A co-culture model of mesenchymal stem cells (MSCs) and fibroblasts is an efficient and rapid method to evaluate the anti-fibrotic effects of MSCs-based cell therapy. Transforming growth factor (TGF)-β1 plays a key role in promotion of fibroblast activation and differentiation which can induce collagen deposition, increase ECM production in lung tissue, eventually resulted in pulmonary fibrosis. Here, we use this co-culture system and examine the ECM production in activated fibroblasts by western blot and quantitative real-time analysis to understand the therapeutic effects of MSCs.
Phage display has been applied successfully for the rapid isolation of monoclonal antibodies against various targets including infectious diseases, autoantigens, cancer markers, and even small molecules. The main component in any phage display experiment is the availability of an antibody library to carry out the selection process of target-specific antibodies through an iterative process termed as biopanning. To generate human antibody libraries, the antibody repertoire can be obtained from human peripheral blood mononuclear cell (PBMC) or directly from cell-sorted B-cell populations. The choice of antibody isotype is dictated by the nature of the library. Naïve libraries would utilize IgM repertoires, whereas the IgG repertoire is commonly used for immune libraries. Antibody genes are amplified through polymerase chain reaction (PCR) and paired in a combinatorial fashion to expand the diversity of the cloned library repertoire. The protocol here describes the use of a two-step cloning method that can be applied for the construction of either a naïve or immune human antibody library in Fab format followed by the subsequent panning.
Analysis of protein-protein interactions is important for better understanding of molecular mechanisms involved in immune regulation and has potential for elaborating avenues for drug discovery targeting T-cell motility. Currently, only a small fraction of protein-protein interactions have been characterized in T-lymphocytes although there are several detection methods available. In this regard, computational approaches garner importance, with the continued explosion of genomic and proteomic data, for handling protein modeling and protein-protein interactions in large scale. Here, we describe a computational method to identify protein-protein interactions based on in silico protein design.
Polymerase chain reaction (PCR) is a laboratory technique used to amplify a targeted region of DNA, demarcated by a set of oligonucleotide primers. Long-range PCR is a form of PCR optimized to facilitate the amplification of large fragments. Using the adapted long-range PCR protocol described in this chapter, we were able to generate PCR products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples. For some of the long PCRs, successful amplification was not possible without the use of PCR enhancers. Thus, we also evaluated the impact of some enhancers on long-range PCR and included the findings as part of this updated chapter.
Aerosol-based cell delivery technique via intratracheal is an effective route for delivering transplant cells directly into the lungs. An aerosol device known as the MicroSprayer(®) Aerosolizer is invented to transform liquid into an aerosol form, which then can be applied via intratracheal administration for drug delivery. The device produces a uniform and concentrated distribution of aerosolized liquid. Using the capability of MicroSprayer(®) Aerosolizer to transform liquid into aerosol form, our group has designed a novel method of cell delivery using an aerosol-based technique. We have successfully delivered skin-derived fibroblast cells and airway epithelial cells into the airway of a rabbit with minimum risk of cell loss and have uniformly distributed the cells into the airway. This chapter illustrates the application of aerosol device to deliver any type of cells for future treatment of lung diseases.
Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.
Protein microarray is a miniaturized multi-analyte, solid-phased immunoassay where thousands of immobilized individual protein spots on a microscopic slide bind are bound to specific antibodies (immunoglobulins) from serum samples, which are then detected by fluorescent labeling. The image processing and pattern recognition are then quantitatively analyzed using advanced algorithms. Here, we describe the use of an in-house-produced complex protein microarray containing extracts and pure proteins that has been probed with antibodies present in the horse sera and detection by fluorophore-conjugated antibody and data analysis. The flexibility of the number and types of proteins that can be printed on the microarray allows different set of specific IgE immunoassay analysis to be carried out.
The biomass concentration of microalgae growth in photobioreactor was predicted using the Monod-based growth models. Kinetic parameters such as maximum specific growth rate and saturation constant of light intensity were evaluated by nonlinear least squares methods that focused on minimizing the sum of squares error (SSE). The importance of good initial guess for the nonlinear least squares method was also discussed. The optimal control problem of the microalgae growth model was determined based on parameter sensitivity. Therefore, a dynamic optimization approach was used where an optimal input design method was formulated to obtain a control function of a problem. The dynamic state equations, additional state equations, cost function, and Hamiltonian function were used to establish a control function of microalgae growth in a photobioreactor. Hence, the biomass production of microalgae can be predicted using numerical methods such as the Taylor series method.
Dengue is an arthropod-borne viral disease that has become endemic and a global threat in over 100 countries. The increase in prevalence would require a long-term measure to control outbreaks. Sanofi Pasteur has licensed the tetravalent dengue vaccine (Dengvaxia) in certain dengue endemic countries. However, the efficacy of the vaccine is limited against certain dengue serotypes and can only be used for individuals from the age from 9 to 45 years old. Over the years, there has been intense research conducted on the development of antivirals against dengue virus (DENV) through either inhibiting the virus replication or targeting the host cell mechanism to block the virus entry. However, no approved antiviral drug against dengue is yet available. In this chapter, we describe the dengue antiviral development workflow including (i) prophylactic, (ii) virucidal, and (iii) postinfection assays that are employed in the antiviral drug screening process against DENV. Further, we demonstrate different methods that can be used to enumerate the reduction in virus foci number including foci-forming unit reduction assay (FFURA), estimation of viral RNA copy number through quantitative real-time PCR, and a high-throughput enzyme linked immunosorbent assay (ELISA)-based quantification of virus particles.
