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Gluten tespitindeki ilerlemeler: Antikor gelişimi ve yanal akışlı immünodeney için altın nanopartikül konjugasyonu
Objective: Gluten-related disorders, like celiac disease (CD), affect 5% of the global population, with CD impacting 1.4% of individuals. Consuming gluten triggers inflammation in the intestines for those with CD. Following a gluten-free (GF) diet is crucial despite challenges like cross-contamination. Immunoassays like enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) are preferred for detecting gluten due to their simplicity and cost-effectiveness. LFIA kits offer a portable option for analyzing gluten in food. The demand for reliable gluten detection methods highlights the need for accurate solutions for both food manufacturers and individuals with gluten-related disorders. Our research focuses on developing specific techniques using antibodies targeting gliadin, one of the main parts of gluten, to confirm the absence of gluten. Method: The polyclonal antibodies obtained from rabbits were purified and tested for reactivity to gliadin using ELISA and Western blot techniques. They were then conjugated with gold nanoparticles (AuNPs) to develop an LFIA, with optimal conditions determined by pH and antibody concentration. The efficacy of the LFIA in detecting wheat gliadin, including the limit of detection, was investigated. Gliadin was extracted and analyzed from various food samples to establish a reliable method for detecting it in different products. Results: Antibodies from three rabbits (R1, R2, and R3) were sensitive to gliadin in an ELISA. Western blot analysis confirmed common gliadin-specific epitopes in these antibodies. They did not react to GF food samples. The LFIA effectively detected wheat gliadin using a selected membrane and fibers, with a detection limit of 50 µg/mL. The LFIA consistently produced reliable results when testing samples from different food sources, making it a dependable tool for gliadin detection in various food samples. Conclusion: Our study presents a technique for identifying gluten, including the development of antibodies, improving AuNP attachment, and establishing a reliable LFIA protocol. This methodology has the potential to ensure food safety and facilitate gluten analysis. Keywords: Antibody development, Antibody conjugation, Lateral flow immunoassay
İnsan fibroblast hücre hatlarından uyarılmış pluripotent kök hücre eldesi
Objective: Induced pluripotent stem cells (iPSCs) possess the remarkable abilities to differentiate into cell types from all three germ layers and to self-renew indefinitely. These features make them highly valuable for in vitro modeling of human biology and gene and cell therapies. However, current methods for iPSC generation are often inefficient and require prolonged culture periods, necessitating improved protocols with enhanced safety and efficacy. In this study, we aimed to reprogram HFF-1 human fibroblasts into iPSCs using integration-deficient lentiviral vectors (IDLV-OSKs) encoding the reprogramming factors Oct4, Sox2, and Klf4, along with sodium butyrate as a supporting small molecule to increase efficiency. Methods: In our study, IDLV-OSK vectors encoding the transcriptional regulators Oct4, Sox2, and Klf4 were used to reprogram HFF-1 fibroblast cells into pluripotency. In addition to the viral vector, the epigenetic modulator sodium butyrate, which is a histone deacetylase inhibitor, was used as a supportive molecule. With our applied reprogramming protocol, colonies suitable for passaging were obtained within 9–12 days. The resulting iPSCs were characterized morphologically and also by the expression of pluripotency markers expressed on the cell surface (alkaline phosphatase, Tra-1-81, Tra-1-60) and those located in the nucleus (Oct4, Klf4, c-Myc, Nanog). Results: We generated iPSCs using IDLV-OSKs with the support of a histone deacetylase inhibitor. The resulting colonies exhibited the expected morphological characteristics. The expression of surface and nuclear pluripotency markers was demonstrated in the colonies. Conclusion: Our findings demonstrate that IDLV-OSK vectors, along with sodium butyrate, constitute an efficient and potentially safe approach for reprogramming human fibroblasts into iPSCs with our protocol. This method holds promise for efficient and safer applications in disease modeling and the development of experimental therapeutic strategies. Keywords: Induced pluripotent stem cells, Sodium butyrate, Lentiviral vectors
