Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells.

Background: Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another Antimicrobial Peptides (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells. Methods: In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Auto-cleavage induction of inteins1/2 was performed based on IMPACT® manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay. Results: The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 mM of IPTG, at 22°C). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 kDa was observed on tricine-SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 µM, respectively. Conclusion: In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide.

Lipocalin-2 Upregulation in Nasopharyngeal Carcinoma: A Novel Potential Diagnostic Biomarker.

Background: Lipocalin-2 (LCN2) deregulation has been reported in several types of cancer and is implicated in the proliferation, migration, angiogenesis, and progression of tumors. However, its aberrant expression has been rarely studied in nasopharyngeal carcinoma (NPC). In the present study, we investigated the expression of LCN2 in NPC patients. Methods: In this descriptive cross-sectional study, 29 NPC and 20 non-cancerous control paraffin pathology blocks were obtained from the seven-year (2011 to 2018) archive of Razi Laboratory in Rasht, Iran. LCN2 mRNA expression was evaluated through quantitative real-time PCR. In addition, immunohistochemistry was performed to evaluate LCN2 expression at the protein level. The fold change value and total immunostaining score (TIS) were applied for quantitative evaluation. The nonparametric Mann-Whitney U test and Fisher's exact test were used through GraphPad Prism 8.3.0 software. P<0.05 was considered statistically significant. Results: Our results revealed that LCN2 mRNA and protein levels in NPC tissues were significantly higher than control tissues (P=0.028 and P=0.002, respectively). At the protein level, 65.51% (19/29) of NPC patients were categorized as having high LCN2 expression (TIS>3) and 34.47% (10/29) as low expression (TIS≤3). While in the control group, 25% (5/20) of subjects represented a high expression of LCN2 (TIS>3), and 75% (15/20) showed no or weak expression (TIS≤3). No significant correlation was found between the overexpression of LCN2 at the protein level and the demographic features of the patients. Conclusion: Our findings suggest that LCN2 might be considered a potential new diagnostic marker for NPC. However, this warrants further studies.

Human umbilical cord-derived mesenchymal stem cells-harvested mitochondrial transplantation improved motor function in TBI models through rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation.

Each year, traumatic brain injury (TBI) causes a high rate of mortality throughout the world and those who survive have lasting disabilities. Given that the brain is a particularly dynamic organ with a high energy consumption rate, the inefficiency of current TBI treatment options highlights the necessity of repairing damaged brain tissue at the cellular and molecular levels, which according to research is aggravated due to ATP deficiency and reactive oxygen species surplus. Taking into account that mitochondria contribute to generating energy and controlling cellular stress, mitochondrial transplantation as a new treatment approach has lately reduced complications in a number of diseases by supplying healthy and functional mitochondria to the damaged tissue. For this reason, in this study, we used this technique to transplant human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)-derived mitochondria as a suitable source for mitochondrial isolation into rat models of TBI to examine its therapeutic benefit and the results showed that the successful mitochondrial internalisation in the neuronal cells significantly reduced the number of brain cells undergoing apoptosis, alleviated astrogliosis and microglia activation, retained normal brain morphology and cytoarchitecture, and improved sensorimotor functions in a rat model of TBI. These data indicate that human umbilical cord-derived mesenchymal stem cells-isolated mitochondrial transplantation improves motor function in a rat model of TBI via rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation, maybe as a result of restoring the lost mitochondrial content.

Novel N-α-amino acid spacer-conjugated phthalimide-triazine derivatives: synthesis, antimicrobial and molecular docking studies.

To design and develop novel antimicrobial agents, a series of phthalimide-triazine-based derivatives (6a-6e) were synthesized, characterized and evaluated for their potential antibacterial activities. The compounds were prepared through reaction of 6-phenyl-1,3,5-triazine-2,4-diamine with phthalimide moiety containing aliphatic amino acid. Structural analysis of the synthesized compounds was carried out by various characterization techniques such as FT-IR, 1H and 13C-NMR and mass spectroscopy. After the confirmation of the structure, the antibacterial screening of the synthesized compounds was performed against two strains of Gram-positive (Staphylococcus aureus, and Bacillus subtilis) and two strains of Gram-negative (Escherichia coli and Salmonella enteritidis) bacteria. The results of antimicrobial activity showed that compound 6d was the most active against all the tested strains of microorganisms with the MIC value 1.25 µg/µl. The synthesized compounds were docked into the binding sites of E. coli-DNA gyrase B and S. aureus-DNA gyrase complex to explore their theoretically binding mode and possible interactions of these ligands with these two targets. Docking study showed the importance of both hydrogen bonding and hydrophobic interactions as a key interaction with the targets. Based on the obtained results, the hybrid derivatives of triazine and phthalimide could be regarded as efficient candidates for further molecular developments of antimicrobial agents.

