Small molecules, growth factors, and cytokines have been used to induce differentiation of stem cells into different lineages. Similarly, demethylating agents can trigger differentiation in adult stem cells. Here, we investigated the in vitro differentiation of rat bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes by a demethylating agent, zebularine, as well as neuronal-like cells by β-mercaptoethanol in a growth factor or cytokines-free media. Isolated bone marrow-derived MSCs cultured in Dulbecco's Modified Eagle's Medium exhibited a fibroblast-like morphology. These cells expressed positive markers for CD29, CD44, and CD117 and were negative for CD34 and CD45. After treatment with 1 μM zebularine for 24 hours, the MSCs formed myotube-like structures after 10 days in culture. Expression of cardiac-specific genes showed that treated MSCs expressed significantly higher levels of cardiac troponin-T, Nkx2.5, and GATA-4 compared with untreated cells. Immunocytochemical analysis showed that differentiated cells also expressed cardiac proteins, GATA-4, Nkx 2.5, and cardiac troponin-T. For neuronal differentiation, MSCs were treated with 1 and 10 mM β-mercaptoethanol overnight for 3 hours in complete and serum-free Dulbecco's Modified Eagle's Medium, respectively. Following overnight treatment, neuron-like cells with axonal and dendritic-like projections originating from the cell body toward the neighboring cells were observed in the culture. The mRNA expression of neuronal-specific markers, Map2, Nefl, Tau, and Nestin, was significantly higher, indicating that the treated cells differentiated into neuronal-like cells. Immunostaining showed that differentiated cells were positive for the neuronal markers Flk, Nef, Nestin, and β-tubulin.
In the present work, a concise library of 1,3,5-triaryl-2-pyrazolines (2a-2q) was designed and synthesized by employing a multistep strategy, and the newly synthesized compounds were screened for their urease and α-glucosidase inhibitory activities. The compounds (2a-2q) were characterized using a combination of several spectroscopic techniques including FT-IR, 1H NMR, 13C NMR, and EI-MS. All the synthesized compounds, except compound 2i, were potent against urease as compared to the standard inhibitor thiourea (IC50 = 21.37 ± 0.26 μM). These analogs disclosed varying degrees of urease inhibitory activities ranging from 9.13 ± 0.25 to 18.42 ± 0.42 μM. Compounds 2b, 2g, 2m, and 2q having IC50 values of 9.36 ± 0.27, 9.13 ± 0.25, 9.18 ± 0.35, and 9.35 ± 0.35 μM, respectively, showed excellent inhibitory activity as compared to standard thiourea (IC50 = 21.37 ± 0.26 μM). A kinetic study of compound 2g revealed that compound 2g inhibited urease in a competitive mode. Among the synthesized pyrazolines, the compounds 2c, 2k, 2m, and 2o exhibited excellent α-glucosidase inhibitory activity with the lowest IC50 values of 212.52 ± 1.31, 237.26 ± 1.28, 138.35 ± 1.32, and 114.57 ± 1.35 μM, respectively, as compared to the standard acarbose (IC50 = 375.82 ± 1.76 μM). The compounds (2a-2q) showed α-glucosidase IC50 values in the range of 114.57 ± 1.35 to 462.94 ± 1.23 μM. Structure-activity relationship revealed that the size and electron-donating or -withdrawing effects of substituents influenced the activities, which led to the urease and α-glucosidase inhibiting properties. Compound 2m was a dual potent inhibitor against urease and α-glucosidase due to the presence of 2-CF3 electron-withdrawing functionality on the phenyl ring. To the best of our knowledge, these synthetic compounds were found to be the most potent dual inhibitors of urease and α-glucosidase with minimum IC50 values. The cytotoxicity of the compounds (2a-2q) was also investigated against human cell lines MCF-7 and HeLa. Compound 2l showed moderate cytotoxic activity against MCF-7 and HeLa cell lines. Moreover, in silico studies on most active compounds were also performed to understand the binding interaction of most active compounds with active sites of urease and α-glucosidase enzymes. Some compounds exhibited drug-like characteristics due to their lower cytotoxic and good ADME profiles.
Background and Objectives: Citrobacter freundii (C. freundii) is an emerging and opportunistic Gram-negative bacteria of the human gastrointestinal tract associated with nosocomial and severe respiratory tract infections. It has also been associated with pneumonia, bloodstream, and urinary tract infections. Intrinsic and adaptive virulence characteristics of C. freundii have become a significant source of diarrheal infections and food poisoning among immune-compromised patients and newborns. Impulsive usage of antibiotics and these adaptive virulence characteristics has modulated the C. freundii into multidrug-resistant (MDR) bacteria. Conventional approaches are futile against MDR C. freundii. Materials and Methods: The current study exploits the modern computational-based vaccine design approach to treat infections related to MDR C. freundii. A whole proteome of C. freundii (strain: CWH001) was retrieved to screen pathogenic and nonhomologous proteins. Six proteins were shortlisted for the selection of putative epitopes for vaccine construct. Highly antigenic, nonallergen, and nontoxic eleven B-cell, HTL, and TCL epitopes were selected for mRNA- and peptide-based multi-epitope vaccine construct. Secondary and tertiary structures of the multi-epitope vaccine (MEVC) were designed, refined, and validated. Results: Evaluation of population coverage of MHC-I and MHC-II alleles were 72% and 90%, respectively. Docking MEVC with TLR-3 receptor with the binding affinity of 21.46 (kcal/mol) occurred through the mmGBSA process. Further validations include codon optimization with an enhanced CAI value of 0.95 and GC content of about 51%. Immune stimulation and molecular dynamic simulation ensure the antibody production upon antigen interaction with the host and stability of the MEVC construct, respectively. Conclusions: These interpretations propose a new strategy to combat MDR C. freundii. Further, in vivo and in vitro trials of this vaccine will be valuable in combating MDR pathogens.