Improved anti-biofilm activity and long-lasting effects of novel serratiopeptidase immobilized on cellulose nanofibers.

Today, enzymatic treatment is a progressive field in combating biofilm producing pathogens. In this regard, serratiopeptidase, a medicinally important metalloprotease, has been recently highlighted as an enzyme with proved anti-biofilm activity. In the present study, in order to increase the long-lasting effects of the enzyme, serratiopeptidase and the novel engineered forms with enhanced anti-biofilm activity were immobilized on the surface of cellulose nanofibers (CNFs) as a natural polymer with eminent properties. For this, recombinant serratiopeptidases including the native and previously designed enzymes were produced, purified and conjugated to the CNF by chemical and physical methods. Immobilization was confirmed using different scanning and microscopic methods. The enzyme activity was assessed using casein hydrolysis test. Enzyme release analysis was performed using dialysis tube method. Anti-biofilm activity of free and immobilized enzymes has been examined on Staphylococcus aureus and Pseudomonas aeruginosa strains. Finally, cytotoxicity of enzyme-conjugated CNFs was performed by MTT assay. The casein hydrolysis results confirmed fixation of all recombinant enzymes on CNFs by chemical method; however, inadequate fixation of these enzymes was found using cold atmospheric plasma (CAP). The AFM, FTIR, and SEM analysis confirmed appropriate conjugation of enzymes on the surface of CNFs. Immobilization of enzymes on CNFs improved the anti-biofilm activity of serratiopeptidase enzymes. Interestingly, the novel engineered serratiopeptidase (T344 [8-339ss]) exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms. In conclusion, incorporation of serratiopeptidases into CNFs improves their anti-biofilm activities without baring any cytotoxicity. KEY POINTS: • Enzymes were successfully immobilized on cellulose nanofibers using chemical method. • Immobilization of enzymes on CNFs improved their anti-biofilm activity. • T344 [8-339ss] exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms.

A Survey of Gasoline Ameliorator, Methyl-Tert-Butyl Ether (MTBE) on Bovine Serum Albumin: A Spectroscopy and Molecular Dynamic Simulation Study.

Background: Methyl-Tert-Butyl Ether (MTBE) as a gasoline modifier is frequently added to fuels and used in plenty of worldwide applications. MTBE biodegradation in groundwater occurs slowly and produces water miscibility; therefore, it causes diverse environmental and human health concerns. Objectives: The interaction of MTBE with bovine serum albumin (BSA) as a model protein at physiological conditions is investigated to illustrate the possible interactions of MTBE with the body's proteins. Materials and Methods: Uv-visible, fluorescence, circular dichroism (CD) spectroscopy methods, and molecular modeling were used to analyze the MTBE's effect on BSA structure and dynamics. The constant protein concentration and various MTBE contents were used for possible interactions. Results: The protein structural analysis shows that MTBE binds to BSA via positive enthalpy and entropy via hydrophobic interactions. Molecular docking shows the participation of several amino acids in the MTBE-BSA interaction. The CD spectroscopy results show that the BSA structure was not changed in the MTBE concentrations utilized in the study. Molecular dynamics (MD) simulation results suggest that MTBE can slightly change protein structure in the last 50ns. Conclusion: Comparing experimental and MD simulation results demonstrated that the BSA secondary structure was maintained in the low concentration of the MTBE. The entropy and enthalpy parameters asserted the hydrophobic interaction was the major force in the interaction between the BSA and MTBE.

