Optimization of plasmid electrotransformation into Bacillus subtilis using an antibacterial peptide.

Bacillus subtilis can potentially serve as an efficient expression host for biotechnology due to its ability to secrete extracellular proteins and enzymes directly into the culture medium. One of the important challenges in the biotechnology industry is to optimize the transformation conditions of B. subtilis bacteria. This study aims to provide a new method to optimize the transformation conditions and improve the transformation efficiency of B. subtilis WB600. To increase the transformation efficiency in B. subtilis, two methods of adding CM11 antibacterial peptides to the bacterial medium along with electroporation and optimizing the variables including the growth medium composition, time to adding CM11 peptide, electroporation voltage, recovery medium, and cell recovery time are used. The results of this study showed that the addition of antimicrobial peptides (AMPs) with a concentration of 2 µg/ml increases the transformation efficiency by 4 times compared to the absence of AMP in the bacterial medium. Additionally, the findings from our study indicated that the most optimal rate of transformation for B. subtilis was observed at a voltage of 7.5 kV/cm, with a recovery period of 12 h. With the optimized method, the transformation efficiency came up to 1.69 × 104 CFU/µg DNA. This improvement in transformation efficiency will be attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for transformation of wild-type B. subtilis strains.

Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

BACKGROUND: Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. METHODS AND RESULTS: First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. CONCLUSIONS: In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Schizophyllan promotes osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

BACKGROUND: Bioactive polysaccharides are a promising way for bone disease prevention with high efficiency. Schizophyllan (SPG) is a polysaccharide derived from a species of fungus with anticancer, antitumor, and anti-inflammatory effects. In the present study, for the first time, the cell proliferation, osteogenic markers, mineral deposition, and osteogenic gene expression of human adipose tissue-derived mesenchymal stem cells (hADMSCs) grown on SPG were evaluated by in vitro assays. METHODS AND RESULTS: The cytotoxicity of SPG was measured using the MTT assay and acridine orange staining. Differentiation of hADMSCs was assessed using alkaline phosphatase (ALP) activity test, cellular calcium content assay, and mineralized matrix staining. To this end, Alizarin red S, von Kossa staining, and the expression of bone-specific markers, including ALP, Runx2, and osteonectin, were used by real-time RT-PCR over a 2-week period. According to the results, SPG at 10 µg/ml concentration was determined as the optimal dosage for differentiation studies. The results of osteogenic differentiation tests showed that compared to the control groups in vitro, SPG enhanced the osteogenic markers and mineralization as well as upregulation of the expression of bone specific genes in differentiated hADMSCs during differentiation. CONCLUSIONS: The results revealed that SPG could be applied as effective factor for osteogenic differentiation in the future. The current study provides insights into the hADMSC-based treatment and introduces promising therapeutic material for individuals who suffer from bone defects and injuries.

Osteogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells on Composite Polymeric Scaffolds: A Review.

The mesenchymal stem cells (MSCs) are the fundamental part of bone tissue engineering for the emergence of reconstructive medicine. Bone tissue engineering has recently been considered a promising strategy for treating bone diseases and disorders. The technique needs a scaffold to provide an environment for cell attachment to maintain cell function and a rich source of stem cells combined with appropriate growth factors. MSCs can be isolated from adipose tissue (ASCs), bone marrow (BM-MSCs), or umbilical cord (UC-MSCs). In the present study, the potential of ASCs to stimulate bone formation in composite polymeric scaffolds was discussed and it showed that ASCs have osteogenic ability in vitro. The results also indicated that the ASCs have the potential for rapid growth, easier adipose tissue harvesting with fewer donor site complications and high proliferative capacity. The osteogenic differentiation capacity of ASCs varies due to the culture medium and the addition of factors that can change signaling pathways to increase bone differentiation. Furthermore, gene expression analysis has a significant impact on improving our understanding of the molecular pathways involved in ASCs and, thus, osteogenic differentiation. Adding some drugs, such as dexamethasone, to the biomaterial composite also increases the formation of osteocytes. Combining ASCs with scaffolds synthesized from natural and synthetic polymers seems to be an effective strategy for bone regeneration. Applying exopolysaccharides, such as schizophyllan, chitosan, gelatin, and alginate in composite scaffolds enhances the osteogenesis potential of ASCs in bone tissue regeneration.

