Biofunctional Agents Research Articles

ISOLASI DAN PEMANFAATAN BAKTERI KITINOLITIK DARI CANGKANG UDANG PUTIH (PENAEUS MERGUENSIS) DALAM MENGHASILKAN ENZIM KITINASE

Indonesia is listed as the third largest shrimp producing country in the world. Shrimp is one of the leading export commodities. Shrimp is one of the main commodities in the fishing industry. Thus encouraging Indonesia to become one of the shrimp producing countries. In the shrimp processing process, usually the heads and shells of the shrimp are thrown away or used as animal feed. Traditionally, shrimp shell waste is generally used as a mixture for making crackers, paste, shrimp paste and as additional animal feed and returned to the shrimp cultivation itself. One use of shrimp shell waste that has prospects for development and has quite high economic value is processing it into chitin, chitosan and glucosamine. The aim of this research is to obtain isolates of chitinolytic bacteria from isolation and to utilize chitinolytic bacteria from white shrimp (Penaeus merguensis) shells to produce chitinase enzymes. Chitinolytic bacteria are bacteria that produce chitinase enzymes which play a role in degrading chitin into oligomers and chitin derivative compounds. Chitinolytic bacteria can be obtained from various sources such as the rhizosphere, phyllosphere, soil or from water environments such as seas, lakes, ponds or shrimp ponds and so on. Chitin that exists in nature is easily degraded by microorganisms. There are two pathways in the chitin degradation process. First, degradation by chitinolytic which hydrolyzes the ?-1,4-glycoside bond. Second, the polymer undergoes first deacetylation and then hydrolysis by chitosanase. Chitinase has many benefits, including as a bioinsecticide and biofunctional agent in controlling plant pests and is used to process chitin waste in the shrimp and crab freezing industry. It can also be used in the medical and food fields. From the test results, three types of bacteria were obtained, namely Vibrio sp, Bacillus sp, and Pseudomonas sp.

Semi-enzymatic acceleration of oxidative protein folding by N-methylated heteroaromatic thiols.

We report the first example of a synthetic thiol-based compound that promotes oxidative protein folding upon 1-equivalent loading to the disulfide bonds in the client protein to afford the native form in over 70% yield. N-Methylation is a central post-translational processing of proteins in vivo for regulating functions including chaperone activities. Despite the universally observed biochemical reactions in nature, N-methylation has hardly been utilized in the design, functionalization, and switching of synthetic bioregulatory agents, particularly folding promotors. As a biomimetic approach, we developed pyridinylmethanethiols to investigate the effects of N-methylation on the promotion of oxidative protein folding. For a comprehensive study on the geometrical effects, constitutional isomers of pyridinylmethanethiols with ortho-, meta-, and para-substitutions have been synthesized. Among the constitutional isomers, para-substituted pyridinylmethanethiol showed the fastest disulfide-bond formation of the client proteins to afford the native forms most efficiently. N-Methylation drastically increased the acidity and enhanced the oxidizability of the thiol groups in the pyridinylmethanethiols to enhance the folding promotion efficiencies. Among the isomers, para-substituted N-methylated pyridinylmethanethiol accelerated the oxidative protein folding reactions with the highest efficiency, allowing for protein folding promotion by 1-equivalent loading as a semi-enzymatic activity. This study will offer a novel bioinspired molecular design of synthetic biofunctional agents that are semi-enzymatically effective for the promotion of oxidative protein folding including biopharmaceuticals such as insulin in vitro by minimum loading.

Bioactive and Topographically-Modified Electrospun Membranes for the Creation of New Bone Regeneration Models

