3D Printing for Tissue Regeneration

Tissue engineering is an interdisciplinary field and 3D bioprinting has emerged to be the holy grail to fabricate artificial organs. This chapter gives an overview of the latest advances in 3D bioprinting technology in the commercial space and academic research sector. It explores the commercially available 3D bioprinters and commercially printed products that are currently available in the market. It provides a brief introduction to bioinks and the latest developments in 3D bioprinting various organs. The chapter also discusses the advancements in tissue regeneration from 3D printing to 4D printing

Thermodynamics of i-tetraplex formation in the nuclease hypersensitive element of human c-myc promoter

More than 85% of c-myc transcription is controlled by the nuclease hypersensitive element III₁ upstream of the P1 promoter of this oncogene. The purine-rich sequence in the anti-sense strand forms a G-quadruplex, which has been recently implicated in colorectal cancer, and is proposed as a silencer element [Proc. Natl. Acad. Sci. USA 101 (2004) 6140]. This prompted us to characterize the thermodynamics and proton/counterion effect of the complementary pyrimidine-rich sequence, which forms a C-tetraplex. We report the thermodynamic parameters for folding of the pyrimidine-rich DNA fragment from this region into a C-tetraplex. At 20°C, we observed a ΔG of −10.36 ± 0.13 kcal mol⁻¹ with favorable enthalpy (ΔH = −75.99±0.99 kcal mol⁻¹) and unfavorable entropy (TΔS = −65.63±0.88 kcal mol⁻¹) at pH 5.3 in 20 mM NaCl for tetraplex folding. Similar characteristic stabilizing enthalpy and destabilizing entropy were observed at other pH and ionic strengths. Folding was induced by uptake of about two to three protons per mole of tetraplex while a marginal (0.5–1 mol/mol) counterion uptake was observed. In the context of current understanding of c-myc transcription we envisage a role of the i-motif in remodeling the G-quadruplex silencer

Quadruplex-duplex competition in the nuclease hypersensitive element of human c-myc promoter: C to T mutation in C-rich strand enhances duplex association

The nuclease hypersensitive element NHE IIII is an important anti-cancer target as the transcription of oncogene c-myc is largely regulated by it. It has been postulated that regulatory control is mediated by G-quadruplex formation in the NHE anti-sense strand through a competition between the duplex and the quadruplex states. A mutation in the NHE has been implicated in cancer. In this study, the reported mutation has been characterized vis-a-vis the kinetics of i-tetraplex formation (in the sense strand) and its effect on duplex formation. We found that i-tetraplex formation was destabilized by ∼1.4 kcal/mol (ΔΔG at 20°C, pH 5.8). Observed hysteresis allowed us to analyze the kinetics of folding for the mutant (M3). Though we observed higher association (ΔE_on ≈ −23.4 kcal/mol) and dissociation (ΔE_off ≈ 22.1 kcal/mol) activation energies (at pH 5.3) for the wild-type (P1) tetraplex folding, the kinetics of folding and unfolding for M3 was somewhat faster at pH 5.3 and 5.8. Interestingly, Surface plasmon resonance (BIAcore) analysis of hybridization at pH 6.6 indicated a higher association constant for M3 (∼22.5 × 10⁴ M⁻¹ s⁻¹) than P1 (∼3.2 × 10⁴ M⁻¹ s⁻¹). The equilibrium dissociation constants also indicated favorable duplex association for M3 (∼22.2 and ∼190.6 nM for M3 and P1, respectively). We envisage that the increased affinity for the duplex state due to the mutation could play a functional role in the aberrant regulation of c-myc

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue96 in the Plasmodial Enzyme

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residuephenylalanineat this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms