Lysozyme Degradation Research Articles

Water-Soluble Star Polymer as a Potential Photoactivated Nanotool for Lysozyme Degradation.

The development of nanotools for chemical sensing and macromolecular modifications is a new challenge in the biomedical field, with emphasis on artificial peptidases designed to cleave peptide bonds at specific sites. In this landscape, metal porphyrins are attractive due to their ability to form stable complexes with amino acids and to generate reactive oxygen species when irradiated by light of appropriate wavelengths. The issues of hydrophobic behavior and aggregation in aqueous environments of porphyrins can be solved by using its PEGylated derivatives. This work proposes the design of an artificial photo-protease agent based on a PEGylated mercury porphyrin, able to form a stable complex with l-Tryptophan, an amino acid present also in the lysozyme structure (a well-known protein model). The sensing and photodegradation features of PEGylated mercury porphyrin were exploited to detect and degrade both l-Trp and lysozyme using ROS, generated under green (532 nm) and red (650 nm) light lasers. The obtained system (Star3600_Hg) and its behavior as a photo-protease agent were studied by means of several spectroscopies (UV-Vis, fluorescence and circular dichroism), and MALDI-TOF mass spectrometry, showing the cleavage of lysozyme and the appearance of several short-chain residues. The approach of this study paves the way for potential applications in theranostics and targeted bio-medical therapies.

A Bacterial Nanomedicine Combines Photodynamic-Immunotherapy and Chemotherapy for Enhanced Treatment of Oral Squamous Cell Carcinoma.

Bacterial therapy is an emerging hotspot in tumor immunotherapy, which can initiate antitumor immune activation through multiple mechanisms. Porphyromonas gingivalis (Pg), a pathogenic bacterium inhabiting the oral cavity, contains a great deal of pathogen associated molecular patterns that can activate various innate immune cells to promote antitumor immunity. Owing to the presence of protoporphyrin IX (PpIX), Pg is also an excellent photosensitizer for photodynamic therapy (PDT) via the in situ generation of reactive oxygen species. This study reports a bacterial nanomedicine (nmPg) fabricated from Pg through lysozyme degradation, ammonium chloride lysis, and nanoextrusion, which has potent PDT and immune activation performances for oral squamous cell carcinoma (OSCC) treatment. To further promote the tumoricidal efficacy, a commonly used chemotherapeutic drug doxorubicin (DOX) is efficiently encapsulated into nmPg through a simple incubation method. nmPg/DOX thus prepared exhibits significant synergistic effects on inhibiting the growth and metastasis of OSCC both in vitro and in vivo via photodynamic-immunotherapy and chemotherapy. In summary, this work develops a promising bacterial nanomedicine for enhanced treatment of OSCC.

Effective Endotoxin Removal from Chitosan That Preserves Chemical Structure and Improves Compatibility with Immune Cells.

Chitosan is one of the most researched biopolymers for healthcare applications, however, being a naturally derived polymer, it is susceptible to endotoxin contamination, which elicits pro-inflammatory responses, skewing chitosan's performance and leading to inaccurate conclusions. It is therefore critical that endotoxins are quantified and removed for in vivo use. Here, heat and mild NaOH treatment are investigated as facile endotoxin removal methods from chitosan. Both treatments effectively removed endotoxin to below the FDA limit for medical devices (<0.5 EU/mL). However, in co-culture with peripheral blood mononuclear cells (PBMCs), only NaOH-treated chitosan prevented TNF-α production. While endotoxin removal is the principal task, the preservation of chitosan's structure is vital for the synthesis and lysozyme degradation of chitosan-based hydrogels. The chemical properties of NaOH-treated chitosan (by FTIR-ATR) were significantly similar to its native composition, whereas the heat-treated chitosan evidenced macroscopic chemical and physical changes associated with the Maillard reaction, deeming this treatment unsuitable for further applications. Degradation studies conducted with lysozyme demonstrated that the degradation rates of native and NaOH-treated chitosan-genipin hydrogels were similar. In vitro co-culture studies showed that NaOH hydrogels did not negatively affect the cell viability of monocyte-derived dendritic cells (moDCs), nor induce phenotypical maturation or pro-inflammatory cytokine release.

Chondrogenic differentiation of mouse CD105+ stem/progenitor cells on amino-group-functionalized biosilica-hydrogel scaffolds

Mesenchymal stem/progenitor cells (MSPCs) are critical for tissue regeneration. Moreover, the CD105 antigen identifies early MSPCs with increased chondrogenic differentiation ability. We hypothesized that amine-(NH2)-functionalized biosilica incorporating hydrogel scaffolds, seeded with mCoSPCs105+ would contribute to creating tissue-engineered scaffolds, capable of de novo cartilage synthesis. Scaffolds were characterized by water uptake, lysozyme degradation, axial compression, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. Differentiation stimulus of scaffold functionalization was evaluated using Alcian blue staining. Cartilage-forming abilities of mCoSPCs105+ were evaluated using Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Biosilica particle incorporation into scaffolds resulted in increased water uptake capacity and compression force withstanding abilities. Amine-(NH2)-group functionalization of biosilica led to significantly increased stem cell differentiation potential, by Alcian blue staining, in the first 3 weeks. Scaffold attachment and viable cell proliferation were observed for 6 weeks under chondrogenic differentiation. Downregulation of Runx2, an increase of Col10a1, Ihh, and maintenance of Sox9, was seen under these culture conditions. mCoSPCs105+ gene expression pattern was defined by the significant upregulation of Col1a1, Col2a1, Prg4, and Agc-1 over 6 weeks of incubation compared to the unsorted control. Immunostaining of cell-seeded scaffolds revealed significantly higher secretion of proteins relevant to cartilage extracellular matrix. The preselecting of CD105+ phenotype in MSPCs may enhance tissue regeneration of fibrocartilage and biosilica nanoparticles may be a beneficial additive in tissue engineering of scaffolds.

Biomimetic Organic-Inorganic Nanocomposite Scaffolds to Regenerate Cranial Bone Defects in a Rat Animal Model.

While bone regenerates itself after an injury, a critical bone defect requires external interventions. Engineering approaches to restore bone provide a temporary scaffold to support the damage and provide beneficial biological cues for bone repair. Biomimetically generated scaffolds replicate the naturally occurring phenomena in bone regeneration. In this study, a gelatin-calcium phosphate nanocomposite was synthesized by an efficient and cost-effective double-diffusion biomimetic approach. Calcium and phosphate ions are impregnated in the gelatin, mimicking the natural bone mineralization process. Glutaraldehyde from 0.5 to 2 w/v% was used for gelatin cross-linking and mechanical properties of the scaffold, and its biological support for rat bone marrow mesenchymal stromal cells was analyzed. Analysis of scanning electron microscopy images of the nanocomposite scaffolds and Fourier transform infrared (FTIR) and X-ray diffraction (XRD) characterizations of these scaffolds confirmed precipitation of calcium phosphates in the gelatin. Moreover, lysozyme degradation assay showed that scaffold degradation reversely correlates with the concentration of the cross-linking agent. Increased glutaraldehyde concentrations enhanced the mechanical properties of the scaffolds, bringing them closer to those of cancellous bone. Rat bone marrow mesenchymal stromal cells maintained their viability on these scaffolds compared to standard cell culture plates. In addition, these cells showed differentiation into bone lineage as evaluated from alkaline phosphatase activity up to 21 days and Alizarin red staining of the cells over 28 days. Eventually, scaffolds were implanted in a cranial defect in a rat animal model with a 5 mm diameter. Bone regeneration was studied over 90 days. Analysis of histological sections of the injury and computer tomography images revealed that nanocomposite scaffolds cross-linked with 1% w/v glutaraldehyde provide the maximum bone regeneration after 90 days. Collectively, our data show that nanocomposite scaffolds developed here provide effective regeneration for extensive bone defects in vivo.

