There is also a need to redesign the currently available substitutes to make them user friendly, commercially affordable, and viable with longer shelf life. The present review focuses on providing an overview of advances in the field of tissue engineered skin substitute development, the availability of various types, and their application.

Future Prospects for Scaffolding Methods and Biomaterials in Skin Tissue Engineering: A Review.

Over centuries, the field of regenerative skin tissue engineering has had several advancements to facilitate faster wound healing and thereby restoration of skin. Skin tissue regeneration is mainly based on the use of suitable scaffold matrices. There are several scaffold types, such as porous, fibrous, microsphere, hydrogel, composite and acellular, etc., with discrete advantages and disadvantages. These scaffolds are either made up of highly biocompatible natural biomaterials, such as collagen, chitosan, etc., or synthetic materials, such as polycaprolactone (PCL), and poly-ethylene-glycol (PEG), etc. Composite scaffolds, which are a combination of natural or synthetic biomaterials, are highly biocompatible with improved tensile strength for effective skin tissue regeneration. Appropriate knowledge of the properties, advantages and disadvantages of various biomaterials and scaffolds will accelerate the production of suitable scaffolds for skin tissue regeneration applications. At the same time, emphasis on some of the leading challenges in the field of skin tissue engineering, such as cell interaction with scaffolds, faster cellular proliferation/differentiation, and vascularization of engineered tissues, is inevitable. In this review, we discuss various types of scaffolding approaches and biomaterials used in the field of skin tissue engineering and more importantly their future prospects in skin tissue regeneration efforts.

Immunogenicity of RSV F DNA Vaccine in BALB/c Mice.

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease leading to numerous hospitalizations and deaths among the infant and elderly populations worldwide. There is no vaccine or a less effective drug available against RSV infections. Natural RSV infection stimulates the Th1 immune response and activates the production of neutralizing antibodies, while earlier vaccine trials that used UV-inactivated RSV exacerbated the disease due to the activation of the allergic Th2 response. With a focus on Th1 immunity, we developed a DNA vaccine containing the native RSV fusion (RSV F) protein and studied its immune response in BALB/c mice. High levels of RSV specific antibodies were induced during subsequent immunizations. The serum antibodies were able to neutralize RSV in vitro. The RSV inhibition by sera was also shown by immunofluorescence analyses. Antibody response of the RSV F DNA vaccine showed a strong Th1 response. Also, sera from RSV F immunized and RSV infected mice reduced the RSV infection by 50% and 80%, respectively. Our data evidently showed that the RSV F DNA vaccine activated the Th1 biased immune response and led to the production of neutralizing antibodies, which is the desired immune response required for protection from RSV infections.

Novel cationic peptide TP359 down-regulates the expression of outer membrane biogenesis genes in Pseudomonas aeruginosa: a potential TP359 anti-microbial mechanism.

BACKGROUND: Antimicrobial peptides (AMPs) are a class of antimicrobial agents with broad-spectrum activities. Several reports indicate that cationic AMPs bind to the negatively charged bacterial membrane causing membrane depolarization and damage. However, membrane depolarization and damage may be insufficient to elicit cell death, thereby suggesting that other mechanism(s) of action could be involved in this phenomenon. In this study, we investigated the antimicrobial activity of a novel antimicrobial peptide, TP359, against two strains of Pseudomonas aeruginosa, as well as its possible mechanisms of action. RESULTS: TP359 proved to be bactericidal against P. aeruginosa as confirmed by the reduced bacteria counts, membrane damage and cytoplasmic membrane depolarization. In addition, it was non-toxic to mouse J774 macrophages and human lung A549 epithelial cells. Electron microscopy analysis showed TP359 bactericidal effects by structural changes of the bacteria from viable rod-shaped cells to those with cell membrane damages, proceeding into the efflux of cytoplasmic contents and emergence of ghost cells. Gene expression analysis on the effects of TP359 on outer membrane biogenesis genes underscored marked down-regulation, particularly of oprF, which encodes a major structural and outer membrane porin (OprF) in both strains studied, indicating that the peptide may cause deregulation of outer membrane genes and reduced structural stability which could lead to cell death. CONCLUSION: Our data shows that TP359 has potent antimicrobial activity against P aeruginosa. The correlation between membrane damage, depolarization and reduced expression of outer membrane biogenesis genes, particularly oprF may suggest the bactericidal mechanism of action of the TP359 peptide.