Combination Therapy of Metadichol Nanogel and Lipocalin-2 Engineered Mesenchymal Stem Cells Improve Wound Healing in Rat Model of Excision Injury.

Purpose: Currently, several disorders including burns, trauma, excisional and diabetic wounds, and bedsores threaten the human health. Application of mesenchymal stem cells (MSCs) is recommended for treatment of skin disorders. However, because of oxidative stress and inflammation after skin injury, survival of transplanted MSCs is low which in turn negatively affects the efficiency of the MSCs-based therapy. In an attempt to address the aforementioned challenge and introducing a novel potential therapeutic strategy, we employed combination therapy by lipocalin 2 (Lcn2)-engineered MSCs and a Metadichol (an inverse agonist of vitamin D receptor (VDR)) nanogel in a rat model of excisional wound. Methods: First, human umbilical cord MSCs (hUC-MSCs) was transfected by a recombinant plasmid encoding Lcn2 gene. Next, a combination of Metadichol nanogel and the engineered MSCs was co-applied on wound in rat model of excision injury. Finally the improvement of wound healing in experimental groups was evaluated by photography and histological assessments (hematoxylin and eosin staining). Results: Our findings revealed that the repair rate was higher in the group received combination therapy comparing to control groups. Notably, Metadichol+Lcn2-MSCs showed significantly higher wound contraction rate compared to control group at all time points (P value < 0.001). Furthermore, wound repair rate was 95% 14 days after surgery, and 100% after 21 days in the treatment groups. Our results also revealed that the combination therapy improved and accelerated the wound healing process. Conclusion: Our findings suggest a novel potential therapeutic strategy i.e. Lcn2-engineered MSCs and Metadichol for wound healing. However, further preclinical and clinical studies are required.

SA/G hydrogel containing NRF2-engineered HEK-293-derived CM improves wound healing efficacy of WJ-MSCs in a rat model of excision injury.

AIM OF THE STUDY: Skin wounds are a major public health issue due to the lack of real effective remedies. Mesenchymal stem cells (MSCs) are considered as a promising therapeutic strategy for wound injuries; however, low survival rate following transplantation limited their application. In an attempt to introduce a novel potential wound dressing and improve wound healing properties, the current study was conducted. MATERIAL AND METHODS: we prepared conditioned medium (CM) harvested from HEK-293 cells overexpressing nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant genes expression. Then, the CM was loaded in a biodegradable hydrogel. Next, in an animal model of full-thickness excision wound, wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) were transplanted at the margins of the wound followed by application of the hydrogel on injury site. Finally, wound healing characteristics were evaluated by proper methods. RESULTS: Our findings revealed that, the NRF2-CM protected the WJ-MSCs against H2O2-induced toxicity in vitro. Furthermore, in vivo results showed that, SA/G hydrogel containing NRF2-CM significantly (P < 0.01) promoted WJ-MSCs survival, increased angiogenesis, accelerated wound contraction, and promoted wound healing compared to other groups. CONCLUSION: Though further preclinical and clinical studies regarding mechanisms behind the protection and also safety of the strategy are needed, our findings strongly suggest that the prepared wound dressing enhanced the efficacy of therapeutic potential of WJ-MSCs by providing an enriched/antioxidant niche support.

Expressing of Recombinant VEGFR2-specific Nanobody in Baculovirus Expression System.