Bone injuries that arise from trauma, cancer treatment, or infection are a major and growing global challenge. An increasingly ageing population plays a key role in this, since a growing number of fractures are due to diseases such as osteoporosis, which place a burden on healthcare systems. Current reparative strategies do not sufficiently consider cell-substrate interactions that are found in healthy tissues; therefore, the need for more complex models is clear. The creation of in vitro defined 3D microenvironments is an emerging topographically-orientated approach that provides opportunities to apply knowledge of cell migration and differentiation mechanisms to the creation of new cell substrates. Moreover, introducing biofunctional agents within in vitro models for bone regeneration has allowed, to a certain degree, the control of cell fate towards osteogenic pathways. In this research, we applied three methods for functionalizing spatially-confined electrospun artificial microenvironments that presented relevant components of the native bone stem cell niche. The biological and osteogenic behaviors of mesenchymal stromal cells (MSCs) were investigated on electrospun micro-fabricated scaffolds functionalized with extracellular matrix (ECM) proteins (collagen I), glycosaminoglycans (heparin), and ceramic-based materials (bioglass). Collagen, heparin, and bioglass (BG) were successfully included in the models without modifying the fibrous structures offered by the polycaprolactone (PCL) scaffolds. Mesenchymal stromal cells (MSCs) were successfully seeded in all the biofunctional scaffolds and they showed an increase in alkaline phosphatase production when exposed to PCL/BG composites. This research demonstrates the feasibility of manufacturing smart and hierarchical artificial microenvironments for studying stem cell behavior and ultimately the potential of incorporating these artificial microenvironments into multifunctional membranes for bone tissue regeneration

Bovine Serum Albumin (BSA)/polyacrylonitrile (PAN) biohybrid nanofibers coated with a biomineralized calcium deficient hydroxyapatite (HA) shell for wound dressing.

Here, for the first time, a nanofibrous (NF) wound dressing comprising biomineralized polyacrylonitrile (PAN) nanofibers is developed. In contrast to the majority of the currently available nanofibrous wound dressings that are based on natural polymers, PAN is a synthetic, industrial polymer, which has been rarely considered for this purpose. PAN NFs are first hydrolyzed to allow for tethering of biofunctional agents (here Bovine Serum Albumin (BSA)). Later, the biofunctionlized PAN NFs are biomineralized by immersion in simulated body fluid (SBF). As a result, core-shell, calcium deficient hydroxyapatite (HA)/BSA/PAN nanofibers form, that are mechanically stronger (elastic modulus; 8.5 vs. 6MPa) compared to the untreated PAN NFs. The biomineralized PAN NFs showed promising bioactivity as reflected in the cell biology tests with fibroblast and keratinocyte cells. Hs68 fibroblasts and HaCat keratinocytes were found to be more viable in the presence of the biomineralized NFs than when they were co-cultured with the neat PAN NFs. Such mechanical and biological characteristics of the biomineralized PAN NFs are favorable for wound dressing applications.

Synthesis, anti-microbial and anti-inflammatory activities of 18β-glycyrrhetinic acid derivatives

Thirteen 18β-glycyrrhetinic acid (GA) derivatives were obtained by reduction at C-11 position, oxidation at C-3 position and condensation at C-2 position of GA. Anti-microbial activity evaluation indicated that compounds 04, 05, 10, 13 and 14 showed more potent inhibitory activity against Staphylococcus aureus subsp. aureus, Staphylococcus epidermidis, Staphylococcus aureus than GA, especially compound 10, the inhibitory activity against Staphylococcus epidermidis was equaled with Ampicillin. Moreover, in vivo experiments exhibited that compound 10 also has anti-inflammatory effect, which could decrease about 59.69% TPA-induced ear edema of mice with the gavage treatment of 40.0 mg/mL. Immunohistochemistry results revealed that the effect of inhibition was related to inhibition of TPA-induced upregulation of the pro-inflammatory cytokines TNF-α and IL-1β. Furthermore, compound 10 also significantly decreased the expression level of p65 in NF-κB signaling pathway. In general, compound 10, both with antibacterial and anti-inflammatory activities, was expected to become a promising bio-functional agent.