Elimination and irrigation therapy to optimize the state of the nasal microbiome in children

The article summarizes the results of the work of domestic and foreign researchers on the study of homeostasis of the nasal cavity, including the state of the microbiota of the nasal mucosa in healthy children and in inflammatory diseases of the upper respiratory tract. The normal microbiota of the nasal cavity is represented by corynebacteria (diphtheroids), neisseria, coagulasenegative staphylococci, alpha-hemolytic streptococci. From among the transient species, such species as Staphylococcus aureus, Escherihia coli, beta-hemolytic streptococci would be found in the specified biotope. The main attention is paid to the features of the nasal microbiota in newborns and young children (dependence of the nasal microbiota on the type of feeding, age and season). The role of hypercolonization by opportunistic microflora and its persistent potential (ability to biofilm formation, degradation of lysozyme, interferon) in the pathogenesis of inflammatory diseases of the upper respiratory tract is noted. The pathogenetic mechanisms providing multilevel protection of the organism from pathogens are considered. The expediency of using elimination-irrigation therapy as a hygienic means of sanitizing the nasal cavity in young children has been determined. The questions about the advantages of the use of isotonic solutions, their effectiveness for ensuring normal colonization resistance of the nasal mucosa are discussed. In conclusion, we must say that for hygienic care and sanitation purposes it is recommended only to rinse the nasal mucosa in newborns and infants using drops of isotonic saline solution and an aspirator to avoid aspiration and inflow of liquid into the cavity of Eustachian tube and middle ear, and also for the preventive purposes, as it allows to maintain homeostasis of the nasal mucosa and prevent the development of acute respiratory diseases.

Calorimetric observation of lysozyme degradation at elevated temperature in water and DMSO-water mixtures

The reversibility of protein denaturation is an essential factor for biotechnology. Previous differential scanning calorimetry (DSC) studies demonstrated the development of the low-temperature shoulder on the calorimetric denaturation peak of lysozyme in successive heating-cooling cycles, which implies irreversible denaturation. However, this effect was not thoroughly investigated. In the present work, we have quantitatively studied the effect of incubation at the elevated temperature on the state of lysozyme in water and mixtures of water with dimethyl sulfoxide (DMSO) using DSC. The changes to the state of the lysozyme molecule indicated by DSC thermograms were also evaluated by circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) techniques. It is noted that with the increase of duration or temperature of incubation, the low-temperature peak on DSC thermograms grows, while the height of the unfolding peak of native structure decreases. The increase in the height of the low-temperature peak in DSC scans correlates with the development of the sideband associated with the absorption of the carboxyl group in the infrared spectra. This result suggests that the low-temperature endothermic peak corresponds to the unfolding of the deamidated protein. At the same time, DLS measurements indicate absence of aggregation, while FTIR and CD data demonstrate that deamidated protein maintains a native-like structure. The evaluation of the DSC thermograms allowed to determine the rates and activation energy of the degradation of protein molecules at the elevated temperatures. The addition of DMSO slows down the protein degradation but has little effect on the apparent activation energy of the process.

Tuning the properties of carboxymethylchitosan-based porous membranes for potential application as wound dressing

Wound repair is a complex process that calls for strategies to allow a rapid and effective regeneration of injured skin, which has stimulated the research of advanced wound dressings. Herein, highly porous membranes of N,O-carboxymethylchitosan (CMCh), and poly (vinyl alcohol) (PVA) were successfully prepared via a green and facile freeze-drying method of blend solutions containing CMCh/PVA at weight ratio 25/75. Membranes composed only by CMCh were also prepared and genipin was used for crosslinking. Different contents of TiO2 nanoparticles were incorporated to both type of membranes, which were characterized in terms of morphology, porosity (Φ), swelling capacity (S.C.), mechanical properties, susceptibility to lysozyme degradation and in vitro cytotoxicity toward human fibroblast (HDFn) and keratinocytes (HaCaT) cells. Larger apparent pores were observed in the surface of the genipin-crosslinked CMCh membrane, which resulted in higher porosity (Φ ≈ 76%) and swelling capacity (S.C. ≈ 1720%) as compared to CMCh/PVA membrane (Φ ≈ 68%; S.C. ≈ 1660%). The porosity of both types of membranes decreased upon the addition of TiO2 nanoparticles while swelling capacity increased. Due to their high porosity and swelling capacity, adequate mechanical properties, controlled degradability, and cytocompatibility, such carboxymethylchitosan-based membranes are potentially useful as wound dressings.