Development of Streptococcus equisimilis Group G Mutant Strains with Ability to Produce Low Polydisperse and Low-Molecular-Weight Hyaluronic Acid

Background: Background: Hyaluronic acid (HA), a natural polymer with wide applications in biomedicine and cosmetics, is mainly produced by Streptococcal fermentation at industrial scale. In the present study, chemical random mutagenesis was used for development of Streptococcus equisimilis group G mutant strains with high HA productivity. Methods: Methods: The optimum of the pH of culture condition and cultivation time for HA production by wild strain group G were assessed. At first, two rounds of mutation at different concentrations of NTG was used for mutagenesis. Then, the nonhemolytic and hyaluronidase-negative mutants were screened on the blood and HA agar. HA productivity and molecular weight were determined by carbazole assay, agarose gel electrophoresis and specific staining. Moreover, stability of the high producer mutants was evaluated within 10 generations. Results: Results: The results showed that the wild-type strain produced 1241 ± 2.1 µg/ml of HA at pH 5.5 and 4 hours of cultivation, while the screened mutants showed a 16.1-45.5% increase in HA production. Two mutant strains, named Gm2-120-21-3 (2470 ± 8.1 µg/ml) and Gm2-120-21-4 (2856 ± 4.2 µg/ml), indicated the highest titer and a consistent production. The molecular weight (Mw) of HA for the mutants was less than 160 kDa, considering as a low Mw HA. Conclusion: Conclusion: The mutant strains producing a low polydisperse, as well as low Mw of HA with high titer might be regarded as potential industrial strains for HA production after further safety investigations.

Mesenchymal stem cells encapsulation in chitosan and carboxymethyl chitosan hydrogels to enhance osteo-differentiation.

BACKGROUND: Recently biomaterials utilized for designing scaffolds in tissue engineering are not cost-effective and eco-friendly. As a result, we design and develop biocompatible and bioactive hydrogels for osteo-tissue regeneration based on the natural polysaccharide chitosan. Three distinct hydrogel components were used for this. METHODS: Hydrogels networks were created using chitosan 2% (CTS 2%), carboxymethyl chitosan 2% (CMC 2%), and 50:50 mixtures of CTS and CMC (CTS/CMC 50:50). Furthermore, scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FTIR), degradation, and swelling behavior of design hydrogels were studied. Also, the cytocompatibility and osteo-differentiation potency were examined by encapsulating mesenchymal stem cells derived from adipose tissue (AMSCs) on the designed hydrogels. RESULTS: According to the findings, our results showed an acceptable pore structure, functional groups, and degradation rate of the designed hydrogels for in vitro evaluation. In addition, employing CMC instead of CTS or adding 50% CMC to the hydrogel component could improve the hydrogel's osteo-bioactivity without the use of external osteogenic differentiation agents. CONCLUSION: The CMC-containing hydrogel not only caused early osteogenesis but also accelerated differentiation to the maturity phase of osteoblasts.

Anti-dermatophytic activity of cold atmospheric plasma against Trichophyton rubrum via affecting fungal growth, morphology, drug susceptibility and HSP90 gene expression.

Trichophyton rubrum, a major human pathogenic dermatophyte, is responsible for the most recurrent dermatophytoses as globally important superficial fungal infections. Typical chemotherapy is used to handle such infections; however, emerging drug resistance and side effects necessitate the new remedial method development. Cold atmospheric plasma (CAP) is an emerging technology, consisted of neutral and charged particles and photons newly developed as a potent and safe antimicrobial technique to combat drug-resistant microbial pathogens. In the present study, the vast effects of CAP irradiation containing oxygen (2%) and helium (98%) on T. rubrum growth and pathogenicity were explored. After exposure of T. rubrum to CAP jet for 90, 120, 150, 180, and 210 s in 96-well microtiter plates, cell morphology and viability, ergosterol content of fungal hyphae, HSP90 gene expression, and the pattern of drug susceptibility were studied by using electron microscopy, RT-qPCR, spectrophotometry, disk diffusion and CLSI microbroth dilution methods. CAP irradiation significantly inhibited the fungal growth by 25.83 to 89.10%, reduced fungal cell viability by 11.68 to 87.71%, disrupted cellular membranous organelles and structures of the fungal hyphae, and suppressed efficiently the expression of HSP90 gene by 2 folds in 210 s exposure. Taken together, our results demonstrated that CAP is an efficient tool with potential in-vivo therapeutic applications against chronic dermatophytosis caused by T. rubrum due to its effectiveness, harmless, and ease of access.

Relationship between blood clots and COVID-19 vaccines: A literature review.