Comparative proteomic analysis on acquired enamel pellicle at two time points in caries-susceptible and caries-free subjects

ObjectivesThe aim of this study was to analyse the protein profiles of acquired enamel pellicle (AEP) at two time points (5 min and 2 h) between caries-susceptible and caries-free subjects using label-free and parallel reaction monitoring (PRM) proteomic approaches, and to discover potential biomarkers for dental caries. MethodsSixty participants (30 caries-susceptible (DMFT>5) and 30 caries-free subjects (DMFT = 0)) were included. Their AEP at 5 min and 2 h was separately sampled, and the AEP materials were quantitatively analysed using label-free proteomics. Bioinformatics analysis of differentially expressed proteins was subsequently conducted and target proteins were verified using PRM analysis. ResultsA total of 82 and 39 differentially expressed proteins were identified at the 5-minute and 2 -h points, respectively, between the caries-susceptible and caries-free groups. Some proteins related to immune response and antibacterial activity were identified, and the characteristic enriched biological processes of up- and down-regulated proteins were presented using bioinformatics analysis. Subsequently, five intriguing target proteins of each group were chosen for PRM analysis and their presence was successfully verified in the 5-minute and 2 -h AEPs, respectively. ConclusionsThe proteins in the AEPs of caries-susceptible and caries-free subjects at both time points presented unique protein profiles related to immune response and antibacterial activity. Mucin-7 can be regarded as an important potential AEP biomarker for dental caries. Clinical significanceThe results of the study identified potential biomarkers, which can facilitate the design of new bio-functional agents for the diagnosis and treatment of dental caries in future.

Antimicrobial Biohybrid Nanofibrous Wound Dressings

Globally, chronic wounds impose a notable burden to patients and healthcare systems. Such non-healing wounds are readily subjected to bacteria that provoke inflammation and hence challenge the healing process. Furthermore, bacteria induce infection impeding re-epithelialization and collagen synthesis, thereby increasing hospital stay and costs. Appropriate wound dressing materials alleviate the infection incidence and accelerate the healing process. In this regard, biopolymeric nanofibers show promising potential for prevention of infection and promotion of the healing rate. They are biocompatible and mostly biodegradable and biomimic the extracellular matrix structure. Also, they provide a high surface area, surface nanoporosity, and the ability to deliver drugs and antibiotics locally into the wound milieu. Drug delivery by nanofibers has proved efficient in the control of infection. Yet, due to emergence of antibiotic resistant bacteria, there is an urgent need to replacing the antibiotic delivery systems with other reliable options such as bionanohybrids containing antimicrobial nanoparticles/ions, nature-derived compounds and biofunctional agents. Here, the most recent (since 2015) developments of antimicrobial biopolymeric nanofibrous wound dressings, particularly those based on bionanohybrids, are reviewed and evaluated in terms of antimicrobial efficiency. Lastly, the prospects and challenges are discussed to draw a roadmap for further progresses in this area.

Generating Cyan Fluorescence with De Novo Tripeptides: An In Vitro Mutation Study on the Role of Single Amino Acid Residues and Their Sequence.

Amino acids are natural choices as building blocks when developing biofunctional entities owing to their superior diversity and versatile physicochemical properties compared to nucleotide bases. A simple permutation of the amino acids creates a broad palette of proteins and these have been successfully engineered into useful biofunctional agents. For example, the intrinsic ultraviolet fluorescence of phenylalanine and tryptophan has been engineered to emit in the visible spectrum, which has broad applications for imaging/sensing probes, photothermal therapy agents, optogenetic switches, etc. Nature produces more colorful coats/furs, feathers/hairs, and eyes through various biochemical modifications of tyrosine-based pigmentation. However, it is challenging to modulate the fluorescence wavelength from the UV to the visible region through oligopeptides. Herein, we report an innovative approach to obtain cyan fluorescence by using de novo tripeptides containing glycine, tyrosine, and lysine, which form robust dimer structures under moderate oxidizing conditions. Through an in vitro mutation approach, we deduce that both the amino acids and their sequence play significant roles in modulating the fluorescence. We believe this work holds great promise for developing novel cell imaging and resonance energy-transfer-based fluorescent probes.

Lactobionic Acid: Significance and Application in Food and Pharmaceutical

Lactose has long been used as a precursor for the manufacture of high-value derivatives with emerging applications in the food and pharmaceutical industries. This review focuses on the main characteristics, manufacturing methods, physiological effects and applications of lactobionic acid. Lactobionic acid is a product obtained from lactose oxidation, with high potential applications as a bioactive compound. Recent advances in tissue engineering and application of nanotechnology in medical fields have also underlined the increased importance of this organic acid as an important biofunctional agent.