A novel γ-PGA composite gellan membrane containing glycerol for guided bone regeneration

An ideal barrier membrane design should incorporate the function of a delivery vehicle for transporting drugs and osteoinductive factors to where the body is under inflammation. In the present study, a functional hydrogel-based barrier membrane is fabricated using calcium-form poly-γ-glutamic acid (γ-PGA) and glycerol blending into gellan gum. The concentration of the calcium-form poly-γ-glutamic acid (γ-PGA) and the glycerol ratio are studied for improving practicability in easy-handling and expanding the coverage area. Gellan gum-based membranes with uniformly distributed calcium aggregates are not only successfully manufactured but also providing excellent characteristics for protein adsorption, bioactivity, and bone cell maturation. Our composite gellan gum-based membranes were tested including to their morphology, mechanical properties, swelling behavior, protein adsorption, drug diffusion, and lysozyme degradation. The biocompatibility, proliferation, and osteoblastic response of membranes were examined by osteoblast-like (MG63) cells. Our results indicate that adequate physical cross-linking with γ-PGA improves the original mechanical properties and delays degradation. Growing glycerol ratio not only enhances the elongation at break and diffusion rate, but it also changes the tensile strength and the remaining weight. In vitro biocompatibility tests, an adequate ratio of γ-PGA modification significantly enhances the proliferation, the secretion of alkaline phosphatase (ALP) and mineralization. However, worth noting is the glycerol-modified membrane cannot bear a close resemblance with the non-glycerol group in the high level of osteoblastic response. In general, these tunable materials with biocompatibility, biodegradability, and positive osteoblastic responses were poised to be possible candidates for bone defect repair.

Inactivation of the sfgtr4 Gene of Shigella flexneri Induces Biofilm Formation and Affects Bacterial Pathogenicity.

Biofilm formation is a significant cause for the environmental persistence of foodborne pathogens. This phenomenon remains misunderstood in Shigella flexneri whose pathogenicity is mainly associated with the virulence plasmid pWR100. Sequence analysis of the latter predicts a putative lipopolysaccharides (LPS) glycosyltransferase (Gtr) encoded by Sfgtr4, which is the second gene of the SfpgdA-orf186-virK-msbB2 locus. We demonstrated here that purified SfGtr4 exhibited a Gtr activity in vitro by transferring glucose to lipid A. To establish the role of SfGtr4 in virulence, we generated a Sfgtr4 mutant and assessed its phenotype in vitro. Sfgtr4 mutant significantly reduced HeLa cells invasion without impairing type III effectors secretion, increased susceptibility to lysozyme degradation, and enhanced bacterial killing by polymorphonuclear neutrophils (PMNs). SfGtr4 is related to proteins required in biofilm formation. We established conditions whereby wild-type Shigella formed biofilm and revealed that its appearance was accelerated by the Sfgtr4 mutant. Additional phenotypical analysis revealed that single SfpdgA and double SfpgdA-Sfgtr4 mutants behaved similarly to Sfgtr4 mutant. Furthermore, a molecular interaction between SfGtr4 and SfPgdA was identified. In summary, the dual contribution of SfGtr4 and SfPgdA to the pathogenicity and the regulation biofilm formation by S. flexneri was demonstrated here.

Designing a novel chitosan-based periofilm containing metronidazole–ciprofloxacin

Periodontitis is the most common periodontal disease. Because of its infectious-inflammatory nature, as well as the difficulty of complete elimination of pathogens from oral cavity, antibiotics have been proposed as adjunctive modalities to control the disease and prevent its recurrence. Systemic antibiotics could lead to a further reduction in probing depth and bleeding on probing. Nowadays, due to their side effects and the emergence of resistant strains of bacteria, there is a tendency to use local antibiotic delivery systems. The aim of this study was to design, develop and test a mucoadhesive film based on chitosan/gelatin containing metronidazole and ciprofloxacin. A mucoadhesive film containing 2% chitosan and gelatin and 0.2% of metronidazole and ciprofloxacin was made by casting method. Laboratory evaluations including drug-releasing pattern through HPTLC, mucoadhesive strength, stability in phosphate buffer, degradation in lysozyme, and water absorption were performed. The results of this study showed that the produced periofilm releases the antibiotics up to 48 h in concentrations higher than MIC. In the presence of lysozyme, a sharp decline in stability observed in the first 4 days, reached to a constant level after seven days. Based on the results of the present study, it could be concluded that applying the proposed system and its drug combination can be a part of the adjunctive periodontal treatment on a daily basis or every 48 h. However, further studies are needed to recommend this system as a routine therapeutic modality.