BACKGROUND: Baculovirus expression system, introduced more than 20 years ago, is considered as a useful tool for large and complex eukaryotic recombinant protein production. A baculovirus expression vector is a recombinant virus which desired foreign protein coding sequences is under control of the virus gene promoter. Baculovirus only infects insect cells and do not normally infect vertebrates therefore, they possess no risk of biological risks for human. OBJECTIVES: The aim of this study was to recombinant expression of vascular endothelial growth factor (VEGF) reseptor-2 specific Nanobody in the baculovirus expression system. MATERIALS AND METHODS: Gene of specific Nanobody against the VEGF reseptor-2 that called 3VGR19 was cloned and expressed in baculovirus system. RESULTS: 3VGR19 Nanobody gene was amplified by Polymerase Chain Reaction (PCR) using the specific primers, and was cloned in pFastBac HTA plasmid. DH10Bac bacteria was transformed with resulted donor plasmid. The cultured Sf9 insect cell line was transfected with recombinant bacmid, and finally, the expression and purification of 3VGR19 was confirmed in insect cells. CONCLUSIONS: In conclusion, Transient infection of insect cells with baculovirus can be a promising technology for expression of antibody fragments.

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.

Design and production of new chimeric reteplase with enhanced fibrin affinity: a theoretical and experimental study.

Plasminogen activators (PAs) are widely used for treatment of disorders caused by clot formation. Fibrin specific PAs are safe drugs from this group because of reducing the incidence of hemorrhage. The newer generation of PAs like tenecteplase, reteplase and desmoteplase were designed with the aim of achieving desirable properties such as improving specificity and affinity to fibrin and increasing half-life. Protein engineering and using of theoretical methods can help to rational and reliable design of new PAs with a set of favorable properties. In the present study, two new chimeric reteplase named M1-chr and M2-chr were designed with the aim of enhancing fibrin affinity also some potential properties include of increasing resistance to plasminogen activator inhibitor-1 and decreasing neurotoxicity. So, finger domain of desmoteplase was added to reteplase as a high fibrin specific domain. Some other point mutations were considering to achieve other mentioned properties. Three dimensional structure of wild-type reteplase and mutants were created by homology modeling and were evaluated by molecular dynamic simulation. Then, mutants docked to fibrin by HADDOCK web tools. Result of theoretical section verified the stability of mutants' structures. Also showed better interaction between M1-chr with fibrin than M2-chr. Wild-type and mutants were produced in bacterial expression system. Experimental assessment showed both mutants have appropriate enzymatic activity also 1.9-fold fibrin binding ability compared to wild-type. Therefore, this study offers new thrombolytic drugs with desirable properties specially enhanced fibrin affinity so they can represent a promising future in cost-effective production of favorable thrombolytic drugs.Communicated by Ramaswamy H. Sarma.

Pseudomonas Exotoxin-Based Immunotoxins: Over Three Decades of Efforts on Targeting Cancer Cells With the Toxin.

Cancer is one of the prominent causes of death worldwide. Despite the existence of various modalities for cancer treatment, many types of cancer remain uncured or develop resistance to therapeutic strategies. Furthermore, almost all chemotherapeutics cause a range of side effects because they affect normal cells in addition to malignant cells. Therefore, the development of novel therapeutic agents that are targeted specifically toward cancer cells is indispensable. Immunotoxins (ITs) are a class of tumor cell-targeted fusion proteins consisting of both a targeting moiety and a toxic moiety. The targeting moiety is usually an antibody/antibody fragment or a ligand of the immune system that can bind an antigen or receptor that is only expressed or overexpressed by cancer cells but not normal cells. The toxic moiety is usually a protein toxin (or derivative) of animal, plant, insect, or bacterial origin. To date, three ITs have gained Food and Drug Administration (FDA) approval for human use, including denileukin diftitox (FDA approval: 1999), tagraxofusp (FDA approval: 2018), and moxetumomab pasudotox (FDA approval: 2018). All of these ITs take advantage of bacterial protein toxins. The toxic moiety of the first two ITs is a truncated form of diphtheria toxin, and the third is a derivative of Pseudomonas exotoxin (PE). There is a growing list of ITs using PE, or its derivatives, being evaluated preclinically or clinically. Here, we will review these ITs to highlight the advances in PE-based anticancer strategies, as well as review the targeting moieties that are used to reduce the non-specific destruction of non-cancerous cells. Although we tried to be as comprehensive as possible, we have limited our review to those ITs that have proceeded to clinical trials and are still under active clinical evaluation.

Conditioned medium harvested from Hif1α engineered mesenchymal stem cells ameliorates LAD-occlusion -induced injury in rat acute myocardial ischemia model.