Gold nanorods inhibit respiratory syncytial virus by stimulating the innate immune response.

Respiratory syncytial virus (RSV) causes severe pneumonia and bronchiolitis in infants, children and older adults. The use of metallic nanoparticles as potential therapeutics is being explored against respiratory viruses like Influenza, Parainfluenza and Adenovirus. In this study, we showed that gold nanorods (GNRs) inhibit RSV in HEp-2 cells and BALB/c mice by 82% and 56%, respectively. The RSV inhibition correlated with marked upregulated antiviral genes due to GNR mediated TLR, NOD-like receptor and RIG-I-like receptor signaling pathways. Transmission electron microscopy of lungs showed GNRs in the endocytotic vesicles and histological analyses indicated infiltration by neutrophils, eosinophils and monocytes correlating with clearance of RSV. In addition, production of cytokines and chemokines in the lungs indicates recruitment of immune cells to counter RSV replication. To our knowledge, this is the first in vitro and in vivo report that provides possible antiviral mechanisms of GNRs against RSV.

A novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide.

BACKGROUND: Due to increasing antibiotic resistance, the use of silver coated single walled carbon nanotubes (SWCNTs-Ag) and antimicrobial peptides (APs) is becoming popular due to their antimicrobial properties against a wide range of pathogens. However, stability against various conditions and toxicity in human cells are some of the major drawbacks of APs and SWCNTs-Ag, respectively. Therefore, we hypothesized that APs-functionalized SWCNTs-Ag could act synergistically. Various covalent functionalization protocols described previously involve harsh treatment of carbon nanotubes for carboxylation (first step in covalent functionalization) and the non-covalently functionalized SWCNTs are not satisfactory. METHODS: The present study is the first report wherein SWCNTs-Ag were first carboxylated using Tri sodium citrate (TSC) at 37 °C and then subsequently functionalized covalently with an effective antimicrobial peptide from Therapeutic Inc., TP359 (FSWCNTs-Ag). SWCNTs-Ag were also non covalently functionalized with TP359 by simple mixing (SWCNTs-Ag-M) and both, the FSWCNTs-Ag (covalent) and SWCNTs-Ag-M (non-covalent), were characterized by Fourier transform infrared spectroscopy (FT-IR), Ultraviolet visualization (UV-VIS) and transmission electron microscopy (TEM). Further the antibacterial activity of both and TP359 were investigated against two gram positive (Staphylococcus aureus and Streptococcus pyogenes) and two gram negative (Salmonella enterica serovar Typhimurium and Escherichia coli) pathogens and the cellular toxicity of TP359 and FSWCNTs-Ag was compared with plain SWCNTs-Ag using murine macrophages and lung carcinoma cells. RESULTS: FT-IR analysis revealed that treatment with TSC successfully resulted in carboxylation of SWCNTs-Ag and the peptide was indeed attached to the SWCNTs-Ag evidenced by TEM images. More importantly, the present study results further showed that the minimum inhibitory concentration (MIC) of FSWCNTs-Ag were much lower (~7.8-3.9 µg/ml with IC50: ~4-5 µg/ml) compared to SWCNTs-Ag-M and plain SWCNTs-Ag (both 62.6 µg/ml, IC50: ~31-35 µg/ml), suggesting that the covalent conjugation of TP359 with SWCNTs-Ag was very effective on their counterparts. Additionally, FSWCNTs-Ag are non-toxic to the eukaryotic cells at their MIC concentrations (5-2.5 µg/ml) compared to SWCNTs-Ag (62.5 µg/ml). CONCLUSION: In conclusion, we demonstrated that covalent functionalization of SWCNTs-Ag and TP359 exhibited an additive antibacterial activity. This study described a novel approach to prepare SWCNT-Ag bio-conjugates without loss of antimicrobial activity and reduced toxicity, and this strategy will aid in the development of novel and biologically important nanomaterials.

Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect.