Induced pluripotent stem cell-derived mesenchymal stem cell seeding on biofunctionalized calcium phosphate cements.

Induced pluripotent stem cells (iPSCs) have great potential due to their proliferation and differentiation capability. The objectives of this study were to generate iPSC-derived mesenchymal stem cells (iPSC-MSCs), and investigate iPSC-MSC proliferati on and osteogenic differentiation on calcium phosphate cement (CPC) containing biofunctional agents for the first time. Human iPSCs were derived from marrow CD34+ cells which were reprogrammed by a single episomal vector. iPSCs were cultured to form embryoid bodies (EBs), and MSCs migrated out of EBs. Five biofunctional agents were incorporated into CPC: RGD (Arg-Gly-Asp) peptides, fibronectin (Fn), fibronectin-like engineered polymer protein (FEPP), extracellular matrix Geltrex, and platelet concentrate. iPSC-MSCs were seeded on five biofunctionalized CPCs: CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets. iPSC-MSCs on biofunctional CPCs had enhanced proliferation, actin fiber expression, osteogenic differentiation and mineralization, compared to control. Cell proliferation was greatly increased on biofunctional CPCs. iPSC-MSCs underwent osteogenic differentiation with increased alkaline phosphatase, Runx2 and collagen-I expressions. Mineral synthesis by iPSC-MSCs on CPC-Platelets was 3-fold that of CPC control. In conclusion, iPSCs showed high potential for bone engineering. iPSC-MSCs on biofunctionalized CPCs had cell proliferation and bone mineralization that were much better than traditional CPC. iPSC-MSC-CPC constructs are promising to promote bone regeneration in craniofacial/orthopedic repairs.

Biofunctionalized nanofibrous membranes as super separators of protein and enzyme from water

Here, we report development of a novel biofunctionalized nanofibrous membrane which, despite its macroporous structure, is able to separate even trace amounts (as low as 2mg/L) of biomolecules such as protein and enzyme from water with an optimum efficiency of ∼90%. Such an extraordinary protein selectivity at this level of pollutant concentration for a nanofibrous membrane has never been reported. In the current study, poly(acrylonitrile-co-glycidyl methacrylate) (PANGMA) electrospun nanofibers are functionalized by a bovine serum albumin (BSA) protein. This membrane is extraordinarily successful in removal of BSA protein and Candida antarctica Lipase B (Cal-B) enzyme from a water based solution. Despite a negligible non-specific adsorption of both BSA and Cal-B to the PANGMA nanofibrous membrane (8%), the separation efficiency of the biofunctionalized membrane for BSA and Cal-B reaches to 88% and 81%, respectively. The optimum separation efficiency at a trace amount of protein models is due to the water-induced conformational change of the biofunctional agent. The conformational change not only exposes more functional groups available to catch the biomolecules but also leads to swelling of the nanofibers thereby a higher steric hindrance for the solutes. Besides the optimum selectivity, the biofunctionalized membranes are highly wettable thereby highly water permeable.

Wettability Control on ZnO Nanowires Driven by Seed Layer Properties

AbstractThis study proposes a way to control the wettability of zinc oxide nanowires (NWs) by properly selecting the kind of seed layer used to promote the growth of the wires. ZnO seed layers were synthesized on silicon and conductive substrates by a physical vapor deposition approach and a wet‐chemical route, namely, the radio frequency magnetron sputtering and the spin‐coating techniques, respectively. ZnO NWs were grown by a hydrothermal method on each kind of seed layer and the results were compared. The morphologies and crystallographic orientations of the seed layers and the resulting NWs were investigated with the aim of correlating the characteristics of the underlying seed layer to those of the resulting NWs. Additional insights were obtained by performing optical contact angle (OCA) measurements on ZnO seed layers to study their wettability behavior immediately after the synthesis processes and two weeks later. Hydrophilic behavior was observed in both sputtered and spin‐coated fresh seed layers. After two weeks of aging, a change in the wettability and a net transition from hydrophilic to hydrophobic behavior was observed in sputtered seed layers, whereas in the spin‐coated films this transition was not so pronounced and was found to be dependent on the precursor concentration. OCA measurements carried out on ZnO NWs showed that the wettability of the NWs is strictly related to the nature of the underlying seed layers and does not depend on the aging time, in contrast to the behavior of the seed layers. Depending on the deposition method, we therefore obtained either highly hydrophilic or superhydrophobic nanowires, which demonstrates the possibility to strongly control the final wetting behavior of these nanostructures for the desired application, such as self‐cleaning surfaces, antireflection coatings, or substrates to anchor biofunctional agents.