Acute myocardial infarction (AMI) is the most common type of ischemic heart diseases with a high mortality rate. Although recent advances in medical cares and therapies have increased the patient's outcomes, but, still there is no real and effective therapeutic modality for AMI. Hence, development of novel therapeutic strategies is under focus of investigations. MSCs-based therapy has been proposed for AMI, though its efficacy is controversial yet. It is believed that MSCs exert their healing effects via secretion of growth factors/cytokines. However, these cells produce a very minute amount of the factors under normal cultivation. Here, in an attempt to improve the potential therapeutic effect of MSCs-derived conditioned medium (CM) on AMI, we transfected the cells with a recombinant plasmid encoding Hif1α-3A (a mutant form of Hif1α stable under normoxic condition), so Hif1α expression and secretion into CM (MSCs-Hif1α-CM) could be up-regulated under normoxic condition. The therapeutic potential of the MSCs-Hif1α-3A-CM was investigated in a rat model of AMI and compared to the CM harvested from non-manipulated MSCs. Our results showed that the MSCs-Hif1α-3A-CM mitigated MI-induced tissues injury, decreased fibrosis, reduced apoptosis, and limited infarct area size. These findings propose a potential therapeutic strategy for treatment of AMI. However, further preclinical and clinical investigations in this regard are still needed.

Optimization of solvent media to solubilize TEV protease using response surface method.

BACKGROUND AND PURPOSE: Tobacco etch virus (TEV) protease, a 27 KDa protein, consists of the catalytic domain of nuclear inclusion a (NIa) protein produced by Tobacco etch virus. Because of its unique sequence, TEV protease is used for purging fusion tags from proteins. It also has many advantages such as stability and activity in a board range of temperature and pH and overproduction in Escherichia coli and these benefits make this protease valuable. Despite all these benefits, TEV protease has problems like low solubility (less than 1 mg/mL). There are methods for enhancing protein solubility and in this study, the effect of additives during cell lysis was studied. EXPERIMENTAL APPROACH: Eleven different additives that made twelve different lysis buffers were used and their effect on TEV protease solubility analyzed by Plackett-Burman and response surface methodology methods. FINDINGS / RESULTS: Three best effective additives on TEV solubility (L-proline, sodium selenite, and CuCl2) were selected according to software analysis and the best concentration of them was applied to optimize TEV protease solubility. CONCLUSION AND IMPLICATIONS: The obtained results provided the composition of an optimum solvent for obtaining soluble TEV protease.

Mesenchymal stem cells-derived mitochondria transplantation mitigates I/R-induced injury, abolishes I/R-induced apoptosis, and restores motor function in acute ischemia stroke rat model.

Acute ischemia stroke (AIS) is one of the leading causes of mortality and disability worldwide, and its neurological impacts are devastating and permanent. There is no efficient and real treatment for acute ischemia stroke so far. Therefore, development of efficient therapeutic strategies is under focus of investigations by basic and clinical scientists. Brain is one of the organs with high energy consumption and metabolism. Hence, its functionality is highly dependent on mitochondrial activity and integrity. Therefore, mitochondria play a vital homeostatic role in neurons physiology and mitochondrial dysfunction implications have been reported in a variety of nervous system diseases including acute ischemia stroke. In an attempt to investigate and introduce a novel potential therapeutic strategy for AIS, we isolated healthy mitochondria from human umbilical cord derived mesenchymal stem cells (hUC-MSCs) followed by their intracerebroventricular transplantation in a rat model of ischemia, i.e. middle cerebral artery occlusion (MCAO). Here we report that the mitochondrial transplantation ameliorated the reperfusion/ischemia-induced damages as reflected by declined blood creatine phosphokinase level, abolished apoptosis, decreased astroglyosis and microglia activation, reduced infarct size, and improved motor function. Although further preclinical and clinical studies are required, our findings strongly suggest that transplantation of MSCs-derived mitochondria is a suitable, potential and efficient therapeutic option for acute ischemia stroke.

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains.

BACKGROUND AND PURPOSE: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli (E. coli) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and Origami™ 2 (DE3). EXPERIMENTAL APPROACH: The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method. FINDINGS / RESULTS: A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl ß- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 104 IU/µg was obtained. CONCLUSION AND IMPLICATIONS: The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties (i.e., containing disulfide binds or cysteine).

