Detection of Nail Oncometabolite SAICAR in Oral Cancer Patients and Its Molecular Interactions with PKM2 Enzyme.

Oncometabolites are known to drive metabolic adaptations in oral cancer. Several oncometabolites are known to be shared between cancer cells and non-cancer cells including microbiotas to modulate the tumor microenvironment. Among potential oncometabolites, succinylaminoimidazolecarboxamide ribose5'-phosphate (SAICAR) supports the growth and invasiveness of cancer cells by pyruvate kinase M2 (PKM2) enzyme in a glucose starved tumor microenvironment. There is a significant gap that shows the detection of SAICAR in biological samples including nails of oral cancer patients. Metabolite identification of SAICAR was investigated in the nails of oral cancer patients using novel vertical tube gel electrophoresis (VTGE) and LC-HRMS. Further molecular docking and molecular dynamics simulations (MDS) were employed to determine the nature of molecular interactions of SAICAR (CHEBI ID:18319) with PKM2 (PDB ID: 4G1N). Molecular docking of SAICAR (CHEBI ID:18319) was performed against pyruvate kinase M2 (PDB ID: 4G1N). Data suggest the presence of oncometabolite SAICAR in nails of oral cancer. Molecular docking of SAICAR with PKM2 showed appreciable binding affinity (-8.0 kcal/mol) with residues including ASP407, THR405, GLU410, ARG443, GLY321, ARG436, HIS439, LYS266, and TYR466. Furthermore, MDS confirmed the specific binding of SAICAR within the activator site of PKM2 and the stability of SAICAR and PKM2 molecular interactions. In conclusion, SAICAR is a promising oncometabolite biomarker present in the nails of oral cancer patients. A significant activation potential of SAICAR exists with the PKM2 enzyme.

Effect of Graphene on Differentiation and Mineralization of Dental Pulp Stem Cells in Poly(4-vinylpyridine) Matrix in Vitro.

We have investigated the influence of graphene nanoplatelet scaffolds for dental pulp cells (DPSCs) made from poly(4-vinylpyridine) (P4VP) either via spin-casting flat films or electrospinning nano- and microscale fibers. We found that graphene predominated over other factors in promoting differentiation of DPSCs. In the absence of graphene, real-time-polymerase chain reaction (RT-PCR) and energy dispersive X-ray (EDX) analyses indicated that the DPSCs differentiated along odontogenic lineages only on the nano- and microelectrospun scaffolds. Closer scanning electron microscopy (SEM) imaging revealed formation of banded collagen structures, which nucleated on the electrospun fibers in the absence of graphene. Biomineral deposition was templated on these fibers, with mineral to protein ratios similar to dentin. In the microfibers, the graphene was completely encapsulated and appeared to hinder biomineralization. Previously minimal biomineralization and banded collagen were observed on flat spun cast substrates. Addition of graphene appeared to induce nucleation of banded collagen fibers and template biomineral deposition. Addition of graphene did not affect the outcome of the DPSCs cultured on the nanofibers, which biomineralized regardless of graphene inclusion. Based on these results, we hypothesize that direct contact with graphene is the primary factor determining differentiation of the DPSCs. On the flat surface and nanoscale electrospun fibers, the graphene protrudes from the sample enabling direct contact with the extracellular matrix (ECM) and cells, while on the microfibers, the graphene is fully encapsulated within the matrix. TUNA imaging with scanning force microscopy showed enhanced conductivity on fibers with encapsulated graphene, which we hypothesize may change the conformation of adsorbed ECM proteins, affecting DPSCs differentiation.

A High-Throughput Bioluminescent Assay to Monitor the Deamidation of Asparagine and Isomerization of Aspartate Residues in Therapeutic Proteins and Antibodies.

Since the introduction of Herceptin and Rituximab in 1986, therapeutic antibodies have gained tremendous momentum in the treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions that may result in compromised bioactivity and safety profile. The nonenzymatic post-translational modification of both deamidation of asparagine and isomerization of aspartate residues is one of the major chemical reactions occurring in proteins during production and storage resulting in formation of protein variants that may affect the quality, safety, and functionality of the therapeutic proteins. Current methods (HPLC and liquid chromatography and mass spectrometry) for monitoring isoaspartate (isoAsp) formation are time consuming, require specialized equipment and trained personnel, and are not amenable to high-throughput scaling. We have developed a robust, homogenous, high-throughput formatted, and sensitive assay to accurately monitor the formation of isoAsp under several conditions, such as new formulations, storage periods, and temperature.