Potential therapeutic effects and nano-based delivery systems of mesenchymal stem cells and their isolated exosomes to alleviate acute respiratory distress syndrome caused by COVID-19.

The severe respiratory effects of the coronavirus disease 2019 (COVID-19) pandemic have necessitated the immediate development of novel treatments. The majority of COVID-19-related fatalities are due to acute respiratory distress syndrome (ARDS). Consequently, this virus causes massive and aberrant inflammatory conditions, which must be promptly managed. Severe respiratory disorders, notably ARDS and acute lung injury (ALI), may be treated safely and effectively using cell-based treatments, mostly employing mesenchymal stem cells (MSCs). Since the high potential of these cells was identified, a great deal of research has been conducted on their use in regenerative medicine and complementary medicine. Multiple investigations have demonstrated that MSCs and their products, especially exosomes, inhibit inflammation. Exosomes serve a critical function in intercellular communication by transporting molecular cargo from donor cells to receiver cells. MSCs and their derived exosomes (MSCs/MSC-exosomes) may improve lung permeability, microbial and alveolar fluid clearance, and epithelial and endothelial repair, according to recent studies. This review focuses on COVID-19-related ARDS clinical studies involving MSCs/MSC-exosomes. We also investigated the utilization of Nano-delivery strategies for MSCs/MSC-exosomes and anti-inflammatory agents to enhance COVID-19 treatment.

Carbachol, along with calcium, indicates new strategy in neural differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

Introduction: Over the past few years, stem cells have represented a promising treatment in neurological disorders due to the well-defined characteristics of their capability to proliferate and differentiate into any cell type, both in vitro and in vivo. Additionally, previous studies have shown that calcium signaling modulates the proliferation and differentiation of neural progenitor cells. The present study investigated the effect of carbachol (CCh), a cholinergic agonist activating acetylcholine receptors, with and without calcium, on the neural differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) in neural media, including forskolin and 3-isobutyl-1-methyl-xanthine and retinoic acid. Methods: For this purpose, first, the MTT assay and acridine orange staining were studied to obtain the optimal concentration of CCh. Next, the differentiation tests, such as cellular calcium assay as well as evaluation of qualitative and quantitative expression of neuronal index markers through immunofluorescence staining and gene expression analysis, respectively, were performed on days 7 and 14 of the differentiation period. Results: According to the results, CCh at 1 µM concentration had no cytotoxicity on hADSCs and also induced cell proliferation. Furthermore, CCh with and without calcium increased the expression of neural-specific genes (NSE, MAP2, ß-III-tubulin, and MAPK3) and proteins (γ-enolase, MAP2, and ß-III-tubulin) as well as the amount of calcium in differentiated hADSCs at 7 and 14 days after induction. Conclusions: In conclusion, the findings suggest that CCh acts as an influential therapeutic factor in the field of neural regenerative medicine and research.

A Novel Nanocomposite Scaffold Based on Polyurethane (PU) Containing Cobalt Nanoparticles (CoNPs) for Bone Tissue Engineering Applications.