Phosphate functionalized and lactic acid containing graft copolymer: synthesis and evaluation as biomaterial for bone tissue engineering applications

Polyvinyl alcohol (PVA) and polylactic acids (PLA) are biocompatible materials possessing some inherent contrasting limitations which have reduced the scope of their individual applicability. Specifically, overcoming strong hydrophobicity and introducing chemical groups for biofunctionalization are unmet challenges for PLA whilst chemical endeavors to render adequate aqueous stability and cell adhesion properties to PVA have not produced completely intended results. Objective of the present work is to explore synthesis of a graft polymer as an approach towards coupling biofunctional groups with PLA materials. In a two-step reaction, PPVA (phosphorylated polyvinyl alcohol or PVA pre-functionalized with phosphate) is esterified with lactic acid followed by polymerization into PLA in presence of stannous chloride as catalyst to obtain phosphorylated polyvinyl alcohol-graft-polylactic acid (PPVA-g-LA) copolymer. Product is characterized by nuclear magnetic resonance, X-ray diffraction, and thermogravimetric analysis. PPVA-g-LA shows an increase in uniaxial elongation compared to parent PPVA under condition of tensile loading. The graft copolymer also exhibits higher water contact angles compared to PPVA, but a more hydrophilic surface compared to PLA. Culture of MG-63 cells on solvent cast films of polymers demonstrates that PPVA-g-LA as a cell substrate can significantly (p < 0.05) improve proliferation and differentiation of cells compared to PPVA substrate whereas in comparison to PLA can significantly ameliorate osteoblast function of cultured cells. Overall, results illustrate the feasibility of PVA to act as a carrier for biofunctional agents to be coupled to lactic acid-based biomaterials with subsequent improvement in cell response on the polymers. In this attempt, it also affords materials with tunable surface or bulk properties of relevance for tissue engineering applications.

Calcium phosphate cement with biofunctional agents and stem cell seeding for dental and craniofacial bone repair

ObjectiveCalcium phosphate cement (CPC) can be injected to harden in situ and is promising for dental and craniofacial applications. However, human stem cell attachment to CPC is relatively poor. The objectives of this study were to incorporate biofunctional agents into CPC, and to investigate human umbilical cord mesenchymal stem cell (hUCMSC) seeding on biofunctionalized CPC for osteogenic differentiation for the first time. MethodsFive types of biofunctional agents were used: RGD (Arg-Gly-Asp) peptides, human fibronectin (Fn), fibronectin-like engineered polymer protein (FEPP), extracellular matrix Geltrex, and human platelet concentrate. Five biofunctionalized CPC scaffolds were fabricated: CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets. The hUCMSC attachment, proliferation, osteogenic differentiation and mineral synthesis were measured. ResultsThe hUCMSCs on biofunctionalized CPCs had much better cell attachment, proliferation, actin fiber expression, osteogenic differentiation and mineral synthesis, compared to the traditional CPC control. Cell proliferation was increased by an order of magnitude via incorporation of biofunctional agents in CPC (p<0.05). Mineral synthesis on biofunctionalized CPCs was 3–5 folds of those of control (p<0.05). hUCMSCs differentiated with high alkaline phosphatase, Runx2, osteocalcin, and collagen I gene expressions. Mechanical properties of biofunctionalized CPC matched the reported strength and elastic modulus of cancellous bone. SignificanceA new class of biofunctionalized CPCs was developed, including CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets. hUCMSCs on biofunctionalized CPCs had cell density, cell proliferation, actin fiber density, and bone mineralization that were dramatically better than those on traditional CPC. Novel biofunctionalized CPC scaffolds with greatly enhanced human stem cell proliferation and differentiation are promising to facilitate bone regeneration in a wide range of dental, craniofacial and orthopedic applications.