12th Annual Biotechnology Congress (BAC Girona 2018): abstract collection

Background

Fibroblast growth factor 9 (FGF9) is a heparin-binding growth factor, secreted by both mesothelial and epithelial cells, which participates in hair follicle regeneration, wound healing, and bone development. A suitable source of recombinant human FGF9 (rhFGF9) is needed for research into potential clinical applications. We present that expression of oleosin-rhFGF9 fusion protein in safflower (Carthamus tinctorius L.) seeds stimulates hair growth and wound healing.

Results

The oleosin-rhFGF9 expressed in safflower seeds, in which it localizes to the surface of oil bodies. The expression of oleosin-rhFGF9 was confirmed by polyacrylamide gel electrophoresis and western blotting. According to BCA and Enzyme-linked immunosorbent assay (ELISA) assay, the results show that the expression level of oleosin-rhFGF9 was 0.14% of oil body protein. The oil body bound oleosin-rhFGF9 showed mitogenic activity towards NIH3T3 cells in a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The efficacy of oil body bound oleosin-rhFGF9 in promoting hair growth and wound healing was investigated in C57BL/6 mice. In a hair regeneration experiment, 50 μg/μl oil body bound oleosin-rhFGF9 was applied to the dorsal skin of mice in the resting phase of the hair growth cycle. After 15 days, thicker hair and increased number of new hairs were seen compared with controls. Furthermore, the number of new hairs was greater compared with rhFGF9-treated mice. The hair follicles of mice treated with oil body bound oleosin-rhFGF9 expressed β-catenin more abundantly. In a wound healing experiment, dorsal skin wounds were topically treated with 50 μg/μl oil body bound oleosin-rhFGF9. Wound healing was quicker compared with mice treated with rhFGF9 and controls, especially in the earlier stages of healing.

Conclusions

The oil body bound oleosin-rhFGF9 promotes both hair growth and wound healing. It appears to promote hair growth, at least in part, by up-regulating β-catenin expression. The potential of oil body bound oleosin-rhFGF9 as an external drug can treat the alopecia and wounds or use in further clinical application.

     

Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli

Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity >99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 × 10⁵ IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results.     

β-catenin and transforming growth factor β have distinct roles regulating fibroblast cell motility and the induction of collagen lattice contraction

Background  

β-catenin and transforming growth factor β signaling are activated in fibroblasts during wound healing. Both signaling pathways positively regulate fibroblast proliferation during this reparative process, and the effect of transforming growth factor β is partially mediated by β-catenin. Other cellular processes, such as cell motility and the induction of extracellular matrix contraction, also play important roles during wound repair. We examined the function of β-catenin and its interaction with transforming growth factor β in cell motility and the induction of collagen lattice contraction.     

Bioproduction of hydrolytic enzymes using apple pomace waste by A. niger: applications in biocontrol formulations and hydrolysis of chitin/chitosan

Studies were carried out for β-glucosidase production using apple pomace (AP) in solid-state fermentation using 2⁴ factorial design and response surface methodology. The influence of four independent variables including initial moisture level and inducers [veratryl alcohol (VA), lactose (LAC) and copper sulfate (CS)] was studied. The experimental design showed that initial moisture level had significant negative effect on the response. Higher β-glucosidase activity of 64.18 IU/gram fermented substrate (gfs) was achieved in solid-state tray fermentation with optimum conditions having initial moisture level 55% (v/w), pH 4.5, 2 mM/kg VA, 2% (w/w) LAC and 1.5 mM/kg CS concentration, respectively,. The non-specific chitinase 70.28 ± 6.34 IU/gfs and chitosanase activities 60.18 ± 6.82 to 64.20 ± 7.12 IU/gfs were observed. The study demonstrated that AP can be potentially used for the β-glucosidase production by Aspergillus niger. Moreover, β-glucosidase can be used for the hydrolysis of chitin/chitosan to depolymerized products and in the formulation of biocontrol agents for enhanced entomotoxicity levels.     

A unique post-translational processing of an exo-β-1,3-glucanase of Penicillium sp. KH10 expressed in Aspergillus oryzae

To characterize an exo-β-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-β-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS–polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104 kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47 kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modification.     

Molecular assembly of Zn porphyrin complexes using synthetic light-harvesting model polypeptides

Synthetic single α-helix hydrophobic polypeptides, which have similar amino acid sequences to the hydrophobic core in the native light-harvesting 1-β polypeptide from Rhodobacter sphaeroides, formed Zn porphyrin complexes on a gold electrode, as well as in n-octyl-β-glucoside micelles: this process is dependent on the structure of the pigments and the polypeptides. Interestingly, an enhanced photoelectric current was observed when Zn mesoporphyrin monomer complexed with the synthetic light-harvesting model polypeptide in an α-helical configuration was assembled with a defined orientation onto the electrode. Analog of these light-harvesting model complexes are also useful in providing insights into the effect of polypeptide structure on the formation of light-harvesting complexes on and off electrodes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro and in vivo wound healing-promoting activities of beta-lapachone

Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. β-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of β-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of β-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without β-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in β-lapachone-treated animals than in those treated with vehicle only. In addition, β-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that β-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that β-lapachone may have potential for therapeutic use for wound healing. cell proliferation; mitogen-activated protein kinase signaling pathways     

Truncation of N- and C-terminal regions of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> dextranase enhances catalytic activity

Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23–70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCGΔ) or N-VR/C-VR (TM-ΔCGΔ) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-ΔCGΔ did not accept any further protease-degradation during long storage. TM-NCGΔ and TM-ΔCGΔ enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.     

