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It is known that native oil-degrading microorganisms are ubiquitous. They can be isolated from contaminated soils. In this study, biodegradation experiments were carried out to evaluate the efficiency of pollutant removal by adding the selected microorganisms. Five mixed cultures and 3 single bacteria strains, Pseudomonas sp., Arthrobacter sp. and Mycobacterium sp. were isolated from hydrocarbon-contaminated soils by enrichment on either crude oil or individual hydrocarbons, as the sole carbon sources. The strains were selected based on their ability to grow in medium containing crude oil, used engine oil or both. Their ability to degrade hydrocarbon contamination in the environment was investigated using soil samples contaminated with used engine oil. The mixed starter culture #1 degraded 66 % of aliphatic compounds in the engine oil, after 60 days of incubation. The mixed starter culture #5 removed 47 % of aromatic compounds during 60 days of incubation, which is the maximum efficiency among the starter cultures, in this study. [ABSTRACT FROM AUTHOR]

The liver is one of the most important organs, with a complex physiology. Current in-vitro approaches are not accurate for disease modeling and drug toxicity research. One of those features is liver zonation, where cells display different physiological states due to different levels of oxygen and nutrient supplements. Organ-on-a-chip technology employs microfluidic platforms that enable a controlled environment for in-vitro cell culture. In this study, we propose a microfluidic design embedding a gas channel (of ambient air), creating an oxygen gradient. We numerically simulate different flow rates and cell densities with the COMSOL Multiphysics package considering cell-specific consumption rates of oxygen and glucose. We establish the cell density and flow rate for optimum oxygen and glucose distribution in the cell culture chamber. Furthermore, we show that a physiologically relevant concentration of oxygen and glucose in the chip is reached after 24 h and 30 min, respectively. The proposed microfluidic design and optimal parameters we identify in this paper provide a tool for in-vitro liver zonation studies. However, the microfluidic design is not exclusively for liver cell experiments but is foreseen to be applicable in cell studies where different gas concentration gradients are critical, e.g., studying hypoxia or toxic gas impact., (© 2023. International Federation for Medical and Biological Engineering.)

This study aimed to improve the mechanical and biological properties of alginate-based hydrogels. For this purpose, in-situ forming hydrogels were prepared by dual crosslinking of Alginate (Alg)/Oxidized Alginate (OAlg)/Silk Fibroin (SF) through simultaneous ionic gelation using CaCO 3 -GDL and Schiff-base reaction. The resulting hydrogels were characterized by FTIR, SEM, compressive modulus, and rheological tests. Compared to the physically-crosslinked alginate hydrogel, the compressive modulus of dual-crosslinked Alg/OAlg/SF hydrogel increased from 28 to 67 kPa, due to the covalent imine bond formation. Then, MTT and DAPI staining assays were performed to demonstrate the biocompatibility of hydrogel. Furthermore, the differentiation potential of bone marrow mesenchymal stem cells encapsulated in hydrogel scaffolds to bone tissue was tested by ALP activity, Alizarin Red staining, and real-time PCR. The overall results showed the potential of Alginate/Oxidized Alginate/Silk Fibroin hydrogel scaffold for bone tissue engineering applications., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Purpose: Bone tissue engineering aims to create a three-dimensional, matured, angiogenic scaffold with a suitable thickness that resembles a natural bone matrix. On the other hand, electrospun fibers, which researchers have considered due to their good biomimetic properties, are considered 2D structures. Due to the highly interwoven network and small pore size, achieving the desired thickness for bone lesions has always been challenging. In bone tissue engineering, bioreactors are crucial for achieving initial tissue maturity and introducing certain signals as flow parameters for differentiation., Methods: In the present study, Human bone marrow mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were co-cultured in a perfusion bioreactor on treated (improved pore size by gelatin sacrification and subsequent ultrasonication) 5-layer polycaprolactone-nano hydroxyapatite-nano zinc oxide (T-PHZ) scaffolds to investigate osteogenesis and angiogenesis simultaneously. The flow parameters and stresses on the cells were studied using two patterns of parallel and vertical scaffolds relative to the flow of the culture medium. In dynamic vertical flow (DVF), the culture medium flows perpendicular to the scaffolds, and in dynamic parallel flow (DPF), the culture medium flows parallel to the scaffolds. In all evaluations, static samples (S) served as the control group., Results: Live/dead, and MTT assays demonstrated the biocompatibility of the 5-layer scaffolds and the suitability of the bioreactor's functional conditions. ALP activity, EDAX analysis, and calcium content measurements exhibited greater osteogenesis for T-PHZ scaffolds in DVF conditions. Calcium content increased by a factor of 2.2, 1.8, and 1.6 during days 7 to 14 of culture under DVF, DPF and S conditions, respectively. After 21 days of co-culturing, an immunohistochemistry (IHC) test was performed to investigate angiogenesis and osteogenesis. Five antibodies were investigated in DVF, CD31, VEGFA, and VEGFR2 for angiogenesis, osteocalcin, and RUNX2 for osteogenesis. Compressive stress applied in DVF mode has increased osteogenic activity compared to DPF., Conclusion: The results indicated the development of ideal systems for osteogenesis and angiogenesis on the treated multilayer electrospun scaffolds in the perfusion bioreactor., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Biofilm formation and its resistance to various antibiotics is a serious health problem in the treatment of wound infections. An ideal wound dressing should have characteristics such as protection of wound from microbial infection, suitable porosity (to absorb wound exudates), proper permeability (to maintain wound moisture), nontoxicity, and biocompatibility. Although silver nanoparticles (AgNPs) have been investigated as antimicrobial agents, their limitations in penetrating into the biofilm, affecting their efficiency, have consistently been an area for further research., Results: Consequently, in this study, the optimal amounts of natural and synthetic polymers combination, along with AgNPs, accompanied by iron oxide nanoparticles (IONPs), were utilized to fabricate a smart bionanocomposite that meets all the requirements of an ideal wound dressing. Superparamagnetic IONPs (with the average size of 11.8 nm) were synthesized through co-precipitation method using oleic acid to improve their stability. It was found that the addition of IONPs to bionanocomposites had a synergistic effect on their antibacterial and antibiofilm properties. Cytotoxicity assay results showed that nanoparticles does not considerably affect eukaryotic cells compared to prokaryotic cells. Based on the images obtained by confocal laser scanning microscopy (CLSM), significant AgNPs release was observed when an external magnetic field (EMF) was applied to the bionanocomposites loaded with IONPs, which increased the antibacterial activity and inhibited the formation of biofilm significantly., Conclusion: These finding indicated that the nanocomposite recommended can have an efficient properties for the management of wounds through prevention and treatment of antibiotic-resistant biofilm., (© 2023. The Author(s).)

