Genetically encoded biocompatible anti-coagulant protein-coated coronary artery stents drive endothelialization.

In response to the critical demand for advancements in coronary artery stents, this study addresses the challenges associated with arterial recoil and restenosis post-angioplasty and the imperative to encourage rapid re-endothelialization for minimizing thrombosis risks. We employed an innovative approach inspired by mussel adhesion, incorporating placental anticoagulant protein (AnnexinV) on stent design. The introduction of a post-translationally modified catecholic amino acid L-3,4-dihydroxyphenylalanine (L-Dopa), mimicking mussel characteristics, allowed for effective surface modification of Stainless steel stents through genetic code engineering in AnnexinV (AnxDopa). The efficacy of AnxDopa was analyzed through microscale thermophoresis and flow cytometry, confirming AnxDopa's exceptional binding with phosphatidylserine and activated platelets. AnxDopa coated stainless steel demonstrates remarkable bio-, hemo-, and immuno-compatibility, preventing smooth muscle cell proliferation, platelet adhesion, and fibrin formation. It acts as an interface between the stent and biological fluid, which facilitates the anticoagulation and rapid endothelialization. Surface modification of SS verified through XPS analysis and contact angle measurement attests to the efficacy of AnxDopa mediated surface modification. The hydrophilic nature of the AnxDopa-coated surface enhanced the endothelialization through increased protein absorption. This approach represents a significant stride in developing coronary stents with improved biocompatibility and reduced restenosis risks, offering valuable contributions to scientific and clinical realms alike.

Photocrosslinkable triple helical protein with enhanced higher-order formation for biomaterial applications.

Bacterial collagen, produced via recombinant DNA methods, offers advantages including consistent purity, customizable properties, and reduced allergy potential compared to animal-derived collagen. Its controlled production environment enables tailored features, making it more sustainable, non-pathogenic, and compatible with diverse applications in medicine, cosmetics, and other industries. Research has focused on the engineering of collagen-like proteins to improve their structure and function. The study explores the impact of introducing tyrosine, an amino acid known for its role in fibril formation across diverse proteins, into a newly designed bacterial collagen-like protein (Scl2), specifically examining its effect on self-assembly and fibril formation. Biophysical analyses reveal that the introduction of tyrosine residues didn't compromise the protein's structural stability but rather promoted self-assembly, resulting in the creation of nanofibrils-a phenomenon absent in the native Scl2 protein. Additionally, stable hydrogels are formed when the engineered protein undergoes di-tyrosine crosslinking under light exposure. The hydrogels, shown to support cell viability, also facilitate accelerated wound healing in mouse fibroblast (NIH/3T3) cells. These outcomes demonstrate that the targeted inclusion of functional residues in collagen-like proteins enhances fibril formation and facilitates the generation of robust hydrogels using riboflavin chemistry, presenting promising paths for research in tissue engineering and regenerative medicine.

Biomimetic design of fibril-forming non-immunogenic collagen like proteins for tissue engineering.

Collagen, a key component of extracellular matrix serves as a linchpin for maintaining structural integrity and functional resilience. Concerns over purity and immunogenicity of animal-derived collagens have spurred efforts to develop synthetic collagen-based biomaterials. Despite several collagen mimics, there remains limited exploration of non-immunogenic biomaterials with the capacity for effective self-assembly. To combat the lacuna, collagen like protein (CLP) variants were rationally designed and recombinantly expressed, incorporating human telopeptide sequences (CLP-N and CLP-NC) and bioactive binding sites (CLP-NB). Circular dichroism analyses of the variants confirmed the triple helical conformation, with variations in thermal stability and conformation attributed to the presence of telopeptides at one or both ends of CLP. The variants had propensity to form oligomers, setting the stage for fibrillogenesis. The CLP variants were biocompatible, hemocompatible and supported cell proliferation and migration, particularly CLP-NB with integrin-binding sites. Gene expression indicated a lack of significant upregulation of inflammatory markers, highlighting the non-immunogenic nature of these variants. Lyophilized CLP scaffolds maintained their triple-helical structure and offered favorable biomaterial characteristics. These results accentuate the potential of designed CLP variants in tissue engineering, regenerative medicine and industrial sectors, supporting the development of biocompatible scaffolds and implants for therapeutic and cosmetic purposes.