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

Background  

Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants.     

Aedes aegypti: characterization of hemocyte polypeptide synthesis during wound healing and immune response to inoculated microfilariae

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80-90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.     

Modification of collagen by 3-deoxyglucosone alters wound healing through differential regulation of p38 MAP kinase

Background

Wound healing is a highly dynamic process that requires signaling from the extracellular matrix to the fibroblasts for migration and proliferation, and closure of the wound. This rate of wound closure is impaired in diabetes, which may be due to the increased levels of the precursor for advanced glycation end products, 3-deoxyglucosone (3DG). Previous studies suggest a differential role for p38 mitogen-activated kinase (MAPK) during wound healing; whereby, p38 MAPK acts as a growth kinase during normal wound healing, but acts as a stress kinase during diabetic wound repair. Therefore, we investigated the signaling cross-talk by which p38 MAPK mediates wound healing in fibroblasts cultured on native collagen and 3DG-collagen.

Methodology/Principal Findings

Using human dermal fibroblasts cultured on 3DG-collagen as a model of diabetic wounds, we demonstrated that p38 MAPK can promote either cell growth or cell death, and this was dependent on the activation of AKT and ERK1/2. Wound closure on native collagen was dependent on p38 MAPK phosphorylation of AKT and ERK1/2. Furthermore, proliferation and collagen production in fibroblasts cultured on native collagen was dependent on p38 MAPK regulation of AKT and ERK1/2. In contrast, 3DG-collagen decreased fibroblast migration, proliferation, and collagen expression through ERK1/2 and AKT downregulation via p38 MAPK.

Conclusions/Significance

Taken together, the present study shows that p38 MAPK is a key signaling molecule that plays a significantly opposite role during times of cellular growth and cellular stress, which may account for the differing rates of wound closure seen in diabetic populations.     

Metal ion-dependent,reversible, protein filament formation by designed beta-roll polypeptides

Background  

A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca²⁺. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide.     

Dermal fibroblasts derived from fetal and postnatal humans exhibit distinct responses to insulin like growth factors

Background  

It has been well established that human fetuses will heal cutaneous wounds with perfect regeneration. Insulin-like growth factors are pro-fibrotic fibroblast mitogens that have important roles in both adult wound healing and during development, although their relative contribution towards fetal wound healing is currently unknown. We have compared responses to IGF-I and -II in human dermal fibroblast strains derived from early gestational age fetal (<14 weeks) and developmentally mature postnatal skin to identify any differences that might relate to their respective wound healing responses of regeneration or fibrosis.     

Non-adenine based purines accelerate wound healing

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5–2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8–3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.     

Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes,a consequence of common growth factors

In addition to acting as a physical barrier against microorganisms, the skin produces antimicrobial peptides and proteins. After wounding, growth factors are produced to stimulate the regeneration of tissue. The growth factor response ceases after regeneration of the tissue, when the physical barrier protecting against microbial infections is re-established. We found that the growth factors important in wound healing, insulin-like growth factor I and TGF-alpha, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, human beta-defensin 3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor in human keratinocytes. Both an individual and a synergistic effect of these growth factors were observed. These findings offer an explanation for the expression of these peptides/polypeptides in the skin disease psoriasis and in wound healing and define a host defense role for growth factors in wound healing.     

Fermentation and microencapsulation of the nematophagous fungus <Emphasis Type="Italic">Hirsutella rhossiliensis</Emphasis> in a novel type of hollow beads

In this work, fermentation and formulation aspects of the nematophagous fungus Hirsutella rhossiliensis BBA were investigated. When incubated in 2% (w/w) glucose and 0.5% (w/w) yeast extract medium in a 1-L Erlenmeyer flask without baffles, heavy pellet formation was observed. Only 40% of the mycelium had a size less than 500 μm. When a flask with three baffles was used, the portion of mycelium <500 μm rose to 95%. In the next step, the influence of aeration rate and stirrer speed on production of finely dispersed mycelium in a stirred tank reactor was investigated. The best fermentation results were obtained at 0.4 vvm and 400 rpm stirrer speed with 90% mycelium <500 μm and 5 g/L biomass. Then, mycelium was microencapsulated in hollow beads based on sulfoethylcellulose (SEC). Experiments on the capsule nutrient reservoir showed that 15% (w/w) corn gluten and 0.5% (w/w) yeast extract could be replaced with 3% (w/w) autoclaved baker's yeast which was never used as capsule additive before. Radial growth of mycelium out of dried hollow beads containing 1% (w/w) biomass and 3% (w/w) baker's yeast was faster than for alginate beads containing equivalent amounts of biomass and yeast indicating a higher bio-control potential.     