Combination nanomedicine is a potent strategy for cancer treatment. Exploiting different mechanisms of action, a novel triple drug delivery system of 5-fluorouracil, curcumin, and piperine co-loaded human serum albumin nanoparticles (5FU-CUR-PIP-HSA-NPs) was developed via the self-assembly method for suppressing breast tumor. Both hydrophobic and hydrophilic drugs were successfully encapsulated in the HSA NPs with a high drug loading efficiency (DLE) of 10%. Successful clinical translation of nanomedicines, however, is a challenging process requiring considerable preclinical in vitro and in vivo animal tests. The aim of this study was to develop a homemade preclinical 3D culture model in the standard 96-well plates in a cost and time-effective novel approach for the rapid generation of homogenous compact tumor spheroids for disease modeling, and anticancer therapeutic/nanomedicine screening. The knowledge of drug screening can be enhanced by employing such a model in a high-throughput manner. Accordingly, to validate the formulated drug delivery system and investigate the cellular uptake and cytotoxicity effect of the nanoformulation, 3D tumor spheroids were employed. The practicality of the nanomedicine system was substantiated in different tests. The in vitro uptake of the NPs into the tight 3D tumor spheroids was facilitated by the semi-spherical shape of the NPs with a proper size and surface charge. 5FU-CUR-PIP-HSA-NPs demonstrated high potency of migration inhibition as a part of successful anti-metastatic therapy as well. The remarkable differences in 2D and 3D cytotoxicities emphasize the importance of employing 3D tumor models as an intermediate step prior to in vivo animal experiments for drug/nanomedicine screening., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Abolhassani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Genome-scale metabolic modeling (GEM) is one of the key approaches to unpack cancer metabolism and for discovery of new drug targets. In this study, we report the Transcriptional Regulated Flux Balance Analysis-CORE (TRFBA-), an algorithm for GEM using key growth-correlated reactions using hepatocellular carcinoma (HCC), an important global health burden, as a case study. We generated a HepG2 cell-specific GEM by integrating this cell line transcriptomic data with a generic human metabolic model to forecast potential drug targets for HCC. A total of 108 essential genes for growth were predicted by the TRFBA-CORE. These genes were enriched for metabolic pathways involved in cholesterol, sterol, and steroid biosynthesis. Furthermore, we silenced a predicted essential gene, 11-beta dehydrogenase hydroxysteroid type 2 ( HSD11B2 ), in HepG2 cells resulting in a reduction in cell viability. To further identify novel potential drug targets in HCC, we examined the effect of nine drugs targeting the essential genes, and observed that most drugs inhibited the growth of HepG2 cells. Some of these drugs in this model performed better than Sorafenib, the first-line therapeutic against HCC. A HepG2 cell-specific GEM highlights sterol metabolism to be essential for cell growth. HSD11B2 downregulation results in lower cell growth. Most of the compounds, selected by drug repurposing approach, show a significant inhibitory effect on cell growth in a wide range of concentrations. These findings offer new molecular leads for drug discovery for hepatic cancer while also illustrating the importance of GEM and drug repurposing in cancer therapeutics innovation.

Objectives: In this study, gelatin was chosen as a novel sacrificial agent in co-electrospun with polycaprolacton-nanohydroxyapatite (PCL-nHA)., Materials and Methods: After electrospinnig, gelatin was washed with water, and the prepared scaffold was ultrasonicated. Morphological and structural properties of the prepared scaffolds were studied by SEM. Fourier transform infrared (FTIR) spectroscopy and water contact angle analysis were used to evaluate the removal of gelatin., Results: According to the SEM results, the pore size of the modified scaffolds was increased 3-folds compared to the control sample. For PCL-nHA gelatin: (80:20) after the treatment, the average cell infiltration was 42.7 μm, while there was no infiltration for the control group. The modified electrospun scaffold significantly enhanced the osteogenic differentiation of hBMSCs as verified by increased ALP activity and upregulation of runt-related transcription factor 2 (RUNX2), collagen type 1 (COL1) and osteocalcin (OCN) genes., Conclusion: Co-electrospun PCL-nHA with gelatin as a sacrificial agent in combination with ultrasonication may be an effective, economic and controllable method to increase the pore size in electrospun scaffolds for bone tissue engineering applications., (Copyright: © 2021 The Author(s); Published by Iranian Journal of Biotechnology.)