A Review on Staphylococcus sp. and its pathogens

The genus Staphylococcus is an important microorganism because of its direct and dangerous impact on the lives of other organisms, and Alexander Ogaston was the first to release this designation in 1880 to denote spherical bacteria usually associated with pus in wound infections. The members of this species are found in various environments such as dust, water, air, feces, on the skin, and the mucous membranes of warm-blooded vertebrates, on clothing, and other places. Most species of this species live as natural flora on human skin and mucous membranes, but some are opportunistic pathologies. The species of staphylococcus is characterized by having several virulence factors that increase the efficacy and pathogenicity of its species on the occurrence of infection due to its possession of tequic acid and the adhesion factor and other factors that enable it to get rid of the host defenses as an enzyme (Staphylokinase) or enable it to invade and spread as an enzyme (Hyaluronidase). Its possession of cellular wall proteins, its formation of the capsule and the exogenous polysaccharides and its production of various types of extracellular enzymes and various toxins such as intestinal toxins fixed in the heat, secretion of immune system inhibitors and their containment of antibiotic resistance genes. It shows the total resistance to the degradation of lysozyme. Staphylococcus aureus is resistant to many antimicrobial agents and thus produces treatment problems. The sex of staphylococcus includes 36 species and 80 subtypes, where more is clarified From 20 types of staphylococcus aureus.

Chitin degradation enzyme-responsive system for controlled release of fibroblast growth factor-2

Chitin is widely found in fungal cell walls and arthropod exoskeletons, and is used as a biomedical material. However, chitin is not water-soluble, restricting its use for controlled release materials. We found that water-soluble chitosan can be acetylated to produce a chitin equivalent, or chitin gel. Chitin gel, produced by mixing chitosan solution with acetic anhydride, can be degraded by lysozyme and fetal bovine serum, so could provide an ideal means for controlled release in biological systems. We tested a combination of chitin gel with a chitin binding domain (CBD) fusion protein as a controlled release system regulated by chitin degradation. A fusion protein consisting of fibroblast growth factor 2 (FGF-2) fused to CBD bound the chitin gel, and was released time-dependently rather than as an initial burst during lysozyme degradation, suggesting that this system could provide a means for controlled drug release in biological systems. Contrastingly, the trinitrophenyl residue (TNP-X) covalently bound to chitin gel, and was released by lysozyme degradation with an initial burst. If release of CBD-FGF-2 were simply dependent on lysozyme degradation of the chitin gel, the release behavior of CBD-FGF-2 would be similar to that of TNP-X, with an initial burst. Therefore, we propose that CBD-FGF-2 repeats the cycle of binding, release, and re-binding to the chitin gel during degradation. This system allows for a time-dependent, controlled release of CBD-FGF-2 without an initial burst.

Characterization and Antibiofilm Activity of Mannitol-Chitosan-Blended Paste for Local Antibiotic Delivery System.

Mannitol, a polyalcohol bacterial metabolite, has been shown to activate dormant persister cells within bacterial biofilm. This study sought to evaluate an injectable blend of mannitol, chitosan, and polyethylene glycol for delivery of antibiotics and mannitol for eradication of Staphylococcal biofilm. Mannitol blends were injectable and had decreased dissociation and degradation in the enzyme lysozyme compared to blends without mannitol. Vancomycin and amikacin eluted in a burst response, with active concentrations extended to seven days compared to five days for blends without mannitol. Mannitol eluted from the paste in a burst the first day and continued through Day 4. Eluates from the mannitol pastes with and without antibiotics decreased viability of established S. aureus biofilm by up to 95.5% compared to blends without mannitol, which only decreased biofilm when loaded with antibiotics. Cytocompatibility tests indicated no adverse effects on viability of fibroblasts. In vivo evaluation of inflammatory response revealed mannitol blends scored within the 2–4 range at Week 1 (2.6 ± 1.1) and at Week 4 (3.0 ± 0.8), indicative of moderate inflammation and comparable to non-mannitol pastes (p = 0.065). Clinically, this paste could be loaded with clinician-selected antibiotics and used as an adjunctive therapy for musculoskeletal infection prevention and treatment.