Maggot excretions/secretions induces human microvascular endothelial cell migration through AKT1

Maggot therapy is a simple and highly successful method for healing of infected and necrotic wounds. The increasing evidences indicate that Maggot excretions/secretions (ES) plays important roles in the wounds healing process. But the precise molecular mechanisms remain undefined. Herein, we investigated if ES induced cell migration during wound healing process using microvascular endothelial cells (HMEC-1) as model, and this effect was associated with the activation of AKT1 and ERK1/2. Wound healing and transwell migration assays were performed to study the effects of ES on HMEC-1 cell migration. Our data showed that ES significantly induced HMEC-1 cell migration in both wound healing and transwell assays, and time-dependently (P < 0.05) activated AKT1, but not ERK1/2. Moreover LY294002 (a PI3K inhibitor) partially attenuated (P < 0.05) ES-induced cell migration in wound healing assay while completely inhibited (P < 0.05) ES-induced AKT1 activation. These findings demonstrate that ES directly induces HMEC-1 cell migration and this event is partially mediated by the activation of AKT1.     

Fibroblast growth factor 2-functionalized collagen matrices for skeletal muscle tissue engineering

Fibroblast growth factor 2 (FGF2) protein plays important roles in wound healing and tissue regeneration. Collagen is clinically used for wound care applications. We investigated the potential value of FGF2-functionalized collagen matrices for skeletal muscle tissue engineering. When C2C12 cells were treated with FGF2, cell adhesion increased after 3 and 5 days compared to the control (P < 0.05). Wound healing activity of FGF2 was slightly higher than the control through cell migration. Cell proliferation activity of FGF2-functionalized collagen matrices on C2C12 cells also increased. Taken together, FGF2 stimulated C2C12 myoblast growth by promoting cell adhesion, proliferation and wound healing activity after injury. The potential effect of FGF2-functionalized collagen matrices was also observed. Thus FGF2 stimulates skeletal muscle development and regeneration, thereby leading to potential utility for skeletal muscle tissue engineering.     

A carboxyl-terminal fragment of Plasmodium falciparum gp195 expressed by a recombinant baculovirus induces antibodies that completely inhibit parasite growth.

The major merozoite surface Ag (gp195) of Plasmodium falciparum has been shown to protect monkeys against parasite infection, and gp195-based synthetic peptides and recombinant polypeptides have been evaluated as potential malaria vaccines. A major problem in developing a gp195-based recombinant vaccine has been the difficulty in obtaining a recombinant polypeptide that is immunologically equivalent to the native protein. In this study, the carboxyl-terminal processing fragment (p42) of gp195 was produced in yeast and in a baculovirus recombinant system. Immunologic analyses indicated that the secreted baculovirus p42 (BVp42) expressed native, disulfide-dependent conformational epitopes, whereas these epitopes were poorly represented in the intracellular yeast p42. BVp42, but not yeast p42, was also recognized by the majority of gp195-specific antibodies of animals immunized with purified native gp195, indicating that the anti-gp195 response of these animals was focused on conformational determinants of the p42 processing fragment. Sera against native gp195 of congenic mice of diverse H-2 haplotypes recognized the BVp42 polypeptide, demonstrating that a genetically heterogeneous population is capable of responding to p42 epitopes. BVp42 was highly immunogenic and induced high titers of antibodies that were cross-reactive with purified native gp195 in an ELISA and also reacted with schizonts and merozoites by immunofluorescence. Anti-BVp42 antibodies completely inhibited the in vitro growth of the malaria parasite, whereas anti-yeast p42 antibodies had no effect. These results indicate that native, conformational epitopes of p42 are critical for the induction of gp195-specific, parasite growth-inhibitory antibodies and that the BVp42 polypeptide efficiently induces antibodies specific for these native determinants.     

Growth factors and growth control of heterogeneous cell populations

In an earlier work a model of the autocrine and paracrine pathways of tumor growth control was developed (Michelson and Leith. 1991. Autocrine and paracrine growth factors in tumor growth.Bull. math. Biol. 53, 639–656). The target population, a generic tumor, was modeled as a single, homogeneous population using the standard Verhulst equation of logistic growth. Mitogenic signals were represented by modifications to the Malthusian growth parameter and adaptational signals were represented by modifications to the carrying capacity. Three growth scenarios were described: (1) normal tissue wound healing, (2) unperturbed tumor growth, and (3) tumor growth in a radiation damaged environment, a phenomenon termed the Tumor Bed Effect (TBE). In this paper, we extend those results to include a “triad” of growth factor controls (autocrine, paracrine and endocrine) and heterogeneity of the target population. The heterogeneous factors in the model represent either intrinsic, epigenetic or environmental differences in both normally differentiating tissues and tumors. Three types of growth are modeled: (1) normal tissue differentiation or wound healing, assuming no communication between differentiated and undifferentiated cell compartments; (2) normal wound healing with feedback inhibition, due to signalling from the differentiated compartment; and (3) the development of hypoxia in a spherical tumor. The signal processing within the triad is discussed for each model and biologically reasonable constraints are defined for limits on growth control.