Cell therapy is one of the proposed treatments for diabetes. Cell encapsulation and differentiation inside the biodegradable polymers overcome the limitations such as islet deficiency and the host immune responses. This study was set to encapsulate the mesenchymal stem cells (MSCs) and differentiate them into insulin-producing cells (IPCs). Human bone marrow-mesenchymal stem cells (hBM-MSCs) were encapsulated in alginate/trimethyl chitosan/alginate (Alg/TMC/Alg) coating. At first, morphology and swelling properties of the cell-free microcapsules were investigated. Next, a three-step protocol was used in the presence of exendin-4 and nicotinamide to differentiate hBM-MSCs into IPCs. Viability of the encapsulated cells was investigated using MTT assay. The differentiated cells were analyzed using a real-time RT-PCR to investigate Glut-2, Insulin, Pdx-1, Ngn-3, nestin, and Isl-1 gene expression. The results revealed that differentiation of the encapsulated cells was higher than non-encapsulated cells. Also, dithizone staining in  two-dimensional (2D) environment showed the differentiated cell clusters. In summary, here, hBM-MSCs after encapsulation in Alg/TMC/Alg microcapsules, as a new design, were differentiated properly in the presence of exendin-4 and nicotinamide as main inducers. A three-dimensional (3D) matrix is more similar to the native ECM in the body and prepares higher cell-cell contacts., (© 2022. The Author(s), under exclusive licence to Islamic Azad University.)

Probiotic bacteria are able to produce antimicrobial substances as well as to synthesize green metal nanoparticles (NPs). New antimicrobial and antibiofilm coatings (LAB-ZnO NPs), composed of Lactobacillus strains and green ZnO NPs, were employed for the modification of gum Arabic-polyvinyl alcohol-polycaprolactone nanofibers matrix (GA-PVA-PCL) against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The physicochemical properties of ZnO NPs biologically synthesized by L. plantarum and L. acidophilus, LAB-ZnO NPs hybrids and LAB-ZnO NPs@GA-PVA-PCL were studied using FE-SEM, EDX, EM, FTIR, XRD and ICP-OES. The morphology of LAB-ZnO NPs hybrids was spherical in range of 4.56-91.61 nm with an average diameter about 34 nm. The electrospun GA-PVA-PCL had regular, continuous and without beads morphology in the scale of nanometer and micrometer with an average diameter of 565 nm. Interestingly, the LAB not only acted as a biosynthesizer in the green synthesis of ZnO NPs but also synergistically enhanced the antimicrobial and antibiofilm efficacy of LAB-ZnO NPs@GA-PVA-PCL. Moreover, the low cytotoxicity of ZnO NPs and ZnO NPs@GA-PVA-PCL on the mouse embryonic fibroblasts cell line led to make them biocompatible. These results suggest that LAB-ZnO NPs@GA-PVA-PCL has potential as a safe promising antimicrobial and antibiofilm dressing in wound healing against pathogens., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Miniaturized bubble columns (MBCs) can provide mass transfer characteristics similar to stirred tank bioreactors. In this study, a new application was developed for MBCs to investigate the effect of feeding strategy and medium type on the fed-batch culture of recombinant E. coli. The results showed that the exponential feeding strategy and defined M9 medium were more suitable to achieve the high cell density culture (HCDC). The maximum obtained cell concentration in exponential feeding strategy in the defined medium without induction, was at OD 600 of 169, while glucose concentration was maintained under 2 g/L. To the best of our knowledge, this cell concentration cannot be achieved in lab or pilot scale bubble columns. At the end of the process, adverse effect of the metabolic burden due to induction and mass transfer limitations decreased the obtained final cell concentration to OD 600 of 116. Finally, a comparison of the results for fed-batch culture in the stirred tank bioreactor with those of the MBCs showed that their lower cell concentrations were due to the hydrodynamics limitations of MBCs. Yet, it was found that the MBCs are efficient tools in development of feeding strategies and evaluation of medium components for HCDC of recombinant E. coli., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Gum Arabic (GA) is a biocompatible polymer with the necessary requirements for a wound dressing. However, electrospinning of GA is a bottleneck due to its physico-chemical properties. The aim of this study was to fabricate an antimicrobial nanofibers mat from GA with suitable porosity, water absorption, water vapor permeability and mechanical strength. For this purpose, the composition of polycaprolacton (PCL)-coated GA-polyvinyl alcohol (PVA) nanofibers mat was optimized based on the possible highest porosity, water absorption and water vapor permeability, and then silver nanoparticles (AgNPs) loaded nanofibers mat was prepared based on this composition. The synthesis of AgNPs was supported by UV-vis and ICP analyses. The structure of mat and its constituents were characterized by FE-SEM, XRD and FTIR. The results showed that the average diameter of nanofibers was in the range of 150 to 250 nm with the porosity, water absorption and water vapor permeability of 37.34%, 547.30% and 2235.50 g/m 2 .day, respectively. The antimicrobial activity of mat against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans was proved. Moreover, the cytotoxicity of mat showed the good biocompatibility for the mouse embryonic fibroblast cells. This study introduced PCL-coated GA-PVA-AgNPs as an effective antimicrobial mat alternative for commercial wound dressing., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Electrospun scaffolds are potentially interesting in bone tissue engineering due to a strong structural similarity to the natural bone matrix. To investigate the osteogenic behavior of cells on the scaffolds, dynamic culture of cells is essential to simulate the biological environment. In the present study, human mesenchymal stem cells (hMSCs) were cultured on multilayer nanohydroxyapatite-polycaprolactone electrospun scaffolds at different configurations (horizontal with or without pressure and parallel with the medium flow) and flow rates in a perfusion bioreactor. Alkaline phosphatase (ALP) activity, cell viability, Ca deposition and RUNX2 expression were determined in three different dynamic states, and compared with static culture after 1, 3, 7, and 14 days. Among dynamic groups, RUNX2 gene expression upregulated more in a horizontal state at a low flow rate without mechanical pressure (LF) and parallel flow (PF), than static group on day 7. At a high flow rate with mechanical pressure, Ca deposition and ALP activity increased 2.34 and 1.7 folds more than in static culture over 7 days, respectively. Furthermore, ALP activity, Ca deposition and RUNX2 gene expression increased in PF samples. PF provided longer culture time with higher cell differentiation. Therefore, high flow rate with mechanical pressure and PF are suggested for producing differentiated cell structure for bone tissue engineering., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Large bone defects treatment is one of the challenges in current bone tissue engineering approaches. Various strategies have been proposed to address this issue, among which, prevascularization by coculturing of angiogenic and osteogenic cells on the scaffolds can alleviate this problem. In the present study, modified fibrous scaffolds were prepared by electrospinning and subsequent ultrasonication of polycaprolactone (PCL) containing nano-hydroxyapatite (n-HA), with/without nano-zinc oxide (n-ZnO), and polyethylene oxide [PEO] as a sacrificial agent. The physical, mechanical, and chemical characteristics of the scaffolds were evaluated. The results showed the presence of n-ZnO, which in turn increased Young's module of the scaffolds from 5.5 ± 0.67 to 6.7 ± 1.77 MPa. Moreover, MTT, SEM, alkaline phosphatase (ALP) activity, chicken embryo chorioallantoic membrane (CAM) assay, and real-time RT-PCR were utilized to investigate the biocompatibility, cell adhesion and infiltration, osteoconductivity, angiogenic properties, and expression of osteogenic and angiogenic related genes. ALP assay showed that the highest enzyme activity was noted when the modified scaffolds containing n-ZnO were seeded with HUVEC:hBMSC at the cell ratio of 1:5. CAM assay showed induction of angiogenesis for the scaffolds containing n-ZnO. Real-time RT-PCR results showed significant upregulation of angiogenic related genes. Thus, the scaffolds containing n-ZnO may have great potential for osteogenesis and angiogenesis in tissue engineering applications., (© 2019 Wiley Periodicals, Inc.)