SARS-CoV-2 pandemic is one of the most critical pandemics during human civilization. Several therapeutic strategies for COVID-19 management have been offered; nonetheless, none of them seems to be sufficiently beneficial. In effect, vaccines have been proffered as a viable option. The critical issue now is to concentrate on protecting individuals against illness through immunization. One of the causes for concern among the researchers, physicians, and generally the whole community from the onset of vaccination has been the adverse effects (specifically blood clots) that may be observed after the injection of the COVID-19 vaccine. In some countries, such concerns have even resulted in the temporary or permanent discontinuation or abandonment of the application of some vaccines (especially AstraZeneca and Janssen). By evaluating rigorous studies published on this subject, the present article is aimed at identifying the association between blood clot incidence and COVID-19 vaccination. Various methods for producing the COVID-19 vaccines are analyzed, along with their possible pros and cons as well as common and rare side effects, especially VITT and blood clots. Finally, the differences of various vaccines on thrombotic events, WHO recommendations for VITT treatment, and blood clots statics are discussed.

Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma.

Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150 s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30 s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30 s. The Lys C gene from M14-150 s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.

Cold atmospheric plasma modification and electrical conductivity induction in gelatin/polyvinylidene fluoride nanofibers for neural tissue engineering.

BACKGROUND: This research follows some investigations through neural tissue engineering, including fabrication, surface treatment, and evaluation of novel self-stimuli conductive biocompatible and degradable nanocomposite scaffolds. METHODS: Gelatin as a biobased material and polyvinylidene fluoride (PVDF) as a mechanical, electrical, and piezoelectric improvement agent were co-electrospun. In addition, polyaniline/graphene (PAG) nanoparticles were synthesized and added to gelatin solutions in different percentages to induce electrical conductivity. After obtaining optimum PAG percentage, cold atmospheric plasma (CAP) treatment was applied over the best samples by different plasma variable parameters. Finally, the biocompatibility of the scaffolds was analyzed and approved by in vitro tests using two different PC12 and C6 cell lines. In the present study the morphology, FTIR, dynamic light scattering, mechanical properties, wettability, contact angle tests, differential scanning calorimetric, rate of degradation, conductivity, biocompatibility, gene expression, DAPI staining, and cell proliferation were investigated. RESULTS: The PAG percentage optimization results revealed fiber diameter reduction, conductivity enhancement, young's modulus improvement, hydrophilicity devaluation, water uptake decrement, and degradability reduction in electrospun nanofibers by increasing the PAG concentration. Furthermore, ATR-FTIR, FE-SEM, AFM, and contact angle tests revealed that helium CAP treatment improves scaffold characterizations for 90 s in duration time. Furthermore, the results of the MTT assay, FE-SEM, DAPI staining, and RT-PCR revealed that samples containing 2.5% w/w of PAG are the most biocompatible, and CAP treatment increases cell proliferation and improves neural gene expression in the differentiation medium. CONCLUSIONS: According to the results, the samples with the 2.5% w/w of PAG could provide a suitable matrix for neural tissue engineering in terms of physicochemical and biological.

Preparation, characterization, and evaluation of eosin B-loaded nano-liposomes for growth inhibition of Plasmodium falciparum.

Malaria is a deadly disease in humans caused by the Plasmodium parasite. High prevalence of malaria and resistance of malaria parasite to currently proposed drugs have increased the need to introduce and use new and effective antimalarial agents. In this study, eosin B was used as an effective antimalarial agent, the efficacy of which has already been confirmed by in vitro models. Also, for efficacy and safety improvement of eosin B, liposomal nanocarrier was used because of diversity and adaptability in controlled drug delivery and targeting. Eosin B was trapped inside liposomal nanocarriers by thin layer hydration method and its optimization was performed based on size, polydispersity index, and drug entrapment efficiency. Finally, the eosin B-loaded liposomes were tested on Plasmodium falciparum in culture to evaluate its anti-plasmodial effect. According to the results, the formulation with DSPC:cholesterol 8:1 (molar ratio) and drug concentration of 3 mg/ml was selected as the optimal form. The optimal nano-liposomes showed a size of 163.3 nm, a polydispersity index of 0.250, and an encapsulation efficiency of 69.94%. The process of drug release from nanocarriers was also obtained about 63% at the end of 72 h. Stability studies over 2 months at 25 °C and 4 °C on the optimum sample showed that the samples stored in the refrigerator were more stable in terms of size characteristics, polydispersity index, and drug entrapment efficiency. The results indicate a greater effect of liposomal-formulated eosin B on inhibiting parasite growth compared to the free eosin B.