Transgenic oil palm (Elaeis guineensis Jacq.) plantlets are regenerated after Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli are transformed with an Agrobacterium strain, LBA4404, harboring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium, and physical injury via biolistics are applied. The main reasons for such modifications are to activate the bacterial virulence system and, subsequently, to increase the transformation efficiency. Transgenic oil palm cells are selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants.
Particular attention has been paid to capillary electrophoresis as versatile and environmentally friendly approach for enantioseparations of a wide spectrum of compounds. Cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) is a method of choice to provide effective separation toward hydrophobic and uncharged stereoisomers. The chiral discrimination of the solutes relies upon the partitioning between a given CD in the aqueous phase and micelles formed from a surfactant. Synergistic combinations of chiral selectors, surfactant, and modifier contribute to successful enantioseparations of the enantiomers. In this chapter, an application of CD-MEKC for the enantioseparation of selected imidazole drugs employing a dual CDs system is described.
The separation of enantiomers is one of the important fields of modern analytical chemistry, especially for agrochemical and pharmaceutical products because the stereochemistry has a significant influence on the biological activities of compounds. Cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) has become an important capillary electrophoresis mode for enantioseparations. Here, we describe an example of a CD-MEKC method using hydroxypropyl-γ-cyclodextrin as chiral selector and sodium dodecyl sulfate as micellar solution for enantioseparation of triazole fungicides and the drug econazole.
Life-cycle assessment (LCA) is one of the most attractive tools employed nowadays by environmental policy-makers as well as business decision-makers to ensure environmentally sustainable production/consumption of various goods/services. LCA is a systematic, rigorous, and standardized approach aimed at quantifying resources consumed/depleted, pollutants released, and the related environmental and health impacts through the course of consumption and production of goods/service. Algal fuels are no exception and their environmental sustainability could be well scrutinized using the LCA methodology. In line with that, this chapter is devoted to present guidelines on the technical aspects of LCA application in algal fuels while elaborating on major standards used, i.e., ISO 14040 and 14044 standards. Overall, LCA practitioners as well as technical experts dealing with algal fuels in both the public and private sectors could be the main target audience for these guidelines.
Discovery of tumor antigenic epitopes is important for cancer vaccine development. Such epitopes can be designed and modified to become more antigenic and immunogenic in order to overcome immunosuppression towards the native tumor antigen. In silico-guided modification of epitope sequences allows predictive discrimination of those that may be potentially immunogenic. Therefore, only candidates predicted with high antigenicity will be selected, constructed, and tested in the lab. Here, we described the employment of in silico tools using a multiparametric approach to assess both potential T-cell epitopes (MHC class I/II binding) and B-cell epitopes (hydrophilicity, surface accessibility, antigenicity, and linear epitope). A scoring and ranking system based on these parameters was developed to shortlist potential mimotope candidates for further development as peptide cancer vaccines.
Cancer is a major health problem worldwide. The failure of current treatments to completely eradicate cancer cells often leads to cancer recurrence and dissemination. Studies have suggested that tumor growth and spread are driven by a minority of cancer cells that exhibit characteristics similar to those of normal stem cells, thus these cells are called cancer stem cells (CSCs). CSCs are believed to play an important role in initiating and promoting cancer. CSCs are resistant to currently available cancer therapies, and understanding the mechanisms that control the growth of CSCs might have great implications for cancer therapy. Cancer cells are consist of heterogeneous population of cells, thus methods of identification, isolation, and characterisation of CSCs are fundamental to obtain a pure CSC populations. Therefore, this chapter describes in detail a method for isolating and characterizing a pure population of CSCs from heterogeneous population of cancer cells and CSCs based on specific cell surface markers.
The main strategy for lowering blood cholesterol levels is through the inhibition of the NADPH-dependent HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase). The enzyme catalyses the reduction of HMG-CoA to mevalonate and this process is inhibited by statins that form the bulk of the therapeutic agents to treat high cholesterol since the 1970s. Newer drugs that are safer than statins are constantly being developed. The inhibition of candidate drugs to HMG-CoA reductase remains the mainstay of drug development research. The determination of the enzyme activity is important for the correct assessment of potency of the enzyme as well as determining the inhibition of potential therapeutic agents from the plant and microbial extracts. Also, this chapter covers the use of the popular four-parameter logistics model that can yield accurate estimation of the IC50 values of therapeutic agents and their 95% confidence intervals.
Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.
Evidence on the role of the oral microbiome in health and disease is changing the way we understand, diagnose, and treat ailments. Numerous studies on diseases affecting the oral cavity have revealed a large amount of data that is invaluable for the advancements in diagnosing and treating these diseases. However, the clinical translation of most of these exploratory data is stalled by variable methodology between studies and non-uniform reporting of the data.Understanding the key areas that are gateways to bias in microbiome studies is imperative to overcome this challenge faced by oral microbiome research. Bias can be multifactorial and may be introduced in a microbiome research study during the formulation of the study design, sample collection and storage, or the sample processing protocols before sequencing. This chapter summarizes the recommendations from literature to eliminate bias in the microbiome research studies and to ensure the reproducibility of the microbiome research data.
Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
Several research groups have utilized dental pulp stem cells for numerous studies as treatment modality for Parkinson's disease (PD). However, the roles of dental pulp stem cells in governing the Parkinson's disease inflammatory microenvironment remain to be evaluated. In this article, we elaborate the method where we can investigate the effects of dental pulp stem cells on neurons and microglia in an in vitro inflammatory microenvironment.