Genome-scale metabolic modeling has emerged as a promising way to study the metabolic alterations underlying cancer by identifying novel drug targets and biomarkers. To date, several computational methods have been developed to integrate high-throughput data with existing human metabolic reconstructions to generate context-specific cancer metabolic models. Despite a number of studies focusing on benchmarking the context-specific algorithms, no quantitative assessment has been made to compare the predictive performance of these methods. Here, we integrated various and different datasets used in previous works to design a quantitative platform to examine functional and consistency performance of several existing genome-scale cancer modeling approaches. Next, we used the results obtained here to develop a method for the reconstruction of context-specific metabolic models. We then compared the predictive power and consistency of networks generated by our method to other computational approaches investigated here. Our results showed a satisfactory performance of the developed method in most of the benchmarks. This benchmarking platform is of particular use in algorithm selection and assessing the performance of newly developed algorithms. More importantly, it can serve as guidelines for designing and developing new methods focusing on weaknesses and strengths of existing algorithms., Competing Interests: The authors have declared that no competing interests exist.

Background: Nowadays, production of nanocomposite scaffolds based on natural biopolymer, bioceramic, and metal ions is a growing field of research due to the potential for bone tissue engineering applications., Methods: In this study, a nanocomposite scaffold for bone tissue engineering was successfully prepared using collagen (COL), beta-tricalcium phosphate (β-TCP) and strontium oxide (SrO). A composition of β-TCP (4.9 g) was prepared by doping with SrO (0.05 g). Biocompatible porous nanocomposite scaffolds were prepared by freeze-drying in different formulations [COL, COL/β-TCP (1:2 w/w), and COL/β-TCP-Sr (1:2 w/w)] to be used as a provisional matrix or scaffold for bone tissue engineering. The nanoparticles were characterized by X-ray diffraction, Fourier transforms infrared spectroscopy and energy dispersive spectroscopy. Moreover, the prepared scaffolds were characterized by physicochemical properties, such as porosity, swelling ratio, biodegradation, mechanical properties, and biomineralization., Results: All the scaffolds had a microporous structure with high porosity (~ 95-99%) and appropriate pore size (100-200 μm). COL/β-TCP-Sr scaffolds had the compressive modulus (213.44 ± 0.47 kPa) higher than that of COL/β-TCP (33.14 ± 1.77 kPa). In vitro cytocompatibility, cell attachment and alkaline phosphatase (ALP) activity studies performed using rat bone marrow mesenchymal stem cells. Addition of β-TCP-Sr to collagen scaffolds increased ALP activity by 1.33-1.79 and 2.92-4.57 folds after 7 and 14 days of culture, respectively., Conclusion: In summary, it was found that the incorporation of Sr into the collagen-β-TCP scaffolds has a great potential for bone tissue engineering applications., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest.

Miniaturized bubble columns (MBCs) have different hydrodynamics in comparison with the larger ones, but there is a lack of scientific data on MBCs. Hence, in this study, the effect of gas hold-up, flow regimes, bubble size distribution on volumetric oxygen mass transfer coefficient at different pore size spargers and gas flow rates in MBCs in the presence and absence of microorganisms were investigated. It was found that flow regime transition occurred around low gas flow rates of 1.18 and 0.85 cm/s for small (16-40 µm) and large (40-100 µm) pore size spargers, respectively. Gas hold-up and K L a in MBC with small size sparger were higher than those with larger one, with an increasing effect in the presence of microorganisms. A comparison revealed that the wall effect on the flow regime and gas hold-up in MBCs was greater than bench-scale bubble columns. The K L a values significantly increased up to tenfold using small pore size sparger. In the MBC and stirred tank bioreactors, the maximum obtained cell concentrations were OD 600 of 41.5 and 43.0, respectively. Furthermore, it was shown that in MBCs, higher K L a and lower turbulency could be achieved at the end of bubbly flow regime.