The possible anti-tumoral/anti-metastatic effects of BMP1 inhibitor alone or in combination with doxorubicin on metastatic breast carcinoma
Objective: Preventing distant organ metastases is critical for reducing mortality in metastatic breast cancer. Current treatments are insufficient in preventing spread and are limited by resistance and side effects. Doxorubicin (DOX) suppresses tumor progression but causes cardiotoxicity and resistance. Bone morphogenetic protein-1 (BMP-1), a metalloproteinase involved in tumor microenvironment and extracellular matrix remodeling, supports metastasis and poor prognosis. BMP-1 inhibitors may reshape the tumor microenvironment, reduce metastasis, and enhance chemotherapy effectiveness. This thesis aimed to evaluate the effects of the BMP-1 inhibitor UK-383367, alone and in combination with DOX, on tumor progression, lung and liver metastasis, immune cell subpopulations, and cytokine responses in in vivo breast cancer models. Method: In vivo models were established using 4TBM and 4TLM cell lines in BALB/c female mice, and tumor volume, lung and liver metastatic burden, distribution of immune cell subpopulations, and cytokine levels (TNF-α, IL-6, IL-10, IFN-γ) were evaluated. Results: Treatment with UK alone was found to reduce the lung metastatic burden and support T cell-mediated immune responses; however, it showed limited effects on primary tumor volume. DOX alone resulted in reduced tumor volume and metastatic spread while activating the immune response. The combination treatment of UK and DOX was observed to most effectively reduce tumor volume and metastatic burden, enhance the cytotoxic T cell response, and decrease immunosuppressive cell populations. Cytokine analyses revealed that IL-6 and IFN-γ levels increased in the treatment groups, while IL-10 levels exhibited context-dependent variability. Conclusion: The results of our study demonstrated that targeting BMP-1 represents a promising approach in the treatment of metastatic breast cancer, and that the combination of UK-383367 and DOX has the potential to reduce metastatic spread and support the immune response. Key words: BMP-1, UK-383367, Doxorubicin, Metastatic Breast Cancer, Immune response, Tumor microenvironment
Generation of pancreatic beta cell-like insulin-producing cells from induced pluripotent stem cells
Objective: Studies on generation of pancreatic beta cell-like insulin-producing cells (IPCs) from induced pluripotent stem cells (iPSCs) aim to compensate for beta cell destruction/dysfunction in diabetes. Pancreas or islet transplantations are frequently associated with disadvantages such as insufficient number of donors and requirement for immunosuppression. Although exogenous insulin administration is life-saving, it is still not considered optimal, with possible long-term complications. IPCs generated from iPSCs originating from individuals' own cells have a great potential to provide an infinite beta cell source and eliminate/reduce requirement for immunosuppression; however, optimally efficient and safer strategies are still needed. In our study, we aimed to generate beta cell-like IPCs from IPSCs via an improved approach. Method: We used small molecules activating key signaling pathways throughout beta cell development and the TNF-Related Apoptosis-Inducing Ligand (TRAIL) molecule, the protective effect of which is frequently reported in diabetes. iPSC characterization was done via confirmation of morphological criteria and pluripotency, along with mycoplasma analysis. We identified three different stages (definitive endoderm, pancreatic differentiation, and differentiation into beta cell-like IPCs) via immunocytochemical stainings for specific markers and insulin ELISA assay for detection of the insulin release. Results: We obtained IPCs from human iPSCs via use of specific small molecules and sTRAIL. Differentiation into three distinct stages of beta cell-like IPCs were confirmed by expression of Sox17 and FoxA2 (definitive endoderm); PDX-1 and insulin (pancreatic differentiation); and PDX-1, insulin, NeuroD1, Pax6, and Islet-1 (differentiation into IPCs). Significantly increased insulin secretion was evident by sTRAIL application. Conclusion: The combined approach of IPC generation used in our study may be useful as an improved strategy, with enhanced insulin secretion in cells in response to different doses of sTRAIL stimulation.