Diagnostic advancements: Isolating Mycobacterium avium ssp. paratuberculosis and unveiling its molecular identity with nested-PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis, which is currently prevalent in many parts of Iran and produces severe economic loss. It is hence necessary to identify and isolate the animals infected with this bacterium, so this research aimed to isolate MAP from milk and fecal samples of ELISA-positive animals and determine the molecular identity of isolates. After performing ELISA on 3,700 bovine blood samples, 115 samples of milk and feces were taken from ELISA-positive cattle and were cultured on Herald's egg yolk medium with and without mycobactin-J and then the acid-fastness of positive samples was determined using Ziehl-Neelsen staining. The 16S rRNA-PCR test was performed after DNA extraction to determine the molecular identity of isolates. Primers IS6110 and IS901 were employed to ensure that the isolates were not related to members of M. tuberculosis complex and  M. avium, respectively. Primer IS900 was also used to determine the molecular identity of MAP isolates. Also, expression levels of MAP-related genes (IS900, ISMAP02, F57, MAP2191, MAP4027) were evaluated via qPCR. Finally, positive samples were confirmed based on the Nested-PCR. Results showed that a total of 9 isolates were obtained from the culture of 90 ELISA-positive samples. The results revealed that all grown samples were positive for acid-fastness. The 16S rRNA-PCR test revealed the 543 bp band, which confirms the presence of Mycobacterium in the samples. The PCR test with Primer IS900 generated the 398 bp fragment in the first step and the 298 bp fragment in the second step, indicating the presence of MAP in samples. Also, relative expression analysis revealed that MAP-related genes were significantly higher in ELIZA-positive samples than in negative ones. Based on the study findings, it can be concluded that MAP-infected animals can be identified by ELISA. In addition, mycobacterium can be isolated by culturing the samples on appropriate media and then its molecular identity can be determined by using nested-PCR.

Management of internal root resorption in the maxillary central incisor with fractured root using Biodentine.

Biodentine is desirable to successfully manage internal root resorption, and the MTA apical plug backfilled with thermoplasticized gutta-percha is suggested for the tooth with external apical root resorption.

Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

Vectors used for stem cell transfection must be non-genotoxic, in addition to possessing high efficiency, because they could potentially transform normal stem cells into cancer-initiating cells. The objective of this research was to bioengineer an efficient vector that can be used for genetic modification of stem cells without any negative somatic or genetic impact. Two types of multifunctional vectors, namely targeted and non-targeted were genetically engineered and purified from E. coli. The targeted vectors were designed to enter stem cells via overexpressed receptors. The non-targeted vectors were equipped with MPG and Pep1 cell penetrating peptides. A series of commercial synthetic non-viral vectors and an adenoviral vector were used as controls. All vectors were evaluated for their efficiency and impact on metabolic activity, cell membrane integrity, chromosomal aberrations (micronuclei formation), gene dysregulation, and differentiation ability of stem cells. The results of this study showed that the bioengineered vector utilizing VEGFR-1 receptors for cellular entry could transfect mesenchymal stem cells with high efficiency without inducing genotoxicity, negative impact on gene function, or ability to differentiate. Overall, the vectors that utilized receptors as ports for cellular entry (viral and non-viral) showed considerably better somato- and genosafety profiles in comparison to those that entered through electrostatic interaction with cellular membrane. The genetically engineered vector in this study demonstrated that it can be safely and efficiently used to genetically modify stem cells with potential applications in tissue engineering and cancer therapy.

Practical Issues with the Use of Stem Cells for Cancer Gene Therapy.