Recombinant and genetic code expanded collagen-like protein as a tailorable biomaterial.

Collagen occurs in nature with a dedicated triple helix structure and is the most preferred biomaterial in commercialized medical products. However, concerns on purity, disease transmission, and the reproducibility of animal derived collagen restrict its applications and warrants alternate recombinant sources. The expression of recombinant collagen in different prokaryotic and eukaryotic hosts has been reported with varying degrees of success, however, it is vital to elucidate the structural and biological characteristics of natural collagen. The recombinant production of biologically functional collagen is restricted by its high molecular weight and post-translational modification (PTM), especially the hydroxylation of proline to hydroxyproline. Hydroxyproline plays a key role in the structural stability and higher order self-assembly to form fibrillar matrices. Advancements in synthetic biology and recombinant technology are being explored for improving the yield and biomimicry of recombinant collagen. It emerges as reliable, sustainable source of collagen, promises tailorable properties and thereby custom-made protein biomaterials. Remarkably, the evolutionary existence of collagen-like proteins (CLPs) has been identified in single-cell organisms. Interestingly, CLPs exhibit remarkable ability to form stable triple helical structures similar to animal collagen and have gained increasing attention. Strategies to expand the genetic code of CLPs through the incorporation of unnatural amino acids promise the synthesis of highly tunable next-generation triple helical proteins required for the fabrication of smart biomaterials. The review outlines the importance of collagen, sources and diversification, and animal and recombinant collagen-based biomaterials and highlights the limitations of the existing collagen sources. The emphasis on genetic code expanded tailorable CLPs as the most sought alternate for the production of functional collagen and its advantages as translatable biomaterials has been highlighted.

A deep dive into the darning effects of biomaterials in infarct myocardium: current advances and future perspectives.

Myocardial infarction (MI) occurs due to the obstruction of coronary arteries, a major crux that restricts blood flow and thereby oxygen to the distal part of the myocardium, leading to loss of cardiomyocytes and eventually, if left untreated, leads to heart failure. MI, a potent cardiovascular disorder, requires intense therapeutic interventions and thereby presents towering challenges. Despite the concerted efforts, the treatment strategies for MI are still demanding, which has paved the way for the genesis of biomaterial applications. Biomaterials exhibit immense potentials for cardiac repair and regeneration, wherein they act as extracellular matrix replacing scaffolds or as delivery vehicles for stem cells, protein, plasmids, etc. This review concentrates on natural, synthetic, and hybrid biomaterials; their function; and interaction with the body, mechanisms of repair by which they are able to improve cardiac function in a MI milieu. We also provide focus on future perspectives that need attention. The cognizance provided by the research results certainly indicates that biomaterials could revolutionize the treatment paradigms for MI with a positive impact on clinical translation.

Beyond protein tagging: Rewiring the genetic code of fluorescent proteins - A review.

Fluorescent proteins (FP) are an integral part of modern biology due to its diverse biochemical and photophysical properties. The boundaries of FP have been extended through conventional mutagenesis and directed evolution approaches. Engineering of FP based on the standard genetic code consisting of 20 amino acids with limited functional groups restrict its diversification. Degeneracy of genetic code has helped in covering this substantial gap through genetic code engineering, wherein introduction of unnatural amino acid (UAA) analogues resulted in a collection of FP with varying properties. This review features the work carried till date in the area of FP incorporated with UAAs and explores strategies employed for incorporation, impact of UAAs in chromophore and surrounding residues and changes in inherent properties of FP. The long-standing association of FP as a tool for high throughput screening of orthogonal aaRS/tRNA pairs used in site specific incorporation of UAAs is expounded. Insertion of UAAs in FP has enabled their use in contemporary fields such as biophotovoltaics, bioremediation, biosensors, biomaterials and imaging of acidic vesicles. Thus, expansion of genetic code of FP is envisaged to rejig the existing spectra of colors and future research initiative in this direction is expected to glow brighter and brighter.