Fe3O4 nanoparticles coated with carboxymethyl chitosan containing curcumin in combination with hyperthermia induced apoptosis in breast cancer cells.

Many studies have demonstrated that curcumin has potential anticancer properties. This research aims to study the effect of iron (II, III) oxide (Fe3O4) nanoparticles coated with carboxymethyl chitosan containing curcumin combination with hyperthermia on breast cancer cells. Magnetic nanoparticles coated with carboxymethyl chitosan containing curcumin (MNP-CMC-CUR) were prepared and specified. MCF-7, MDA-MB-231, and human fibroblast cells were treated with free curcumin and MNP-CMC-CUR at concentrations of 0-60 µM and at different time points. A combined therapy of MNP-CMC-CUR and hyperthermia was performed on MCF-7 cells. The cytotoxicity of curcumin and MNP-CMC-CUR combined with hyperthermia was assessed by MTT. The changes in TP53 and CASPASE3 gene expression were evaluated using real-time PCR. Both cell apoptosis and cell cycle were studied by Annexin/PI staining. The results of MTT showed that the IC50 amount of MNP-CMC-CUR has significantly decreased compared to free curcumin (p < 0.05) and MNP-CMC-CUR in combination with the hyperthermia, and significantly reducing the metabolic activity of the cells (p < 0.05). Real-time PCR results revealed the up-regulation of TP53 and CASPASE3 (p < 0.05). The combinational therapy-induced cell apoptosis (64.51%) and sub-G1 cell cycle were arrested in MCF-7 cells. Based on these observations, a combination of MNP-CMC-CUR with hyperthermia could inhibit the proliferation of MCF-7 cells.

Induced genetic and chemical changes in medicinally important plant Catharanthus roseus (L.) G. Don: cold plasma and phytohormones.

BACKGROUND: Catharanthus roseus (L.) G. Donis a medicinal plant species belonging to the Apocynaceae family, which produces vinblastine and vincristine along with 100 other monoterpenoid indole alkaloids. The process of biosynthesis of C. roseus alkaloids is complex, in which many genes, enzymes, and regulators are involved. Induced mutations may be considered as a potential source for producing a higher amount of vinblastine and vincristine in this plant species. Therefore, the objective of the present study was to examine the effects of different treatments utilized on the induced genetic changes in C. roseus plants and enzyme activities. METHODS AND RESULTS: Spermine, jasmonic acid, methyjasmonate, putrescine, and cold plasma treatments were used for seed treatments. Different molecular markers, namely inter simple sequence repeat, inter retrotransposon amplified polymorphism, and retrotransposon microsatellite amplified polymorphism were employed to reveal the induced genetic changes. Antioxidant enzyme activities were also studied. The treated plants showed genetic variability and a significant increase in antioxidant enzyme activity compared to the control plants. The putrescine treatment resulted in the highest level of activity in superoxidase. A significant positive correlation occurred between the molecular markers data and antioxidant enzyme activities in treated plants. CONCLUSION: Our data revealed that the different phytohormones and cold plasma treatments could induce both genetic and chemical content changes in C. roseus plants.