Stem cell-based drug delivery for cancer therapy has steadily gained momentum in the past decade as several studies have reported stem cells' inherent tropism towards tumors. Since this science is still in its early stages and there are many factors that could significantly impact tumor tropism of stem cells, some contradictory results have been observed. This review starts by examining a number of proof-of-concept studies that demonstrate the potential application of stem cells in cancer therapy. Studies that illustrate stem cells' tumor tropism and discuss the technical difficulties that could impact the therapeutic outcome are also highlighted. The discussion also emphasizes stem cell imaging/tracking, as it plays a crucial role in performing reliable dose-response studies and evaluating the therapeutic outcome of treatment protocols. In each section, the pros and cons associated with each method are highlighted, limitations are underlined, and potential solutions are discussed. The overall intention is to familiarize the reader with important practical issues related to stem cell cancer tropism and in vivo tracking, underline the shortcomings, and emphasize critical factors that need to be considered for effective translation of this science into the clinic.

Reducing the Visibility of the Vector/DNA Nanocomplexes to the Immune System by Elastin-Like Peptides.

PURPOSE: One of the major hurdles facing nanomedicines is the antibody production against nanoparticles that subsequently results in their opsonization and clearance by macrophages. The objective of this research was to examine and identify the sequence of a low-immunogenic peptide based on recombinant elastin-like polypeptides (ELPs) that does not evoke IgG response and can potentially be used for masking the surfaces of the nanoparticles. METHODS: Biopolymers composed of a DNA condensing domain in fusion with anionic, neutral and cationic elastin-like peptides were genetically engineered. The biopolymers were used to complex with plasmid DNA and form ELP-coated nanoparticles. Then, the potential immunogenicity of nanoparticles in terms of IgM/IgG response after repeated injections was evaluated in Balb/c immunocompetent mice. RESULTS: The results revealed the sequence of a non-immunogenic ELP construct that in comparison to control group did not elicit any significant IgG response, whereas the vector/DNA complexes that were coated with polyethylene glycol (PEG) did elicit significant IgG response under the same conditions. CONCLUSIONS: The identification of the sequence of an ELP-based peptide that does not induce IgG response opens the door to more focused in-depth immunotoxicological studies which could ultimately lead to the production of safer and more effective drug/gene delivery systems such as liposomes, micelles, polymeric nanoparticles, viruses and antibodies.

Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TK(SR39) mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems.

A recombinant biopolymeric platform for reliable evaluation of the activity of pH-responsive amphiphile fusogenic peptides.

Over the past couple of decades, the sequences of several cationic and anionic pH-responsive amphiphile fusogenic peptides (FPs) have been reported in the literature. Due to their endosome membrane disrupting activity, these peptides have been routinely used for enhancing the efficacy of drug/gene delivery systems. However, no accurate comparative study has been performed to establish the precise correlation between FP sequence and its impact on enhancing drug/gene delivery efficiency. Therefore, there has been no clear rationale for selecting one FP over another in the past, and it is still unclear which FP is the most suitable and efficient construct for use in drug/gene delivery system design. To address this shortcoming, we examined the use of a recombinant biopolymeric platform as a tool to assess the pH-dependent membrane disruption activity, cell toxicity and impact on gene transfer efficiency of the five most widely used cationic and anionic pH-responsive FPs, INF7, GALA, KALA, H5WYG, and RALA. We first developed specific expression methods for the production of five identical recombinant biopolymers that were different only in FP sequence in their structures. Through the use of physicochemical and biological assays, the biopolymers were characterized and compared in terms of DNA condensation ability, cell toxicity, pH-dependent cell membrane disruption activity, and gene transfer efficiency. Overall, our data suggests that, among the tested constructs, GALA is the most suitable pH-responsive FP for enhancing the efficiency of gene delivery systems due mostly to its efficient endosomolytic activity and negligible cell toxicity. Most importantly, this study demonstrates the application of an effective biopolymeric tool that facilitates reliable evaluation of the physicochemical and biological activities of any pH-responsive FP independent of its charge. Therefore, whether artificially designed or inspired by nature, the FPs can be screened for their efficacy with a higher degree of accuracy in the future.

Systematic engineering of uniform, highly efficient, targeted and shielded viral-mimetic nanoparticles.