The antimicrobial activity of silver-coated carbon nanotubes (AgCNTs) and their potential mode of action against mucoid and nonmucoid strains of Pseudomonas aeruginosa was investigated in vitro. The results showed that AgCNTs exhibited antimicrobial activity against both strains with minimum inhibitory concentrations of approximately 8 µg/mL, indicating a high sensitivity of P. aeruginosa to AgCNTs. AgCNTs were also bactericidal against both strains at the same minimum inhibitory concentration. Scanning and transmission electron-microscopy studies further revealed that a majority of the cells treated with AgCNTs transformed from smooth rod-shape morphology to disintegrated cells with broken/damaged membranes, resulting in leakage of cytoplasmic contents to produce ghost cells. The molecular effects of AgCNTs on P. aeruginosa genes involved in virulence and pathogenicity, stress response, and efflux pumps were evaluated for changes in their expression. Quantitative real-time PCR (qRT-PCR) showed that after exposure to AgCNTs, the expression levels of the rpoS, rsmZ, and oprD genes were significantly downregulated in both strains of P. aeruginosa compared to the untreated samples. These results suggest that the mechanism of action of AgCNTs may be attributed to their effect on cell-membrane integrity, downregulation of virulence-gene expression, and induction of general and oxidative stress in P. aeruginosa.

Novel pegylated silver coated carbon nanotubes kill Salmonella but they are non-toxic to eukaryotic cells.

BACKGROUND: Resistance of food borne pathogens such as Salmonella to existing antibiotics is of grave concern. Silver coated single walled carbon nanotubes (SWCNTs-Ag) have broad-spectrum antibacterial activity and may be a good treatment alternative. However, toxicity to human cells due to their physico-chemical properties is a serious public health concern. Although pegylation is commonly used to reduce metal nanoparticle toxicity, SWCNTs-Ag have not been pegylated as yet, and the effect of pegylation of SWCNTs-Ag on their anti-bacterial activity and cell cytotoxicity remains to be studied. Further, there are no molecular studies on the anti-bacterial mechanism of SWCNTs-Ag or their functionalized nanocomposites. MATERIALS AND METHODS: In this study we created novel pegylated SWCNTS-Ag (pSWCNTs-Ag), and employed 3 eukaryotic cell lines to evaluate their cytotoxicity as compared to plain SWCNTS-Ag. Simultaneously, we evaluated their antibacterial activity on Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) by the MIC and growth curve assays. In order to understand the possible mechanisms of action of both SWCNTs-Ag and pSWCNTs-Ag, we used electron microscopy (EM) and molecular studies (qRT-PCR). RESULTS: pSWCNTs-Ag inhibited Salmonella Typhimurium at 62.5 µg/mL, while remaining non-toxic to human cells. By comparison, plain SWCNTs-Ag were toxic to human cells at 62.5 µg/mL. EM analysis revealed that bacteria internalized either of these nanocomposites after the outer cell membranes were damaged, resulting in cell lysis or expulsion of cytoplasmic contents, leaving empty ghosts. The expression of genes regulating the membrane associated metabolic transporter system (artP, dppA, and livJ), amino acid biosynthesis (trp and argC) and outer membrane integrity (ompF) protiens, was significantly down regulated in Salmonella treated with both pSWCNTs-Ag and SWCNTs-Ag. Although EM analysis of bacteria treated with either SWCNTs-Ag or pSWCNTs-Ag revealed relatively similar morphological changes, the expression of genes regulating the normal physiological processes of bacteria (ybeF), quorum sensing (sdiA), outer membrane structure (safC), invasion (ychP) and virulence (safC, ychP, sseA and sseG) were exclusively down regulated several fold in pSWCNTs-Ag treated bacteria. CONCLUSIONS: Altogether, the present data shows that our novel pSWCNTs-Ag are non-toxic to human cells at their bactericidal concentration, as compared to plain SWCNTS-Ag. Therefore, pSWCNTs-Ag may be safe alternative antimicrobials to treat foodborne pathogens.

Enhanced intracellular translocation and biodistribution of gold nanoparticles functionalized with a cell-penetrating peptide (VG-21) from vesicular stomatitis virus.

