CONSTRUCTION AND CHARACTERIZATION OF A NOVEL FUSION PROTEIN FROM THE EXTRACELLULAR DOMAIN OF MULT1 AND TRANSMEMBRANE AND INTRACELLULAR DOMAINS OF FAS AND ITS THERAPEUTIC EVALUATION FOR CANCER TREATMENT USING AN ADENOVIRAL DELIVERY SYSTEM

One of the strategies that tumor cells adopt to evade immunosurveillance mounted by elements of the innate immune system, such as NK cells, is to down-regulate certain cell surface molecules through a process also called shedding. Mouse UL16-binding protein-like transcript 1 (MULT1), which can activate NK cells through NK cell receptor NKG2D, is one of such molecules. Tumor cells can also avoid Fas mediated apoptosis by down-regulating its expression, secreting antagonistic `decoy\u27 receptors, or expressing anti-apoptotic molecules. In this study, we report the design and evaluation of the antitumor activity of a novel fusion protein MULT1E/FasTI, consisting of the extracellular domain of MULT1 and transmembrane and intracellular domains of Fas. We hypothesized that this protein, when expressed on a cell, would not only activate NK cells and other NKG2D expressing killer cells through its MULT1E region but also send death signals to induce apoptosis of the cell through the FasTI region. We cloned cDNA encoding the extracellular domain of MULT1 gene from thymus of new born mice and ligated it to the transmembrane and intracellular domains of mouse fas cDNA. The resulting fusion cDNA was inserted into a mammalian cell expressing vector under the control of CMV promoter. The vector was then transfected into mouse TC-1 lung epithelial cancer cells; and stable cell lines expressing the fusion protein were established. In vitro cell culture studies demonstrated that the binding of NKG2D/Fc, a recombinant protein of mouse NK cell receptor, to MULT1E/FasTI expressed on tumor cells was able to elicit apoptosis as assayed by Annexin V-FITC staining and caspase-3 ELISA and also activated NK cells as indicated by enhanced interferon-gamma; expression. In vivo subcutaneous tumor studies demonstrated that tumor cells expressing MULT1E/FasTI grew significantly slower than tumors without the protein. Pulmonary metastasis studies showed that most of the mice completely rejected tumor cells expressing MULT1E/FasTI. We also examined the use of a replication-defective adenovirus as a gene therapy vector to deliver the fusion protein into tumor cells. In vitro and in vivo studies not only demonstrated that the novel fusion protein can be successfully delivered by adenoviral vectors but also confirmed antitumor activity of the fusion protein. Therefore, the reported fusion protein strategy represents a novel and hopeful new anticancer agent for cancer patients

Genomic diversity of bacteriophages infecting Microbacterium spp

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics

Instructional Models for Course-Based Research Experience (CRE) Teaching

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching

Evaluation of Polyethylene Glycol Diacrylate-Polycaprolactone Scaffolds for Tissue Engineering Applications

Polyethylene Glycol Diacrylate (PEGDA) tissue scaffolds having a thickness higher than 1 mm and without the presence of nutrient conduit networks were shown to have limited applications in tissue engineering due to the inability of cells to adhere and migrate within the scaffold. The PEGDA scaffold has been coated with polycaprolactone (PCL) electrospun nanofiber (ENF) membrane on both sides to overcome these limitations, thereby creating a functional PEGDA-PCL scaffold. This study examined the physical, mechanical, and biological properties of the PEGDA and PEGDA-PCL scaffolds to determine the effect of PCL coating on PEGDA. The physical characterization of PEGDA-PCL samples demonstrated the effectiveness of combining PCL with a PEGDA scaffold to expand its applications in tissue engineering. This study also found a significant improvement of elasticity of PEGDA due to the addition of PCL layers. This study shows that PEGDA-PCL scaffolds absorb nutrients with time and can provide an ideal environment for the survival of cells. Furthermore, cell viability tests indicate that the cell adhered, proliferated, and migrated in the PEGDA-PCL scaffold. Therefore, PCL ENF coating has a positive influence on PEGDA scaffold

Biomineralization and Biocompatibility Studies of Bone Conductive Scaffolds Containing Poly(3,4-Ethylenedioxythiophene):Poly(4-Styrene Sulfonate) (PEDOT:PSS)