Plasma-based strategy for inhibiting Candida albicans growth and CaMCA1 gene expression in vitro and reducing fungal pathogenicity in a murine model of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is a common mucosal infection, mainly caused by Candida albicans. The use of common antifungal drugs in treatment of VVC is limited due to emergence of resistant fungal strains and severe side effects. Cold atmospheric plasma (CAP) as a novel therapeutic approach is proven to display strong antifungal activity against C. albicans. In the present study, the effects of CAP treatment on virulence and pathogenicity of C. albicans in a murine model was investigated. Candida albicans was treated with CAP at different time exposures. Fungal cell morphology and the expression profile of CaMCA1 gene in CAP-treated fungus was evaluated using electron microscopy and quantitative RT-PCR. Moreover, the mice model of VVC was developed using CAP-treated and non-treated C. albicans and characterized in terms of vaginal fungal burden, the rate of hyphae formation in the vaginal tissue and fluid and the inflammation degree of mice vaginal tissue. Significant reduction in CaMCA1 expression and remarkable mitochondrial degradation were observed in CAP-treated C. albicans cells. The lowest fungal burden, reduced hyphae formation, poor adherence of yeast cells to vaginal epithelium, and the low degree of inflammation were observed in mice infected with CAP-treated C. albicans. Suppression of CaMCA1 gene and mitochondrial degradation in CAP-treated C. albicans yeast cells may diminish yeast to hyphae transition and reduce fungal pathogenicity in murine model of VVC. CAP treatment can be considered as a novel and efficient therapeutic strategy against C. albicans and related Candida infections in practice. LAY SUMMARY: CAP was successfully used to inhibit fungal growth and CaMCA1 gene expression in C. albicans. It caused morphological alterations in membranous structures of the yeast cells and finally led to the cell death. CAP meaningfully reduced C. albicans virulence and pathogenicity in a murine model of VVC.

Time-dependent behavior of the Staphylococcus aureus biofilm following exposure to cold atmospheric pressure plasma.

OBJECTIVES: Formation of Staphylococcus aureus biofilm leads to persistent infection in tissue or on exter-nal and indwelling devices in patients. Cold atmospheric plasma (CAP) is used for eradication of bacterial biofilms and it has diverse applications in the healthcare system. However, there is not sufficient information on the behavior of biofilms during the CAP exposure period. MATERIALS AND METHODS: Pre-established S. aureus biofilms were exposed to CAP for 0 to 360 sec, then subjected to washing steps and sonication. Subsequently, biomass, number of colonies, vitality of bacteria, structure of colonies, size of produced particles, and viability of bacteria were evaluated by different assays including crystal violet, colony-forming unit, MTT, scanning electron mi-croscopy, confocal laser scanning microscopy, and dynamic light scattering assays. RESULTS: The results showed that the strength of biomass increased in the first 60 sec, then decreased to less than no-CAP treated controls. Moreover, short CAP exposure (≤60 sec) ehances the fusion of the biofilm extracellular matrix and other components, which results in preservation of bacteria during ultra-sonication and washing steps compared with control biofilms. The S. aureus biofilm structure only breaks down following more CAP exposure (> 90 sec) and demolition. Interestingly, the 60 sec CAP exposure could cause the fusion of biofilm compo-nents, and large particles are detectable. CONCLUSION: According to this study, an inadequate CAP exposure period prevents absolute eradication of biofilm and enhances the preservation of bacteria in stronger biofilm compartments.

The Drug Release Kinetics and Anticancer Activity of the GO/PVA-Curcumin Nanostructures: The Effects of the Preparation Method and the GO Amount.

The Graphene Oxide (GO) incorporated polyvinyl alcohol/sodium alginate (PVA-SA) composites with curcumin were prepared by the solvent casting and electro-spinning techniques. The GO was incorporated into PVA-SA nano-fiber and film matrixes, and the performance of these nano-composites as drug carriers was investigated. The effects of production method (film or mat) and GO amount on the water absorption properties and delivery of curcumin behaviors were investigated. The swelling and releasing were studied at the specific interval times in deionized water and phosphate buffer solution (pH = 7.4), respectively. The release kinetics was evaluated to find a suitable mechanism of the release. Finally, the anticancer activity of composite nano-fibers on the cancer cells was investigated. The XRD and FTIR analyses confirmed nanocomposites structures, and the successful incorporation was shown by scanning electron microscopy (SEM). The results showed that addition of the GO to PVA/SA decreased swelling ratio of the films (up to 31%) and increased the swelling ratio of the mats (up to 37.5%). However, for both film and mat, increasing of the GO amount reduced the curcumin release. Drug release decreasing up to 22.5% was observed for film, while a very high release decreasing up to about 70% was seen for mat. Also, both film and mat structures showed significant anti-cancer activity on MCF-7 cells. The lower cell viability was about 40 and 30% for film and mat, respectively. The kinetics evaluations suggested a Korsmeyer-Peppas model and Fickian controlled drug release.