Reduced toxicity and ease of modification make gold nanoparticles (GNPs) suitable for targeted delivery, bioimaging and theranostics by conjugating cell-penetrating peptides (CPPs). This study presents the biodistribution and enhanced intracellular uptake of GNPs functionalized with VG-21, a CPP derived from vesicular stomatitis virus glycoprotein (G). Cell penetrating efficiency of VG-21 was demonstrated using CellPPD web server, conjugated to GNPs and were characterized using, UV-visible and FTIR spectroscopy, transmission electron microscopy, dynamic light scattering and zeta potential. Uptake of VG-21 functionalized GNPs (fGNPs) was tested in eukaryotic cell lines, HEp-2, HeLa, Vero and Cos-7, using flow cytometry, fluorescence and transmission electron microscopy (TEM), and inductively coupled plasmon optical emission spectroscopy (ICP-OES). The effects of nanoparticles on stress and toxicity related genes were studied in HEp-2 cells. Cytokine response to fGNPs was studied in vitro and in vivo. Biodistribution of nanoparticles was studied in BALB/c mice using TEM and ICP-OES. VG-21, GNPs and fGNPs had little to no effect on cell viability. Upon exposure to fGNPs, HEp-2 cells revealed minimal down regulation of stress response genes. fGNPs displayed higher uptake than GNPs in all cell lines with highest internalization by HEp-2, HeLa and Cos-7 cells, in endocytotic vesicles and nuclei. Cytokine ELISA showed that mouse J774 cells exposed to fGNPs produced less IL-6 than did GNP-treated macrophage cells, whereas TNF-α levels were low in both treatment groups. Biodistribution studies in BALB/c mice revealed higher accumulation of fGNPs than GNPs in the liver and spleen. Histopathological analyses showed that fGNP-treated mice accumulated 35 ng/mg tissue and 20 ng/mg tissue gold in spleen and liver respectively, without any adverse effects. Likewise, serum cytokines were low in both GNP- and fGNP-treated mice. Thus, VG-21-conjugated GNPs have enhanced cellular internalization and are suitable for various biomedical applications as nano-conjugates.

Recent advances in diagnosis, prevention, and treatment of human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a common cause of respiratory infection in infants and the elderly, leading to significant morbidity and mortality. The interdisciplinary fields, especially biotechnology and nanotechnology, have facilitated the development of modern detection systems for RSV. Many anti-RSV compounds like fusion inhibitors and RNAi molecules have been successful in laboratory and clinical trials. But, currently, there are no effective drugs for RSV infection even after decades of research. Effective diagnosis can result in effective treatment, but the progress in both of these facets must be concurrent. The development in prevention and treatment measures for RSV is at appreciable pace, but the implementation into clinical practice still seems a challenge. This review attempts to present the promising diverse research approaches and advancements in the area of diagnosis, prevention, and treatment that contribute to RSV management.

Development of a LIBS assay for the detection of Salmonella enterica serovar Typhimurium from food.

Laser-induced breakdown spectroscopy (LIBS) is used for the identification of the presence of hazardous bacteria in food. In this study, our main focus was centered on the identification of S. enterica serovar Typhimurium, a Gram-negative foodborne pathogen, in various liquids such as milk, chicken broth, and brain heart infusion due to the infection being most prevalent in raw meat and dairy products. A Nd:YAG laser of operating wavelength 266 nm was used to obtain the spectra from the artificially inoculated liquid samples. A series of experiments were performed to determine the effectiveness of LIBS to discriminate the bacteria from the background liquids. These results are compared with competing modern molecular methods of detection which include polymerase chain reaction (PCR) and quantitative real-time PCR. In addition to analyzing S. enterica serovar Typhimurium, another common Gram-negative, Escherichia coli, as well as Gram-positive pathogen, Staphlycoccus auerus, were used to determine the specificity of the LIBS technique.

Synthesis of Ag/CNT hybrid nanoparticles and fabrication of their nylon-6 polymer nanocomposite fibers for antimicrobial applications.

Ag-coated CNTs hybrid nanoparticles (Ag/CNTs) were prepared by ultrasonic irradiation of dimethylformamide (DMF) and silver (I) acetate precursors in the presence of CNTs. The morphology of Ag/CNTs was characterized using x-ray diffraction and transmission electron microscopy (TEM) techniques. The Nylon-6 powder and 1 wt% Ag/CNTs mixture was dispersed uniformly using a noncontact spinning technique. The dried mixture was melted in a single screw extrusion machine and then extruded through an orifice. Extruded filaments were later stretched and stabilized by sequentially passing them through a set of tension adjusters and a secondary heater. The Nylon-6/Ag/CNT hybrid polymer nanocomposite (HPNC) fibers, which were of approximately 80 microm size, were tested for their tensile properties. The failure stress and modulus of the extruded HPNC fibers (doped with 1% Ag/CNTs) was about 72.19 % and 342.62% higher than the neat extruded Nylon-6 fiber, respectively. DSC results indicated an increase in the thermal stability and crystallization for HPNC fibers. The antibacterial activity of the Ag-coated CNTs, commercial Ag, neat Nylon-6 and plain CNTs were evaluated. Ag-coated CNTs at 25 microg demonstrated good antimicrobial activity against four common bacterial pathogens as tested by the Kirby-Bauer assay. The mean diameters of the zones of inhibition were 27.9 +/- 6.72 mm, 19.4 +/- 3.64 mm, 21.9 +/- 4.33 mm, and 24.1 +/- 4.14 mm, respectively, for Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Salmonella enterica serovar Typhimurium. By comparison, those obtained using the broad spectrum antibiotic amoxicillin-clavulanic acid were 37.7 +/- 2.13 mm, 28.6 +/- 4.27 mm, 22.6 +/- 1.27 mm, and 27.0 +/- 1.41 mm, respectively, for the same strains. The zones of inhibition obtained for Nylon-6 Ag-coated CNT powder at 25 microg were also high, ranging from 15.2 to 25.3 mm in contrast to commercial silver or neat Nylon-6, which did not inhibit the bacterial strains tested. Further, the Nylon-6 nanocomposite fibers infused with Ag/CNTs inhibited bacterial growth by 11-20%. Our results suggest that nylon nanocomposite fibers infused with Ag-coated CNTs have significant antimicrobial activity.