Background: Pancreatic islet transplantation is one of the most promising strategies for treating patients with type I diabetes mellitus., Objective: We aimed to assess the immunoisolation properties of the multilayer encapsulated islets using alginate-chitosan-PEG for immunoprotection and insulin secretion from the encapsulated islets induced under different glucose concentrations in vitro ., Materials and Methods: In this study, the islets were isolated from Wistar rats. The biological function (insulin secretion) of the immunoisolated islets following to PEGylation and encapsulation in the alginate-chitosan-PEG, separately, in addition to their immuno-protection in a co-culturing with the lymphocytes isolated from the male C57BL/6 mice were investigated, respectively., Results: Alginate-chitosan-PEG decreased IL-2 secretion from the lymphocytes co-cultured with islets. Also, insulin secretion from the encapsulated and PEGylated groups was stimulated by glucose (i.e., 5.6 and 16.7 mM of glucose, respectively); showed insulin secretion similar to the naked islets, without coating, after 30 and 60 min of incubation., Conclusion: In conclusion, encapsulation and PEGylation have no negative effect on the insulin secretion and glucose sensitivity of the islets for all of the groups. Also, encapsulation decreased IL-2 secretion from the lymphocytes.

Recombinant plasminogen activator (reteplase) is a third generation thrombolytic agent which has been used on coronary artery thrombosis and acute myocardial infarction. Clot lysis assay is usually considered as a unique method to evaluate biological activity of reteplase. In this study biological activity of reteplase was determined by APTT (activated partial thromboplastin time) lysis method. Validity of this method was evaluated in comparison with reference method, clot lysis time assay. Results of APTT lysis test showed good reproducibility (relative standard deviation (RSD) 3-5% for within day analysis and 4-7% for between day analysis), and accuracy (101.3-102.7%). APTT lysis responses were linear in range of 0.001-0.1 mg/mL reteplase. Therefore, APTT lysis method is applicable for biological activity determination of reteplase. Although more comprehensive studies are required to approve this test as a reference method, APTT lysis method seems to be valuable to receive more attention due to advantages of technical simplicity, sensitivity, applicability, and cost efficiency.

Nanocarrier-based drug delivery systems have been explored extensively in cancer therapy. Among the vast number of different nanocarrier systems applied to deliver chemotherapeutics to cancer tumor, intelligent systems which deliver drug to various sites in the body have attracted considerable attentions. Finding a specific stimulant that triggers the carrier to release its payload in the target tissue is a key parameter for efficacy of delivery systems. Acidic pH of cancer tumor helps a pH-sensitive carrier to release drug at the tumor site. In this study, a pH-sensitive mixed micellar system was developed using Dextran-Stearic Acid (Dex-SA) and Dextran-Histidine (Dex-His) conjugated polymers to deliver doxorubicin (DOX) to cancer cells. Drug release from this micellar system showed higher release rate at acidic pH than that of in neutral environment, where the release was 56 and 76% at pH 7.4 and acidic pH, respectively. Finally, the in vitro cytotoxicity and cell uptake of DOX-loaded micelles and free DOX on U87 MG cell line showed that micellar systems had more anti-proliferation effect and uptake compared to free drug., (Copyright © 2018. Published by Elsevier Inc.)

Today, cell therapy is known as an important tool in the treatment of chronic diseases where cells lose their normal function. Immunoisolation systems using microencapsulation or PEGylation have been developed to evade the problem of rejection by the immune system. The aim of the present study was to investigate a combination of microencapsulation and PEGylation methods in coating mouse embryonic stem cells (mESCs) to determine its effect in reducing the host's immune response. Therefore, methoxy polyethylene glycol (mPEG) binding on alginate-trimethyl chitosan (TMC) microcapsules was investigated using FTIR. Furthermore, survival of the microencapsulated mESCs was confirmed using AO/PI staining and MTT assays. In addition, the effect of mESCs co-cultured with foreign lymphocytes was evaluated. Overall, interleukin-2 (IL-2) secretions as a response of the immune system revealed that mESCs microencapsulation in alginate-TMC-PEG, reduced the immune system response. The results suggested that IL-2 secretion was reduced to 62% at seventh day.

One of the major problems associated with the electrospun scaffolds is their small pore size, which limits the cellular infiltration for bone tissue engineering. In this study, the effect of increasing the pore size on cellular infiltration was studied in poly/nanohydroxyapatite electrospun scaffolds, which were modified using ultrasonication, co-electrospinning with poly (ethylene oxide), and a combination of both. Ultrasonic process was optimized by central composite design. The ultrasonic output power and time of the process were considered as the effective parameters. The pore size of the scaffolds was evaluated by scanning electron microscope. The optimum conditions, according to the pore area and mechanical properties of the scaffolds were selected, and finally the groups that had the highest pore size and mechanical strength were selected for the combined method. Increasing the pore size enhanced the cellular proliferation, extension and infiltration, as well as the osteodifferentiation of stem cells. At the optimum condition, the average cellular infiltration was 36.51 µm compared to the control group with no cellular infiltration. In addition, alkaline phosphatase activity and the expression of osteocalcin and collagen I (COL I) were, respectively, 1.86, 2.54, and 2.16 fold compared to the control group on day 14. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1887-1899, 2017., (© 2017 Wiley Periodicals, Inc.)

