Growth factor-mimicking 3,4-dihydroxyphenylalanine-encoded bioartificial extracellular matrix like protein promotes wound closure and angiogenesis.

The present work reports a new route to prepare a "smart biomaterial" by mimicking long-acting cellular growth factor showing enhanced cell-material interactions by promoting cell proliferation and angiogenesis. For that, reactive non-proteogenic amino acid 3,4-dihydroxyphenylalanine (DOPA) was genetically introduced into an intrinsic triple-helical hierarchical structure forming protein to initiate hierarchical self-assembly to form a macromolecular structure. The self-assembled scaffold displayed vascular endothelial growth factor mimicking the pro-angiogenic reactive group for repairing and remodeling of damaged tissue cells. We customized the recombinant collagen-like protein (CLP) with DOPA to promote rapid wound healing and cell migrations. Selective incorporation of catechol in variable and C-terminal region of CLP enhanced interaction between inter- and intra-triple-helical collagen molecules that resulted in a structure resembling higher-order native collagen fibril. Turbidity analysis indicated that the triple-helical CLP self-assembled at neutral pH via a catechol intra-crosslinking mechanism. After self-assembly, only DOPA-encoded CLP formed branched filamentous structures suggesting that catechol mediated network coordination. The catechol-encoded CLP also acted as a "smart material" by mimicking long-acting cellular growth factor showing enhanced cell-material interactions by promoting cell proliferation and angiogenesis. It eliminates release rate, stability, and shelf-life of hybrid growth factor conjugated biomaterials. The newly synthesized CLP has the potential to promote accelerated cell migration, pro-angiogenesis, and biocompatibility and could be used in the field of implantable medical devices and tissue engineering.

Insights into the effect of artificial sweeteners on the structure, stability, and fibrillation of type I collagen.

Artificial sweeteners (AS) are widely used as sugar substitutes because natural sweetener (sugar) leads to a number of health issues, including diabetes, obesity, and tooth decay. Since natural sugar (sucrose), diabetes and skin are highly interlinked, and also sucrose is known to inhibit the fibrillation of collagen, the major protein of the skin, a study on the impact of AS on collagen is important and essential. Herein, we have studied the influence of commonly used AS such as Sucralose (SUC), Aspartame (APM), and Saccharin (SAC) on the structure, stability, and fibrillation of collagen using various spectroscopic methods. The circular dichroism and turbidity results suggest that the AS does not disrupt the triple helix structure and also the fibrillar property of collagen, respectively. The fibrillar morphology was sustained, although there was a trivial difference in the entanglement of fibrils in the presence of SAC, compared to native collagen fibrils. The thermal stability of collagen is maintained in the presence of AS. Fluorescence and STD-NMR results indicate that the interaction between AS and collagen was weak, which supports the intact structure, stability, and fibrillation property of collagen. The current study thus suggests that the chosen AS does not influence collagen properties.

Immunological evaluation of fusion protein of Brugia malayi abundant larval protein transcript-2 (BmALT-2) and Tuftsin in experimental mice model.

INTRODUCTION: Filariasis, a neglected tropical helminth disease needs vaccine besides mass drug administration for its successful eradication. METHODS: An attempt was made to produce a fusion protein (P-TUFT-ALT-2) of abundant larval transcript protein-2 and Tuftsin to enhance its immunogenicity. The fusion construct was expressed in Pichia pastoris, a nonexpensive commercial expression system. This study focused on the evaluation of immunological response produced by P-TUFT-ALT-2 in Balb/c mice. RESULT AND DISCUSSION: P-TUFT-ALT-2 showed an enhanced IgG peak titre compared to E. coli expressed E-ALT-2 and P. pastoris expressed P-ALT-2. IgG2b, IgG2a and IgG1 production were predominant indicating a balanced Th1/Th2 response. P-TUFT-ALT-2 also induced about 28% and 9.5% higher splenocyte proliferation over control and E-ALT-2 respectively. Splenocytes produced predominant IFN-γ followed by IL-5, IL-2 and IL-10 specifying a balanced Th1/Th2 response. P-TUFT-ALT-2 showed 55% to 80% with an average of 65% cytotoxicity in B. malayi L3 larvae in in vitro ADCC assay. CONCLUSION: This experiment validates P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis.

A self-assembly and higher order structure forming triple helical protein as a novel biomaterial for cell proliferation.

Collagen plays a critical role in the structural design of the extracellular matrix (ECM) and cell signaling in mammals, which makes it one of the most promising biomaterials with versatile applications. However, there is considerable concern regarding the purity and predictability of the product performance. At present, it is mainly derived as a mixture of collagen (different types) from animal tissues, where the selective enrichment of a particular type of collagen is generally difficult and expensive. Collagen derived from bovine sources poses the risk of transmitting diseases and can cause adverse immunologic and inflammatory responses. Hence, recombinant collagen can be a good alternative. Nevertheless, the necessity of post-translational hydroxyproline (Hyp) modification limits large-scale recombinant collagen production. Here, we recombinantly expressed the collagen-like protein (CLTP) and genetically introduced the Hyp in the CLTP to form a higher order self-assembled fibril structure, similar to human collagen. During the current study, it was observed that the Hyp incorporated CLTP protein (CLTHP) formed a stable triple helical polyproline-II like structure and self-assembled to form fibrils at neutral pH, which had an initial lag phase followed by a growth phase similar to animal collagen. In contrast, the higher order fibrillar assembly was missing in the nonhydroxylated CLTP. This study demonstrated that CLTHP self-association is based on the common underlying lateral interactions between triple helical structured proteins, where the hydroxyproline forms the significantly stable hydration network. Hence, this work will be the first fundamental empirical research for flexible modifications of recombinant collagen for structural analysis and biomedical applications.