Characterization of Chitosan-Gelatin Blend Scaffolds

Vacuum freeze-drying was used to prepare chitosan-gelatin (CG) scaffolds from hydrogels, with glutaraldehyde (GA) used as a crosslinker. The effects of the changes in volume ratios of the 2 wt% CG and GA solutions on scaffold performance were studied. The ratio of chitosan to gelatin solution volumes, vr(C/G), was adjusted to 1/2 or 1/1, with the 0.25 wt% GA volume at 3, 6, or 8% of the CG/GA volume. Six groups of CG scaffolds were fabricated and the scaffolds performance compared. After the cells were incubated for 4 days, hematoxylin eosin (HE) staining was used to observe the spreading of human skin keratinocyte (HaCaT) cells on these scaffolds, with the MTT method also used to detect the cells proliferation. The inhibition zone method was used on cells cultures to determine the antibacterial properties of the scaffolds against S. aureus and E. coli. Scaffolds were also examined for degradation in lysozyme and their compression properties were tested after degradation. The results showed that the HaCaT cells grew well on these scaffolds and proliferated significantly, indicating that these scaffolds possessed good cytocompatibility. With increased chitosan volume, the antibacterial properties of the scaffolds against S. aureus increased, however, there was no significant change in the antibacterial properties toward E. coli. Increased volumes of chitosan and GA decreased the scaffolds degradation rates and improved the elastic compressive moduli of the scaffolds after degradation. The scaffolds in the vr(C/G) = 1/1, 8% GA group have potential application prospects in the field of skin regeneration.

ZnO-NPs embedded biodegradable thiolated bandage for postoperative surgical site infection: In vitro and in vivo evaluation.

Post-operative surgical site infections (SSI) present a serious threat and may lead to complications. Currently available dressings for SSI lack mucoadhesion, safety, efficacy and most importantly patient compliance. We aimed to address these concerns by developing a bioactive thiolated chitosan-alginate bandage embedded with zinc oxide nanoparticles (ZnO-NPs) for localized topical treatment of SSI. The FTIR, XRD, DSC and TGA of bandage confirmed the compatibility of ingredients and modifications made. The porosity, swelling index and lysozyme degradation showed good properties for wound healing and biodegradation. Moreover, in-vitro antibacterial activity showed higher bactericidal effect as compared to ZnO-NPs free bandage. In-vivo wound healing in murine model showed significant improved tissue generation and speedy wound healing as compared to positive and negative controls. Over all, thiolated bandage showed potential as an advanced therapeutic agent for treating surgical site infections, meeting the required features of an ideal surgical dressing.

In Vitro Degradation of Gelatin/carboxymethylcellulose Scaffolds for Skin Tissue Regeneration

The field of tissue engineering has grown in response to many medical needs for tissue replacement. For the skin replacement, there have to develop the different types of tissue engineered skin substitutes. The artificial scaffold have to be a biodegradable material with appropriate degradation rate for skin regeneration. In this study, gelatin blended with carboxymethylcellulose (CMC) scaffold was fabricated via freeze drying method. The various gelatin and CMC ratios were 100/0, 90/10, 80/20, 70/30 and 60/40, respectively. In vitro degradation of the scaffold was done by degradation in lysozyme in PBS buffer within desired time. The swelling ratio of the scaffolds was done by immersing in PBS buffer for 2 h at 37oC. The results revealed that the gelatin/CMC (80:20) scaffold showed the highest value of swelling ratio and have an appropriate degradation rate with completely degraded after 36 h.