Since the leading cause of death are cardiac diseases, engineered heart tissue (EHT) is one of the most appealing topics defined in tissue engineering and regenerative medicine fields. The importance of EHT is not only for heart regeneration but also for in vitro developing of cardiology. Cardiomyocytes could grow and commit more naturally in their microenvironment rather than traditional cultivation. Thus, this research tried to develop a set up on-a-chip to produce EHT based on chitosan hydrogel. Micro-bioreactor was hydrodynamically designed and simulated by COMSOL and produced via soft lithography process. Chitosan hydrogel was also prepared, adjusted, and assessed by XRD, FTIR and also its degradation rate and swelling ratio were determined. Finally, hydrogels in which mice cardiac progenitor cells (CPC) were loaded were injected into the micro-device chambers and cultured. Each EHT in every chamber was evaluated separately. Prepared EHTs showed promising results that expanded in them CPCs and work as an integrated syncytium. High cell density culture was the main accomplishment of this study., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Elucidation of the molecular mechanism of silver nanoparticle (AgNP) synthesis is necessary to control nanoparticle size, shape, and monodispersity. In this study, the mechanism of AgNP formation by Neurospora intermedia was investigated. The higher production rate of AgNP formation using a culture supernatant heat-treated at 100° and 121°C relative to that with an un-treated culture supernatant indicated that the native form of the molecular species is not essential. The effect of the protein molecular weight (MW) on the nanoparticle size distribution and average size was studied by means of ultraviolet-visible spectroscopy and dynamic light scattering. Using un-treated and concentrated cell-free filtrate passed through 10 and 20 kDa cut-off filters led to the production of AgNPs with average sizes of 25, 30, and 34 nm, respectively. Also, using the permeate fraction of cell-free filtrate passed through a 100 kDa cut-off filter led to the formation of the smallest nanoparticles with the narrowest size distribution (average size of 16 nm and polydispersity index of 0.18). Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of the fungal extracellular proteins showed two notable bands with the MWs of 15 and 23 kDa that are involved in the reduction and stabilisation of the nanoparticles, respectively.

In this study, a method is proposed to reduce immunological response of immune system against Langerhans islets by PEGylation of islets combined with adjuvant therapy. For this purpose, the best composition for a mixture of succinimidyl valeric acid activated mPEG (mPEG-SVA) with different molecular weights (MWs) and for a mixture of succinimidyl carbonate activated mPEG (mPEG-SC) with different MWs was determined separately. Then, the effect of pentoxifylline (PTX) as an adjuvant drug on immunological response against PEGylated islets at best mPEG composition was studied. The extent of mPEGs reaction, the amount of interlukin-2 (IL-2) and perforin secretion, and the viability of lymphocytes and islets in homo and co-cultures in the presence of PTX at different concentrations were considered for the in vitro evaluation of the proposed method. It was found, that a mixture of mPEG-SVA with MWs of 10 and 5 kDa at a composition of 75 and 25%, respectively, was the best formulation. Also, the addition of PTX drug to co-culture medium increased the protection of PEGylated islets against immune system and a concentration of 75 μg mL -1 of PTX was suitable for islet protection with no adverse effect on immune cells.

Correction for 'BiKEGG: a COBRA toolbox extension for bridging the BiGG and KEGG databases' by Oveis Jamialahmadi et al., Mol. BioSyst., 2016, DOI: .

Development of an interface tool between the Biochemical, Genetic and Genomic (BiGG) and KEGG databases is necessary for simultaneous access to the features of both databases. For this purpose, we present the BiKEGG toolbox, an open source COBRA toolbox extension providing a set of functions to infer the reaction correspondences between the KEGG reaction identifiers and those in the BiGG knowledgebase using a combination of manual verification and computational methods. Inferred reaction correspondences using this approach are supported by evidence from the literature, which provides a higher number of reconciled reactions between these two databases compared to the MetaNetX and MetRxn databases. This set of equivalent reactions is then used to automatically superimpose the predicted fluxes using COBRA methods on classical KEGG pathway maps or to create a customized metabolic map based on the KEGG global metabolic pathway, and to find the corresponding reactions in BiGG based on the genome annotation of an organism in the KEGG database. Customized metabolic maps can be created for a set of pathways of interest, for the whole KEGG global map or exclusively for all pathways for which there exists at least one flux carrying reaction. This flexibility in visualization enables BiKEGG to indicate reaction directionality as well as to visualize the reaction fluxes for different static or dynamic conditions in an animated manner. BiKEGG allows the user to export (1) the output visualized metabolic maps to various standard image formats or save them as a video or animated GIF file, and (2) the equivalent reactions for an organism as an Excel spreadsheet.

Background: Monolayer electrospun scaffolds have already been used in bone tissue engineering due to their high surface-tovolume ratio, interconnectivity, similarity to natural bone extracellular matrix (ECM), and simple production., Objectives: The aim of this study was to evaluate the dynamic culture effect on osteogenic differentiation and mineralizationi into a compact cellular multilayer nHA-PCL electrospun construct. The dynamic culture was compared with static culture., Materials and Methods: The calcium content, alkaline phosphatase (ALP) activity and cell viability were investigated on days 3 and 7., Results: When the dynamic culture compared to static culture, the mineralization and ALP activity were increased in dynamic culture. After 7 days, calcium contents were 41.24 and 20.44 μg.(cm 3 ) -1 , and also normalized ALP activity were 0.32 and 0.19 U.mg -1 in dynamic and static culture, respectively. Despite decreasing the cell viability until day 7, the scanning electron microscopy (SEM) results showed that, due to higher mineralization, a larger area of the construct was covered with calcium deposition in dynamic culture., Conclusions: The dynamic flow could improve ALP activity and mineralization into the compact cellular multilayer construct cultured in the perfusion bioreactor after 7 days. Fluid flow of media helped to facilitate the nutrients transportation into the construct and created uniform cellular construct with high mineralization. This construct can be applied for bone tissue engineering.

In this study, an enzyme catalyzed in situ forming hydrogel based on tyramine conjugated high methoxyl content gum tragacanth (TA-HMGT) was prepared and characterized. TA-HMGT was synthesized via heterogeneous ammonolysis of methyl ester groups of HMGT. Then, the hydrogel was prepared via horseradish peroxidase catalyzed coupling reaction in the presence of hydrogen peroxide. Hydrogel properties, such as gelation time, swelling/degradation behavior and rheological properties could be adjusted by tuning the gelation parameters and extent of tyramine conjugation. This system was a soft elastic hydrogel with appropriate biocompatibility. The fast gelation of the hydrogel is desirable for clinical applications. Also, in vitro bovine serum albumin release from the synthesized hydrogel showed good release profile with limited burst release., (© The Author(s) 2015.)