Self-Assembly and Mechanical Properties of Engineered Protein Based Multifunctional Nanofiber for Accelerated Wound Healing.

The present work reports a new route for preparing tunable multifunctional biomaterials through the combination of synthetic biology and material chemistry. Genetically encoded catechol moiety is evolved in a nanofiber mat with defined surface and secondary reactive functional chemistry, which promotes self-assembly and wet adhesion property of the protein. The catechol moiety is further exploited for the controlled release of boric acid that provides a congenial cellular microenvironment for accelerated wound healing. The presence of 3,4-dihydroxyphenylalanine in the nanofiber mat act as a stimulus to trigger cell proliferation, migration, and vascularization to accelerate wound healing. Electron paramagnetic resonance, NMR, FTIR, and circular dichroism spectroscopy confirm the structural integrity, antioxidant property, and controlled release of boric acid. Fluorescent and scanning electron microscopy reveals the 3D architecture of nanofiber mat, which favors fibroblast growth, endothelial cell attachment, and tube formation, which are the desirable properties of a wound-healing material. Animal studies in the murine wound healing model assert that the multifunctional biomaterial significantly improve re-epithelialization and accelerate wound closure.

Esterification of Polymeric Carbohydrate Through Congener Cutinase-Like Biocatalyst.

Cutinase-like enzymes (CLEs) are bi-functional hydrolases, which share the conserved catalytic site of lipase and consensus pentapeptide sequence of cutinase. Here, we have genetically replaced the canonical amino acids (CAA) by their non-canonical fluorinated surrogates to biosynthesize a novel class of congener biocatalyst for esterification of polymeric carbohydrate with long-chain fatty acid. It is a new enzyme-engineering approach used to manipulate industrially relevant biocatalyst through genetic incorporation of new functionally encoded non-canonical amino acids (NCAA). Global fluorination of CLE improved its catalytic, functional, and structural stability. Molecular docking studies confirmed that the fluorinated CLE (FCLE) had developed a binding affinity towards different fatty acids compared with the parent CLE. Importantly, FCLE could catalyze starch oleate synthesis in 24 h with a degree of substitution of 0.3 ± 0.001. Biophysical and microscopic analysis substantiated the efficient synthesis of the ester by FCLE. Our data represent the first step in the generation of an industrially relevant fluorous multifunctional enzyme for facile synthesis of high fatty acid starch esters.

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.

Screening and production of a potent extracellular Arthrobacter creatinolyticus urease for determination of heavy metal ions.

This paper describes the isolation of a potent extracellular urease producing microorganism, identified by 16S rRNA as Arthrobacter creatinolyticus MTCC 5604 and its medium optimization by classical one-factor-at-a-time method and central composite rotatable design (CCRD), a tool of response surface methodology (RSM). An optimal activity of 9.0 U ml(-1) was obtained by classical method and statistical optimization of the medium resulted in an activity of 17.35 U ml(-1) at 48 h and 30 °C. This activity was 4.91 times greater than the initial activity (3.53 U ml(-1) ) from the basal medium and the enzyme showed maximum activity at pH 8.0 and 60 °C and was stable at pH 7.0-9.0 and temperatures up to 50 °C. Furthermore, the enzyme was assessed for its activity reduction by determining the inhibitory concentration (IC50 ) of heavy metal ions and the inhibition of urease was in the order of Cu(II) > Cd(II) > Zn(II) > Ni(II). Urease was highly sensitive to Cu(II) and its inhibition was 94% and 100% in model solutions containing a mixture of Cu(II) with heavy metal ions Cd(II) and Zn(II), respectively. The results of these studies suggested that the enzyme could be utilized as sensors to determine the levels of Cu(II) ions in industrial effluents, contaminated soil and ground water.