Bi-layered α-tocopherol acetate loaded membranes for potential wound healing and skin regeneration

With an increase in the demand for skin regeneration products, there is a noticeable increase in developing materials that encourage, wound healing and skin regeneration. It has been reported that antioxidants play an important role in anti-inflammatory reactions, cellular proliferation and remodeling phase of wound healing. While consideration all these factors, a novel α-tocopherol acetate (vitamin E) (VE) loaded bi-layered electrospun membrane, based on lower polycaprolactone (PCL) layer and upper polylactic acid (PLA) layer, was fabricated through electrospinning. Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), in-vitro degradation studies, swelling studies and VE release studies were performed to evaluate structural, physical and in-vitro behavior of membranes. Biological properties of membranes were evaluated through cell proliferation assay, cell adhesion studies, live/dead cell assay and CAM assay. SEM images showed that the average diameter of nanofibers ranged from 1 to 6 μm, while addition of VE changed the diameter and morphology of fibers. Bi-layered membranes showed significant swelling behavior through water uptake, membranes loaded with 30% VE showed 8.7% and 6.8% degradation in lysozyme and H2O2 respectively. 20% and 30% VE loaded membranes followed Korsmeyer-Peppas and first order drug release kinetics followed by non-fickian drug release kinetics. Membranes showed non-toxic behavior and supported cell proliferation via alamar blue assay, cell adhesion via SEM, cell viability via live/dead assay and wound healing by scratch assay. CAM assay showed that membranes having VE supported angiogenesis and showed significant formation of blood vessels making it suitable for skin regeneration and wound healing. Results showed that large surface area of nanofibers, porous structure and biocompatible nature are suitable for targeted clinical applications.

Use of Bioorthogonal N‐Acetylcysteamine (SNAc) Analogues and Peptidoglycan O‐Acetyltransferase B (PatB) to Label Peptidoglycan

In Gram (−) bacteria, peptidoglycan O‐acetyltransferase B (PatB) is responsible for the O‐acetylation of peptidoglycan to prevent degradation by lysozyme and other lytic transglycosylases generated by its host. In this work, PatB was exploited as a tool to incorporate bioorthogonal functionality on the carbohydrate backbone of the peptidoglycan by using a variety of donors, either p‐nitrophenol (pNP) or N‐acetylcysteamine (SNAc). Using tri‐N‐acetylglucosamine (GlcNAc)3 as a peptidoglycan mimic, a variety of functionalities were installed, including an alkyne and azide handle, on this mimic. Further studies in vitro showed that these modifications have a protective effect on isolated peptidoglycan from B. subtilis, E. coli, P. putida, and V. parahaemolyticus against lysozyme degradation. Intrigued by these results, an alkyne handle was subsequently installed on whole bacterial cells of B. subtilis using the SNAc alkyne derivative and the carbohydrate backbone was fluorescently labeled via click chemistry. Altering the peptidoglycan post‐synthetically with these derivatives will aid in the isolation and identification of immune stimulatory fragments from bacteria, as well as in the development of antibiotics and immune modulators.Support or Funding InformationThe Delaware COBRE and INBRE programs supported this project with a grant from the NIGMS (5 P30GM110758‐02, P20GM104316‐01A1 and P20GM103446) from the NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An investigation of konjac glucomannan-keratin hydrogel scaffold loaded with Avena sativa extracts for diabetic wound healing.

We have developed a novel hydrogel composed of konjac glucomannan (KGM), human hair proteins (KER), and an ethanolic extract of Avena sativa (OAT) and evaluated its potential as a dressing material for diabetic wounds. KGM is an excellent biocompatible gelling agent that stimulates fibroblast proliferation and immunomodulation. Human hair proteins (KER) are biocompatible, biodegradable, and possess abundant cell adhesion sites. KER also promotes fibroblast attachment and proliferation, keratinocyte migration, and collagen expression, which can accelerate wound healing. OAT consists of oat β-glucans and several anti-inflammatory and antioxidant moieties that can reduce prolonged inflammation in chronic wounds. SEM images confirm the highly porous architecture of the scaffolds. When immersed in PBS, KGM+KER+OAT hydrogels absorb 7.5 times their dry weight. These hydrogels display a measured rate of degradation in lysozyme. KGM+KER+OAT hydrogels showed no significant cytotoxicity against NIH/3T3 fibroblasts. DAPI and SEM images obtained after 48h of cell culture illustrate the attachment and infiltration of fibroblasts. In vivo studies performed using a diabetic rat excision wound model showed that KGM+KER+OAT hydrogels significantly accelerated wound healing compared to the control and the KGM+KER hydrogels.