Considering the well-known phenomenon of enhancing bone healing by applying electromagnetic stimulation, manufacturing conductive bone scaffolds is on demand to facilitate the delivery of electromagnetic stimulation to the injured region, which in turn significantly expedites the healing procedure in tissue engineering methods. For this purpose, hybrid conductive scaffolds composed of poly(3,4-ethylenedioxythiophene), poly(4-styrene sulfonate) (PEDOT:PSS), gelatin (Gel), and bioactive glass (BaG) were produced employing freeze drying technique. Concentration of PEDOT:PSS were optimized to design the most appropriate conductive scaffold in terms of biocompatibility and cell proliferation. More specifically, scaffolds with four different compositions of 0, 0.1, 0.3 and 0.6 % (w/w) PEDOT:PSS in the mixture of 10 % (w/v) Gel and 30 % (w/v) BaG were synthesized. Immersing the scaffolds in simulated body fluid (SBF), we evaluated the bioactivity of samples, and the biomineralization were studied in details using scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction analysis and Fourier transform infrared spectroscopy. By performing cytocompatibility analyses for 21 days using adult human mesenchymal stem cells, we concluded that the scaffolds with 0.3 % (w/w) PEDOT:PSS and conductivity of 170 μS/m has the optimized composition and further increasing the PEDOT:PSS content has inverse effect on cell proliferation. Based on our finding, addition of this optimized amount of PEDOT:PSS to our composition can increase the cell viability more than 4 times compared to a nonconductive composition

Antimicrobial Activity of Polycaprolactone Nanofiber Coated with Lavender and Neem Oil Nanoemulsions against Airborne Bacteria

The development of efficient, eco-friendly antimicrobial agents for air purification and disinfection addresses public health issues connected to preventing airborne pathogens. Herein, the antimicrobial activity of a nanoemulsion (control, 5%, 10%, and 15%) containing neem and lavender oils with polycaprolactone (PCL) was investigated against airborne bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Various parameters such as the physicochemical properties of the nanoemulsion, pH, droplet size, the polydispersity index (PDI), the minimum inhibitory concentration (MIC), the minimum bacterial concentration (MBC), and the color measurement of the emulsion have been evaluated and optimized. Our results showed that the antimicrobial activity of PCL combined with neem and lavender oil was found to be the highest MIC and MBC against all tested bacteria. The droplet sizes for lavender oil are 21.86–115.15 nm, the droplet sizes for neem oil are 23.92–119.15 nm, and their combination is 25.97–50.22 nm. The range of pH and viscosity of nanoemulsions of various concentrations was found to be 5.8 to 6.6 pH and 0.372 to 2.101 cP. This study highlights the potential of nanotechnology in harnessing the antimicrobial properties of natural essential oils, paving the way for innovative and sustainable solutions in the fight against bacterial contamination

Evaluation of Polyethylene Glycol Diacrylate-Polycaprolactone Scaffolds for Tissue Engineering Applications

Polyethylene Glycol Diacrylate (PEGDA) tissue scaffolds having a thickness higher than 1 mm and without the presence of nutrient conduit networks were shown to have limited applications in tissue engineering due to the inability of cells to adhere and migrate within the scaffold. The PEGDA scaffold has been coated with polycaprolactone (PCL) electrospun nanofiber (ENF) membrane on both sides to overcome these limitations, thereby creating a functional PEGDA-PCL scaffold. This study examined the physical, mechanical, and biological properties of the PEGDA and PEGDA-PCL scaffolds to determine the effect of PCL coating on PEGDA. The physical characterization of PEGDA-PCL samples demonstrated the effectiveness of combining PCL with a PEGDA scaffold to expand its applications in tissue engineering. This study also found a significant improvement of elasticity of PEGDA due to the addition of PCL layers. This study shows that PEGDA-PCL scaffolds absorb nutrients with time and can provide an ideal environment for the survival of cells. Furthermore, cell viability tests indicate that the cell adhered, proliferated, and migrated in the PEGDA-PCL scaffold. Therefore, PCL ENF coating has a positive influence on PEGDA scaffold

Incorporation of Mycobacteriophage Fulbright into Polycaprolactone Electrospun Nanofiber Wound Dressing

The Genus Mycobacterium includes pathogens known to cause disease in mammals such as tuberculosis (Mycobacterium tuberculosis) and skin infections (M. abscessus). M. smegmatis is a model bacterium that can cause opportunistic infections in human tissues and, rarely, a respiratory disease. Due to the emergence of multidrug-resistant bacteria, phage therapy is potentially an alternative way of treating these bacterial infections. As bacteriophages are specific to their bacterial host, it ensures that the normal flora is unharmed. Fulbright is a mycobacteriophage that infects the host bacteria M. smegmatis. The main goal of this study is to incorporate Mycobacteriophage Fulbright into a polycaprolactone (PCL) nanofiber and test its antimicrobial effect against the host bacteria, M. smegmatis. Stability tests conducted over 7 days showed that the phage titer does not decrease when in contact with PCL, making it a promising vehicle for phage delivery. Antimicrobial assays showed that PCL_Fulbright effectively reduces bacterial concentration after 24 h of contact. In addition, when stored at −20 °C, the phage remains viable for up to eleven months in the fiber. Fulbright addition on the nanofibrous mats resulted in an increase in water uptake and decrease in the mechanical properties (strength and Young’s modulus) of the membranes, indicating that the presence of phage Fulbright can greatly enhance the physical and mechanical properties of the PCL. Cytotoxicity assays showed that PCL_Fulbright is not cytotoxic to Balbc/3T3 mouse embryo fibroblast cell lines; thus, phage-incorporated PCL is a promising alternative to antibiotics in treating skin infections