Transplantation of Umbilical Cord-Derived Mesenchymal Stem Cells Overexpressing Lipocalin 2 Ameliorates Ischemia-Induced Injury and Reduces Apoptotic Death in a Rat Acute Myocardial Infarction Model.

Myocardial infarction (MI) is a leading cause of death worldwide and requires development of efficient therapeutic strategies . Mesenchymal stem cells (MSCs) -based therapy of MI has been promising but inefficient due to undesirable microenvironment of the infarct tissue. Hence, the current study was conducted to fortify MSCs against the unfavorable microenvironment of infarct tissue via overexpression of Lipocalin 2 (Lcn2) as a cytoprotective factor. The engineered cells (Lcn2-MSCs) were transplanted to infarcted heart of a rat model of MI. According to our findings, Lcn2 overexpression resulted in increased MSCs survival in the MI tissue (p < 0.05) compared to non-engineered cells. Furthermore, the infusion of Lcn2-MSCs mitigated Left ventricle (LV) remodeling, decreased fibrosis (p < 0.0001), and reduced apoptotic death of the LVs' cells (p < 0.0001) compared to the control. Our findings suggest a potential novel therapeutic strategy for MI, however, further investigations such as safety and efficacy assessments in large animals followed by clinical trials are required.

In silico design of two novel fusion proteins, p28-IL-24 and p28-M4, targeted to breast cancer cells.

BACKGROUND AND PURPOSE: An anticancer peptide P28, has shown to be cytolethal on various cancer cells including breast cancer. Moreover, p28 can be also used as a targeting moiety in the structure of fusion proteins. IL-24 (or its truncated form, M4) is a cytokine with anticancer activity against a wide range of tumor cells. We aimed at production of a fusion protein consisted of p28 and either IL-24 or M4 to target breast cancer. However, selection of a proper linker to join the two moieties without intervening each other's function is a key factor in the construction of fusion proteins. In the present study, the impact of different linkers on construction of the two chimeric proteins (p28-IL-24 and p28-M4) was assessed in silico. EXPERIMENTAL APPROACH: After selection of some linkers with different lengths and characteristics, a small library of the chimeric proteins was created and assessed. Furthermore, following selection of the most suitable linker, the three-dimensional structures and dynamic behavior of both fusion proteins were evaluated by homology modeling and molecular dynamic simulation, respectively. FINDINGS / RESULTS: Based on the results, a rigid linker having the peptide sequences of AEAAAKEAAAKA showed highest freedom of action for both moieties. CONCLUSION AND IMPLICATIONS: Between the p28-IL-24 and p28-M4 fusion proteins, the former showed better stability as well as solubility and might show stronger anticancer effects in vitro and in vivo, because its peptide moieties showed to exert their activities freely.

Mitochondrial transplantation ameliorates ischemia/reperfusion-induced kidney injury in rat.

No real therapeutic modality is currently available for Acute kidney injury (AKI) and if any, they are mainly supportive in nature. Therefore, developing a new therapeutic strategy is crucial. Mitochondrial dysfunction proved to be a key contributor to renal tubular cell death during AKI. Thus, replacement or augmentation of damaged mitochondria could be a proper target in AKI treatment. Here, in an animal model of AKI, we auto-transplanted normal mitochondria isolated from healthy muscle cells to injured kidney cells through injection to renal artery. The mitochondria transplantation prevented renal tubular cell death, restored renal function, ameliorated kidney damage, improved regenerative potential of renal tubules, and decreased ischemia/reperfusion-induced apoptosis. Although further studies including clinical trials are required in this regard, our findings suggest a novel therapeutic strategy for treatment of AKI. Improved quality of life of patients suffering from renal failure and decreased morbidity and mortality rates would be the potential advantages of this therapeutic strategy.

Transfer of healthy fibroblast-derived mitochondria to HeLa ρ0 and SAS ρ0 cells recovers the proliferation capabilities of these cancer cells under conventional culture medium, but increase their sensitivity to cisplatin-induced apoptotic death.