The most important obstacle in islets transplantation for the treatment of diabetes is graft rejection by the host immune system. To solve this problem, immunosuppressive drugs should be used, but they may have several side effects. To overcome these problems, islets immunoisolation systems such as encapsulation and PEGylation have been developed. The aim of this study was to investigate the possibility of using encapsulation and PEGylation techniques simultaneously (as a novel design) for immunocamouflaging the islets of Langerhans. For this purpose, the attachment of poly-L-ornithine (PLO) onto the surface of alginate microcapsules and activated methoxy polyethylene glycol (mPEG) onto alginate-PLO microcapsules was verified by Fourier transform infrared analysis and scanning electron microscopy. Viability of the free and encapsulated islets up to the 7th day was approved by acridine orange (AO)/propidium iodide (PI). The obtained results from lymphocytes co-culturing with free and encapsulated islets (in different designs of microcapsules with one to three layers) showed that encapsulation generally reduces the immune response against the islets. However, the addition of PLO and mPEG as second and third layers to the surface of alginate microcapsules decreased interleukine-2 (IL-2) secretion against the islets more and more. Finally, two different activated mPEG, mPEG-succinimidyl carbonate (mPEG-SC) and mPEG-succinimidylvaleric acid (mPEG-SVA), used separately on the surface of microcapsules were investigated, and the results showed that IL-2 secretion was reduced 14.3% and 37.5% in comparison with the alginate-PLO microcapsules, respectively. On the other hand, mPEG-SVA was more effective than mPEG-SC, so it decreased IL-2 secretion 27.1% more than mPEG-SC., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: The effectiveness of bioremediation systems for PAH-contaminated soil may be constrained by physicochemical properties of contaminants and environmental factors. Information on what is the most effective factor in bioremediation process is essential in the decision of what stimulations can be taken to assist the biodegradation efficacy., Methods: In this study, four factors of surfactant (Tween 80), humic acid (HA), salinity and nutrients in a 2(4) full factorial design were screened in bioremediation of phenanthrene contaminated soil by using a consortium of bacteria., Results: Between the employed levels of the factors only salinity had not significant effect. Optimal concentrations of surfactant, HA and nutrient were obtained by a response surface design. For phenanthrene biodegradation, a central composite face centred design (CCFD) showed that nutrient, surfactant and HA concentrations had highly significant, significant and insignificant effects, respectively. The best conditions with 87.1% phenanthrene biodegradation were 150 mg HA/Kg soil, 12.68 μg/L surfactant, and nutrients as K2HPO4, 0.8; KH2PO4, 0.2 and KNO3, 1 g/L. A high similarity was between the model prediction and experimental results., Conclusions: This study showed that nutrient with 81.27% efficiency could be considered as the most effective factor for practical implications of bioremediation process for PAHs contaminated soil cleanup strategies.

Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

In the present study, the biosynthesis of silver nanoparticles (AgNPs) using Neurospora intermedia, as a new non-pathogenic fungus was investigated. For determination of biomass harvesting time, the effect of fungal incubation period on nanoparticle formation was investigated using UV-visible spectroscopy. Then, AgNPs were synthesized using both culture supernatant and cell-free filtrate of the fungus. Two different volume ratios (1:100 and 1:1) of the culture supernatant to the silver nitrate were employed for AgNP synthesis. In addition, cell-free filtrate and silver nitrate were mixed in presence and absence of light. Smallest average size and highest productivity were obtained when using equal volumes of the culture supernatant and silver nitrate solution as confirmed by UV-visible spectra of colloidal AgNPs. Comparing the UV-visible spectra revealed that using cell-free filtrate for AgNP synthesis resulted in the formation of particles with higher stability and monodispersity than using culture supernatant. The absence of light in cell-free filtrate mediated synthesis led to the formation of nanoparticles with the lowest rate and the highest monodispersity. The presence of elemental silver in all prepared samples was confirmed using EDX, while the crystalline nature of synthesized particles was verified by XRD. FTIR results showed the presence of functional groups which reduce Ag(+) and stabilize AgNPs. The presence of nitrate reductase was confirmed in the cell-free filtrate of the fungus suggesting the potential role of this enzyme in AgNP synthesis. Synthesized particles showed significant antibacterial activity against E. coli as confirmed by examining the growth curve of bacterial cells exposed to AgNPs.

In this study, pH-sensitive blended polymeric beads were prepared by ionic gelation of mixed alginate and N,O-carboxymethyl chitosan (NOCC) solutions in aqueous media containing calcium chloride. To prepare drug-loaded beads, sulfasalazine (SA) as a model drug was added to the initial aqueous polymer solution. These beads were characterized and evaluated in vitro as potential carriers for colon-specific drug delivery. A 3 2 full factorial experimental design was employed to evaluate the effect of polymer and CaCl 2 concentrations on swelling and drug release behavior of the beads in simulated gastrointestinal tract fluid. It was found that the rate of swelling and drug release decreased significantly with increasing polymer and CaCl 2 concentrations, but polymer concentration was more effective than CaCl 2 concentration. The beads prepared using 4.5% polymer concentration and 4% CaCl 2 concentration retained approximately 60% of the loaded drug before approaching the simulated colonic fluid. Based on the results, the alginate-NOCC beads prepared with high polymer concentration could be potentially suitable polymeric carriers for colon-specific delivery of SA.