Inhibitory effects of cold atmospheric plasma on the growth, virulence factors and HSP90 gene expression in Candida albicans.

In spite of the abundance of antifungal therapies, 75% of women in the world suffer from the second most common cause of vaginal infection named vulvovaginal candidiasis. This complication is characterized with overgrowth of Candida albicans. The low efficacy and side effects of current antifungal therapies have convinced the researchers to look for a non-antibiotic based treatment such as cold atmospheric plasmas (CAP). The aim of this research was to evaluate the effects of CAP on C. albicans growth, ergosterol and biofilm formation. In addition, antibiotic resistance, phospholipase and proteinase activity, and structural properties were examined with different exposure duration. Putative critical effect of CAP on the expression of HSP90 as a target of anti-fungal therapy was investigated. ROS production in C. albicans exposed to CAP was assessed. For this purpose, C. albicans subjected to 0, 90, 120, 150, 180 and 210 s of He/O2 (2%), and non-treated cells as control were examined in terms of the mentioned virulence factors. The results showed that CAP had a significant effect on inhibition of C. albicans growth, Inhibition of biofilm formation, ergosterol content, and fluconazole and amphotericin B antibiotic sensitivity were significant in 210 s treatment group. This effect was validated based on changes of the cell architecture and morphology given the microscopy imaging results. The expression of HSP90 in both C. albicans ATCC 10231 and C. albicans PFCC 9362 was inhibited in 210 s of exposition. CAP exposition induced intracellular ROS, which may cause membrane damage and cell death in C. albicans. Taken together, the potential of CAP for therapeutic purposes in C. albicans-induced fungal infections is supported.

Enhanced Adipose Mesenchymal Stem Cells Proliferation by Carboxymethyl-Chitosan Functionalized Polycaprolactone Nanofiber.

Background: Through combining two synthetic and natural polymers, scaffolds can be developed for tissue engineering and regenerative medicine purposes. Methods: In this work, carboxymethyl chitosan (CMC; 20%) was grafted to Polycaprolactone (PCL) nanofibers using the cold atmospheric plasma of helium. The PCL scaffolds were exposed to CAP, and functional groups were developed on the PCL surface. Results: The results of Fourier Transform Infrared Spectroscopy confirmed CMC (20%) graft on PCL scaffold. The Thiazolyl blue tetrazolium bromide assay showed a significant enhancement (p < 0.05) in the cell affinity and proliferation of adipose-derived stem cells (ADSCs) to CMC20%-graft-PCL scaffolds. After 14 days, bone differentiation was affirmed through alizarin red and calcium depositions. Conclusion: Based on the results, the CMC20%-graft-PCL can support the proliferation of ADSCs and induce the differentiation into bone with longer culture time.

Osteoinductivity of polycaprolactone nanofibers grafted functionalized with carboxymethyl chitosan: Synergic effect of ß-carotene and electromagnetic field.

In this study, carboxymethyl chitosan (CMC) was grafted on polycaprolactone (PCL) nanofibers to fabricate scaffolds for bone tissue engineering. The electrospun PCL nanofibers were treated by cold atmospheric plasma (CAP) of helium to generate the reactive functions necessary for CMC grafting. ß-carotene (ßC) as a biochemical clue and electromagnetic field (EMF, 31.4 µT, 1 h per day) as a biophysical stimulator were used to promote the proliferation and osteodifferentiation of adipose mesenchymal stem cells (ADSCs). Alizarin red staining and calcium content results indicated the generation of nodal calcium on the CMC30%-g-PCL scaffold after 14 days of incubation in the presence or absence of external stimulation factors. Immunocytochemistry (ICC) results confirmed the expression of osteonectin protein for the stem cells seeded on CMC30%-g-PCL with or without using ßC or EMF. These results suggest that the fabricated CMC-grafted scaffolds have the ability to self-differentiate stem cells to osteoblasts due to the osteoinductive effects of the grafted CMC. Furthermore, the osteodifferentiation of ADSCs is promoted by using an external stimulation factor such as ßC or EMF.