Mitochondrial dysfunction is known to contribute to cancer initiation, progression, and chemo-and radio-resistance. However, the precise role of mitochondria in cancer is controversial. Hence, here we tried to further clarify the role of mitochondria in cancer by transferring healthy mitochondria to cancer cells, and also to cells with depleted mitochondrial DNA (ρ0). Healthy mitochondria were isolated from WI-38 cells and were transferred to HeLa, SAS, HeLa ρ0, and SAS ρ0 cells. Then, cell proliferation was verified. In addition, the cells were treated by different concentrations of cisplatin and assessed for apoptosis induction and quantifying the mRNA expression of apoptosis-related genes. Results revealed that incubation of the HeLa, SAS and HeLa ρ0 cells with 5 µg/ml of the isolated mitochondria for 24 h significantly (p < 0.001) increased cell proliferation compared to non-treated controls. Interestingly, the mitochondria transfer rescued the ρ0 cells and made them capable of growing under conventional culture medium. However, the number of apoptotic cells was significantly higher in the HeLa ρ0 cells that received the mitochondria (HeLa-Fibro-Mit) compared to the HeLa ρ0. Furthermore, the expression level of BCL-2 anti-apoptotic gene was down-regulated in both HeLa-Fibro-Mit and SAS-Fibro-Mit cell lines while the expression levels of the BAX, caspase8, caspase9, and AIF pro-apoptotic genes were upregulated. Our findings indicated that although the response of cancer cells to the mitochondria transfer is cancer-type dependent, but the introduction of normal exogenous mitochondria to some cancer cells might be considered as a potential novel therapeutic strategy.

Expression and one step intein-mediated purification of biologically active human G-CSF in Escherichia coli.

Recombinant form of granulocyte colony stimulating factor (G-CSF) was first approved by FDA in 1998 for chemotherapy induced neutropenia. However, despite production of its biosimilars, less expensive production of G-CSF could reduce the overall therapeutic cost. The aim of this study was to evaluate the possibility of producing biologically active recombinant G-CSF via a single step purification procedure mediated by a self-cleavable intein. G-CSF was expressed by E. coli BL21 (DE3) through IPTG induction, followed by its purification using pH optimization on a chitin column. Western blotting, ELISA, size exclusion chromatography, circular diachorism, peptide mapping, and in vitro assays were performed to compare the structural similarity and biological activity of the purified G-CSF with Neupogen™. Protein purification was confirmed by revealing a band of approximately 18.8 kDa on SDS-PAGE. Bioactivity and physicochemical assays based on the US pharmacopeia showed almost identical or acceptable ranges of similarities between recombinant G-CSF and Neopogen™. this study, biologically active soluble recombinant G-CSF was successfully produced with high purity without using chaotropic solvents through a one-step procedure. This shorter and more efficient purification procedure can reduce the cost and time of G-CSF production which makes its industrial production more cost-effective and might be also applicable for production of other biopharmaceuticals.

RGD peptide grafted polybutylene adipate-co-terephthalate/gelatin electrospun nanofibers loaded with a matrix metalloproteinase inhibitor drug for alleviating of wounds: an in vitro/in vivo study.

The objective of the present study was the fabrication of a wound dressing membrane based on RGD modified polybutylene adipate-co-terephthalate (PBAT)/gelatin nanofibrous structures loaded with doxycycline (DOX). This type of nanofiber for wound healing has not been reported so far and is quite novel. PBAT and gelatin nanofibers were separately electrospun using double needles electrospinning setup. Electrospinning variables were optimized to obtain bead-free thin nanofibers. The amount of drug loaded and release were measured in different concentrations of DOX and PBAT. MMPs inhibition was studied by polyacrylamide gel-zymography. Then, surface of the nanofibers was modified with RGD peptide, and their antimicrobial effect was investigated on Staphylococcus aureus and Pseudomonas aeruginosa. Effect of developed nanofibrous membranes on L929 fibroblast cells proliferation, adhesion and closure of excised wounds in rat were also studied. PBAT/gelatin nanofibrous structures with average fiber diameter of 75-529 nm were developed successfully. Drug release study revealed that about 65% of DOX was released from the optimized formulation (P17D1.6) after 20 h. The developed DOX loaded membrane inhibited the MMPs activity and showed no cytotoxicity. RGD surface-modified PBAT/gelatin nanofibers significantly improved the wound closure and histopathological results (re-epithelialization, collagen deposition, and angiogenesis) in rats compared to the control groups. Overall, RGD immobilized PBAT/gelatin nanofibrous membrane may have a potential application for wound healing.