In the past decades, numerous types of nanomedicines have been developed for the efficient and safe delivery of nucleic acid-based drugs for cancer therapy. Given that the destination sites for nucleic acid-based drugs are inside cancer cells, delivery systems need to be both targeted and shielded in order to overcome the extracellular and intracellular barriers. One of the major obstacles that has hindered the translation of nanotechnology-based gene-delivery systems into the clinic has been the complexity of the design and assembly processes, resulting in non-uniform nanocarriers with unpredictable surface properties and efficiencies. Consequently, no product has reached the clinic yet. In order to address this shortcoming, a multifunctional targeted biopolymer is genetically engineered in one step, eliminating the need for multiple chemical conjugations. Then, by systematic modulation of the ratios of the targeted recombinant vector to PEGylated peptides of different sizes, a library of targeted-shielded viral-mimetic nanoparticles (VMNs) with diverse surface properties are assembled. Through the use of physicochemical and biological assays, targeted-shielded VMNs with remarkably high transfection efficiencies (>95%) are screened. In addition, the batch-to-batch variability of the assembled targeted-shielded VMNs in terms of uniformity and efficiency is examined and, in both cases, the coefficient of variation is calculated to be below 20%, indicating a highly reproducible and uniform system. These results provide design parameters for engineering uniform, targeted-shielded VMNs with very high cell transfection rates that exhibit the important characteristics for in vivo translation. These design parameters and principles could be used to tailor-make and assemble targeted-shielded VMNs that could deliver any nucleic acid payload to any mammalian cell type.

Use of biotin targeted methotrexate-human serum albumin conjugated nanoparticles to enhance methotrexate antitumor efficacy.

Biotin molecules could be used as suitable targeting moieties in targeted drug delivery systems against tumors. To develop a biotin targeted drug delivery system, we employed human serum albumin (HSA) as a carrier. Methotrexate (MTX) molecules were conjugated to HSA. MTX-HSA nanoparticles (MTX-HSA NPs) were prepared from these conjugates by cross-linking the HSA molecules. Biotin molecules were then conjugated on the surface of MTX-HSA NPs. The anticancer efficacy of biotin targeted MTX-HSA NPs was evaluated in mice bearing 4T1 breast carcinoma. A single dose of biotin targeted MTX-HSA NPs showed stronger in vivo antitumor activity than non-targeted MTX-HSA NPs and free MTX. By 7 days after treatment, average tumor volume in the biotin targeted MTX-HSA NPs-treated group decreased to 17.6% of the initial tumor volume when the number of attached biotin molecules on MTX-HSA-NPs was the highest. Average tumor volume in non-targeted MTX-HSA NPs-treated mice grew rapidly and reached 250.7% of the initial tumor volume. Biotin targeted MTX-HSA NPs increased the survival of tumor-bearing mice to 47.5 ± 0.71 days and increased their life span up to 216.7%. Mice treated with biotin targeted MTX-HSA NPs showed slight body weight loss (8%) 21 days after treatment, whereas non-targeted MTX-HSA NPs treatment at the same dose caused a body weight loss of 27.05% ± 3.1%.

Targeted delivery of methotrexate to tumor cells using biotin functionalized methotrexate-human serum albumin conjugated nanoparticles.

Systemic toxicity following cancer chemotherapy is an important concern. Targeted drug delivery systems could reduce the toxicity of anticancer drugs. Vitamins have been considered as targeting moieties in novel cancer treatment strategies. In this study, biotin was attached to nanoparticles of conjugated methotrexate-human serum albumin. Biotin functionalized methotrexate-human serum albumin with three different amounts of biotin attached to the nanoparticles were prepared. It was shown that the cytotoxicity of biotin functionalized nanoparticles on T47D and HeLa tumor cells was significantly higher than that of free methotrexate and non-functionalized nanoparticles. The cytotoxicity of biotin functionalized nanoparticles further increased when the number of biotin molecules attached on the surface of nanoparticles increased. The uptake of FITC labeled biotin functionalized nanoparticles by T47D and HeLa cells measured by flow cytometry, was also higher than that of non-functionalized nanoparticles. It can be concluded that the biotin functionalized methotrexate-human serum albumin conjugated nanoparticles could be used as a potent drug for specific delivery of methotrexate to tumors.