Bone morphogenic protein-2 immobilization by cold atmospheric plasma to enhance the osteoinductivity of carboxymethyl chitosan-based nanofibers.

Electrospun polycaprolactone/carboxymethyl chitosan (PCL/CMC) nanofibers treated by helium cold atmospheric plasma (CAP) and grafted with bone morphogenic protein-2 (BMP-2) were used scaffolds for the osteodifferentiation of stem cells to. For in vitro study, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on these scaffolds, and their behaviors were assessed via optical microscopy, MTT assay, and SEM. The osteogenic differentiation of the hMSCs was evaluated by calcium content and alkaline phosphatase assays, Alizarin red and immunofluorescence (ICC) staining, and RT-PCR. The results showed that scaffolds not only can support the proliferation of hMSCs but also can promote their differentiation to osteoblasts without using any external osteogenic differential agent. The RT-PCR and ICC data revealed that the CAP treatment and BMP-2-functionalization have synergic enhancement on the ossification of hMSCs. These fabricated scaffolds can be used as promising candidates for bone tissue engineering applications.

Increment in protease activity of Lysobacter enzymogenes strain by ultra violet radiation.

BACKGROUND AND OBJECTIVES: Increasing the amount of protease from microbial sources is in the focus of attention. Random mutagenesis by physical methods like ultraviolet (UV) radiation is a cost effective and convenient procedure for strain improvement. Therefore, in the present study attempts were made to investigate the effect of UV radiation on Lysobacter enzymogenes in order to increase its protease activity. MATERIALS AND METHODS: UV mutagenesis was induced in L. enzymogenes fresh culture at the distance of 20 cm from light source for different exposure times of 70, 90, 150 and 200 seconds. The mutated isolates were randomly cultured from the nutrient agar medium to casein agar plate, as a selective medium. The primary screening was performed by observing hydrolysis of casein in the plate and the secondary screening was carried out on skim milk agar on the basis of zone of hydrolysis using bacterial supernatants. Quantification of protease activity was done by Anson's method using tyrosine as standard. RESULTS: UV radiation resulted in obtaining 12 mutants out of 100 examined L. enzymogenes strains with increased protease activity. The mutant M2, at 90s exposure time was selected as the best mutant bacterium which produced 1.96 fold more protease over the parent strain. CONCLUSION: Random mutation by UV radiation is a simple and convenient method to increase the protease activity of Lysobacter enzymogenes. Furthermore, it seems that the middle time of exposure to UV, 90 s, was the best time because it can induce mutagenesis but did not hamper the bacteria growth and viability.

Chondroitin sulfate immobilized PCL nanofibers enhance chondrogenic differentiation of mesenchymal stem cells.

Cold Atmospheric Plasma (CAP) is used as a promising method in surface modification for immobilization of chondroitin sulfate functional biomacromolecules on PCL nanofibrous substrates for cartilage tissue engineering. The GAG-grafted scaffolds are able to successfully support the attachment and proliferation of mesenchymal stem cells (MSCs). The seeded scaffolds show the chondro-differentiation of MSCs during a 21-days cell culture in a non-differential medium. Expression of SOX9, Collagen10 and Collagen2 proved the chondro-inductive effect of GAG-grafted scaffolds. Besides, no external chondro-genic differential agent was used in the differentiation of MSCs to chondrocyte. The cells passed the last phase of chondrogenesis after 14 days of incubation. Thus, the GAG-fabricated fibrous scaffolds using CAP are potential candidates for cartilage tissue engineering.