Secondary RNA structure and its role in RNA interference to silence the respiratory syncytial virus fusion protein gene.

RNA interference (RNAi) is a post-transcriptional, gene silencing mechanism which uses small interfering RNA molecules (siRNA) for gene silencing. Respiratory Syncytial Virus (RSV) is an important respiratory pathogen of medical significance that causes high mortality in infants. The fusion (F) protein of RSV is a good target for therapeutic purposes as it is primarily responsible for penetration of the virus into host cells and subsequent syncytium formation during infection. In the present study, four siRNAs were designed and used individually as well as a mixture, to silence the RSV F gene. The relationship between siRNA design, target RNA structure, and their thermodynamics was also investigated. Silencing of F gene was observed using indirect immunofluorescence, western blot, reverse transcription PCR, and progeny viral titers. Our results show F gene silencing by all the four siRNAs individually and collectively. RT-PCR analysis revealed a decrease in mRNA level which corresponded to decreased F protein expression. siRNAs also inhibited RSV progeny as shown by viral titer estimation on infected HEp-2 cells. The present study demonstrates the silencing of the F gene using siRNA. Thermodynamic characteristics of the target RSV mRNA and siRNA seem to play an important role in siRNA gene silencing efficiency.

RSV fusion (F) protein DNA vaccine provides partial protection against viral infection.

The present study was conducted to investigate the feasibility and efficacy of a RSV F DNA vaccine incorporated with a mucosal adjuvant. Two DNA vaccine vectors (DRF-412 and DRF-412-P) were developed containing residues 412-524 of the RSV F gene. These antigenic regions were cloned into the phCMV1 DNA vaccine vector. One of the DNA vaccine vectors, DRF-412, contained the ctxA(2)B region of the cholera toxin gene as a mucosal adjuvant. The in vitro expressions of these DNA vectors were confirmed in Cos-7 cells by indirect immunofluorescence and Western blot analyses. In vivo expression of the cloned gene was further confirmed in mouse muscle tissue by immunohistological analysis. The active transcription of the RSV F gene in mouse muscle cells was confirmed by RT-PCR. The purified DRF-412 and DRF-412-P DNA vectors were used to immunize mice by intramuscular injections. Our results indicated that DRF-412 and DRF-412-P vaccine vectors were as effective as live RSV in inducing neutralization antibody, systemic Ab (IgG, IgG1, IgG2a, and IgG2b) responses, and mucosal antibody responses (Ig A). The Th1 (TNF-alpha, IL-12p70, IFN-gamma, IL-2) and Th2 (IL-10, IL-6) cytokine profiles were analyzed after stimulation of spleen cells from mice immunized with purified RF-412 protein. We observed that mice inoculated with vector DRF-412 induced a higher mixed Th1/Th2 cytokine immune response than DRF-412-P. Reverse transcriptase and quantitative real-time PCR (qRT-PCR) revealed that mice immunized with the DRF-412 vector contained less viral RNA in lung tissue and the lung immunohistology study confirmed that mice immunized with DRF-412 had better protection than those immunized with the DRF-412-P vector. These results indicate that the RSV DRF-412 vaccine vector, which contains the cholera toxin subunit ctxA2B as a mucosal adjuvant may provide a better DNA vaccination strategy against RSV.