Mesenchymal stem cells (MSCs) show a decline in pluripotency and differentiation with increased cell culture passages in 2D cultures. The 2D monolayer culture fails to correctly imitate the architecture and microenvironments of in-vivo cell models. Alternatively, 3D culture may improve the simulations of in-vivo cell microenvironments with wide applications in cell culture and drug discovery. In the present study, we compared various 3D culturing techniques such as 3D micro-well (3D-S), hanging drop (HD), and ultra-low attachment (ULA) plate-based spheroid culture to study their effect on morphology, viability, pluripotency, cell surface markers, immunomodulatory factors, and differentiation capabilities of Wharton's jelly-mesenchymal stem cells (WJ-MSCs). The cell morphology, viability, and senescence of 3D cultured WJ-MSCs were comparable to cells in 2D culture. The expression of pluripotency markers (OCT4, SOX2, and NANOG) was enhanced upto 2-8 fold in 3D cultured WJ-MSCs when compared to 2D culture. Moreover, the immunomodulatory factors (IDO, IL-10, LIF, ANG1, and VEGF) were significantly elevated in ULA based 3D cultured WJ-MSCs. Furthermore, significant enhancement in the differentiation potential of WJ-MSCs towards adipocyte (ADP and C/EBP-α), osteocyte (OPN and RUNX2), and definitive endodermal (SOX17, FOXA2, and CXCR4) lineages in 3D culture conditions were observed. Additionally, the osteogenic and adipogenic differentiation potential of WJ-MSCs over the time points 7 days, 14 days, and 28 days was also significantly increased in 3D culture groups. Our study demonstrates that stemness properties of WJ-MSCs were significantly enhanced in 3D cultures and ULA-based culture outperformed other methods with high pluripotency gene expression and enhanced differentiation potential. This study indicates the efficacy of 3D cultures to bridge the gap between 2D cell culture and animal models in regenerative medicine.

Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase which is expressed in most of the hematopoietic cells and plays an important role in many cellular signaling pathways. B cell malignancies are dependent on BCR signaling, thus making BTK an efficient therapeutic target. Over the last few years, significant efforts have been made in order to develop BTK inhibitors to treat B-cell malignancies, and autoimmunity or allergy/hypersensitivity but limited success has been achieved. Here in this study, 3D QSAR pharmacophore models were generated for Btk based on known IC₅₀ values and experimental energy scores with extensive validations. The five features pharmacophore model, Hypo1, includes one hydrogen bond acceptor lipid, one hydrogen bond donor, and three hydrophobic features, which has the highest correlation coefficient (0.98), cost difference (112.87), and low RMS (1.68). It was further validated by the Fisher’s randomization method and test set. The well validated Hypo1 was used as a 3D query to search novel Btk inhibitors with different chemical scaffold using high throughput virtual screening technique. The screened compounds were further sorted by applying ADMET properties, Lipinski’s rule of five and molecular docking studies to refine the retrieved hits. Furthermore, molecular dynamic simulation was employed to study the stability of docked conformation and to investigate the binding interactions in detail. Several important hydrogen bonds with Btk were revealed, which includes the gatekeeper residues Glu475 and Met 477 at the hinge region. Overall, this study suggests that the proposed hits may be more effective inhibitors for cancer and autoimmune therapy.

Melanoma is one of the most lethal forms of skin cancer and its incidence is continuing to rise in the United States. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. SIRT1, a NAD(+)-dependent class III histone deacetylase, has been implicated in a variety of physiological processes and pathological conditions. We recently demonstrated that SIRT1 is upregulated in melanoma and its inhibition by a small-molecule, tenovin-1, inhibits cell proliferation and clonogenic survival of melanoma cells, possibly via activating p53. Here, we employed a gel free quantitative proteomics approach to identify the downstream effectors and targets of SIRT1 in melanoma. The human malignant melanoma, G361 cells were treated with tenovin-1 followed by protein extraction, in liquid trypsin digestion, and peptide analyses using nanoLC-MS/MS. A total of 1091 proteins were identified, of which 20 proteins showed significant differential expression with 95% confidence interval. These proteins were subjected to gene ontology and Ingenuity Pathway Analysis (IPA) to obtain the information regarding their biological and molecular functions. Real-Time qRT-PCR validation showed that five of these (PSAP, MYO1B, MOCOS, HIST1H4A and BUB3) were differentially expressed at mRNA levels. Based on their important role in cell cycle regulation, we selected to focus on BUB family proteins (BUB3, as well as BUB1 and BUBR1) for subsequent validation. The qRT-PCR and immunoblot analyses showed that tenovin-1 inhibition of SIRT1 resulted in a downregulation of BUB3, BUB1 and BUBR1 in multiple melanoma cell lines. Since tenovin-1 is an inhibitor of both SIRT1 and SIRT2, we employed lentivirus mediated silencing of SIRT1 and SIRT2 in G361 cells to determine if the observed effects on BUB family proteins are due to SIRT1- or SIRT2- inhibition. We found that only SIRT1 inhibition resulted in a decrease in BUB3, BUB1 and BUBR1. Our study identified the mitotic checkpoint regulator BUB family proteins as novel downstream targets of SIRT1. However, further validation is needed in appropriate models to confirm our findings and expand on our observations.

Hyaluronic acid (HA) has a wide range of biomedical applications including the formation of hydrogels, microspheres, sponges, and films. The modeling of HA to understand its behavior and interaction with other biomolecules at the atomic level is of considerable interest. The atomistic representation of long HA polymers for the study of the macroscopic structural formation and its interactions with other polyelectrolytes is computationally demanding. To overcome this limitation, we developed a coarse grained (CG) model for HA adapting the Martini scheme. A very good agreement was observed between the CG model and all-atom simulations for both local (bonded interactions) and global properties (end-to-end distance, a radius of gyration, RMSD). Our CG model successfully demonstrated the formation of HA gel and its structural changes at high salt concentrations. We found that the main role of CaCl₂ is screening the electrostatic repulsion between chains. HA gel did not collapse even at high CaCl₂ concentrations, and the osmotic pressure decreased, which agrees well with the experimental results. This is a distinct property of HA from other proteins or polynucleic acids which ensures the validity of our CG model. Our HA CG model is compatible with other CG biomolecular models developed under the Martini scheme, which allows for large-scale simulations of various HA-based complex systems.