Langerhans islet transplantation is a much less invasive approach compared with the pancreas transplantation to 'cure' diabetes. However, destruction of transplanted islets by the immune system is an impediment for a successful treatment. Chemical grafting of monomethoxy poly(ethylene glycol) onto pancreatic islet capsule is a novel approach in islet immunoisolation. The aim of this study was to determine an optimized condition for grafting of monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) on islets capsule. Independent variables such as reaction time, the percentage of longer mPEG in the mixture, and polymer concentration were optimized using a three-factor, three-level Box-Behnken statistical design. The dependent variable was IL-2 (interleukin-2) secretion of lymphocytes co-cultured with PEGylated or uncoated control islets for 7 days co-culturing. A mathematical relationship is obtained which explained the main and quadratic effects and the interaction of factors which affected IL-2 secretion. Response surface methodology predicted the optimized values of reaction time, the percentage of longer mPEG in the mixture, and polymer concentration of 60 min to be 63.7% mPEG 10 and 22 mg/mL, respectively, for the minimization of the secreted IL-2 as response. Islets which were PEGylated at this condition were transplanted to diabetic rats. The modified islets could survive for 24 days without the aid of any immunosuppressive drugs and it is the longest survival date reported so far. However, free islets (unmodified islets as control) are completely destroyed within 7 days. These results strongly suggest that this new protocol provides an effective clinical means of decreasing transplanted islet immunogenicity.

Islet transplantation is one of the promising ways to treat diabetes. To reduce the immune system response, several methods have been developed, a novel one being the grafting of methoxy polyethylene glycol (mPEG) derivatives onto collagen capsules of islets. In this study, the effects of the first and second generations of activated mPEG on the immunological response of polyethylene glycol (PEG) grafted pancreatic islets were studied. mPEG-Succinimidyl carbonate (mPEG-SC) and mPEG-succinimidyl propionic acid (mPEG-SPA) (with nominal molecular weight 5 kDa), typical of the first and second generations of activated mPEG, were selected, respectively. Both activated mPEGs did not affect the morphology, viability, or functionality of PEGylated islets compared to free islets (naked islets). The amount of IL-2 secreted from lymphocytes co-cultured with mPEG-SPA grafted islets (131.83 ± 15.28 pg/ml) was not significantly different from that with mPEG-SC grafted islets (156.09 ± 27.94 pg/ml). These results indicated that both mPEG-SC and mPEG-SPA had the same effect for camouflaging Langerhans islets, but the former is more suitable due to its easier synthesis process.

Modifying the surface of living cells with specially selected polymers is a new method of protecting them from the immune system. Covalent attachment of polyethylene glycol (PEG) derivatives on the capsule of pancreatic islets in aqueous media under moderate conditions is an example. This study aimed to find out whether higher polymer concentration could protect PEGylated islets from immune systems more efficiently without affecting their viability and morphology. Methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC) with molecular weight of 5 kDa at different concentrations was grafted onto islets capsules. It was found that at polymer concentrations higher than 22 mg/mL, islets lost their viability with changes in their surface structure as compared to free islets. At mPEG-SC concentrations less than 22 mg/mL, there was no change in morphology and viability of PEGylated islets, but PEGylation of islets at polymer concentration 7 mg/mL was not sufficient for their camouflaging. Polymer concentration of 22 mg/mL was more effective in weakening the immunogenicity of islets. It was concluded that increasing the 5kDa mPEG-SC concentration up to 22 mg/ml protected islets from the immune cells more efficiently without affecting their viability and functionality.

The surface modification of Langerhans islets by grafting activated poly(ethylene glycol) on to their capsules in order to prevent immune-system stimulation is a novel method in diabetes cell therapy. In the present study, mPEG [methoxypoly(ethylene glycol)] with two molecular masses of 5 and 10 kDa, activated with SC (succinimidyl carbonate), was grafted on to the surface of pancreatic islets at a polymer concentration of 22 mg/ml. It was found that PEGylated islets were viable and active, and no morphological changes on the collagen capsule of islets were observed. The amount of interleukin-2 secretion from lymphocytes co-cultured with islets PEGylated with mPEG-SC of 5 and 10 kDa was 112.12 ± 23.09 pg/ml and 172.75 ± 27.94 pg/ml respectively. Thus mPEG-SC (SC-activated mPEG) with higher molecular mass was more suitable for camouflaging islets from the immune system.

The expression of rhIL-2 (recombinant human interleukin-2) in bacteria results in the formation of insoluble inclusion-body aggregates. These aggregates were first solubilized under denaturing conditions (sodium phosphate buffer solution containing 8 M urea and 10 mM 2-mercaptoethanol) and then purified using IMAC (immobilized metal-ion-affinity chromatography). IMAC was used to capture rhIL-2. The protein was gradually refolded on the column by a gradient elution (8 M to 0 M urea) in the presence of 10% (v/v) glycerol. Glycerol was used to prevent protein aggregation during the refolding step. Using this method, rhIL-2 was collected at 97% purity and its activity was measured by the lymphocyte transformation test. The measured activity was identical with commercial human interleukin-2.

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. A proposed method to overcome donor-recipient blood group incompatibility is to mask the blood group antigens by the covalent attachment of poly(ethylene glycol) (PEG) to the red blood cell (RBC) membrane. Despite much work in the development of PEG-coating of RBCs, there is a paucity of data on the optimization of the PEG-coating technique; it is the aim of this study to determine the optimum conditions for PEG coating using a cyanuric chloride reactive derivative of methoxy-PEG as a model polymer. Activated PEG of molecular mass 5 kDa was covalently attached to human RBCs under various reaction conditions. Inhibition of binding of a blood-type specific antiserum (anti-D) was employed to evaluate the effect of the PEG-coating, quantified by hemocytometry and flow-cytometry. RBC morphology was examined by light and scanning electron microscopy. Statistical analysis of experimental design together with microscopy results showed that the optimum PEGylation conditions are pH = 8.7, temperature = 14 degrees C, and reaction time = 30 min. An optimum concentration of reactive PEG could not be determined. At high polymer concentrations (>25 mg/mL) a predominance of type III echinocytes was observed, and as a result, a concentration of 15 mg/mL is the highest recommended concentration for a linear PEG of molecular mass 5 kDa.