BACKGROUND: Bone tissue engineering, as a relatively new approach, has focused on combining biodegradable scaffolds, cells, and biologically active molecules for the recovery of different damaged tissues, such as bone defects. Polyurethane (PU), as a synthetic polymer, benefits from a porous structure which impersonates bone's natural environment. However, PU lacks osteoinduction activities. Cobalt nanoparticles (CoNPs) stimulate angiogenesis and biomineralization, which greatly favors osteogenesis. METHODS: Here, we designed a novel scaffold based on PU and combined it with CoNPs for bone regeneration applications. The composition and structure of PU-CoNPs nanocomposite were characterized using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). MTT and AO data showed biocompatibility and enhanced viability and proliferation of fibroblasts on PU-CoNPs scaffold. Ascorbic acid-2-phosphate, ß-glycerophosphate, and dexamethasone-induced osteogenesis for 14 days. RESULTS: The alkaline phosphatase test asserts the increased mineralization of hADSCs cultured on PUCoNPs compared to pure PU scaffold. Further, the results disclosed an elevated osteogenic differentiation at the level of genes and proteins using immunocytochemical analysis (ICC) and quantitative real-time PCR (qPCR). CONCLUSION: These findings provide an evidence that PU-CoNPs nanocomposite might be a promising candidate for bone repair applications.

Combination of natural scaffolds and conditional medium to induce the differentiation of adipose-derived mesenchymal stem cells into keratocyte-like cells and its safety evaluation in the animal cornea.

Keratocytes are the main cellular components of the corneal stroma. This cell is quiescent and cannot be cultured easily. The aim of this study was to investigate differentiate human adipose mesenchymal stem cells (hADSCs) into corneal keratocyte cells by combining natural scaffolds and conditioned medium (CM) and evaluating their safety in the rabbit's cornea. Keratocytes were cultured in an optimal culture medium and this medium was collected and kept as a CM. hADSCs were cultured on the decellularized human small incision lenticule extraction (SMILE) lenticule (SL), amniotic membrane (AM), and collagen-coated plates, and were exposed to keratocyte-CM (KCM) for 7, 14, and 21 days. Differentiation was evaluated using Real-time PCR and immunocytochemistry (ICC). hADSCs were cultured on the SL scaffolds and implanted in the corneal stroma of 8 New Zealand male rabbits. Rabbits were followed for 3 months and the safety was evaluated by clinical and histological variables. Real-time PCR results showed a significant increase in the expression of keratocyte-specific markers on the 21 day of differentiation compared to the control group. ICC also confirmed the induction of differentiation. Implantation of SLs containing differentiated cells in the cornea of animals showed no serious complications including neovascularization, corneal opacity, inflammation, or signs of tissue rejection. Furthermore, the evaluation of the presence of keratocyte-like cells after three months in the rabbit stroma was confirmed by Real-time PCR and immunohistochemistry (IHC) analysis. Our results showed that combination of combination of corneal extracellular matrix and KCM can induced keratocytes differentiation of hADSC and can be introduced as a alternative method to supply the required keratocytes in corneal tissue engineering.

Immunosenescence and inflamm-ageing in COVID-19.

The destructive effects of coronavirus disease 2019 (COVID-19) on the elderly and people with cardiovascular disease have been proven. New findings shed light on the role of aging pathways on life span and health age. New therapies that focus on aging-related pathways may positively impact the treatment of this acute respiratory infection. Using new therapies that boost the level of the immune system can support the elderly with co-morbidities against the acute form of COVID-19. This article discusses the effect of the aging immune system against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the pathways affecting this severity of infection.

Neoadjuvant VS adjuvant chemotherapy in patients with locally advanced breast cancer; a retrospective cohort study.

Background: Breast cancer is one of the most common challenges for women's health. Until now, neoadjuvant chemotherapy is a standard approach in locally advanced breast cancer (LABC), as it increases the probability of breast-conserving surgery (BCS). This study aimed to compare the survival rate in neoadjuvant and adjuvant groups to suggest a better treatment strategy for locally advanced breast cancer. Methods: The study was conducted between 2009 and 2019 on 845 LABC patients at the Cancer Research Center of Shahid Beheshti University of Medical Sciences in Iran. All patients with LABC at stages 3A, 3B, and two were evaluated for treatment with adjuvants (n = 520 female patients) and neoadjuvant (n = 320 female patients) treatment strategies. Patients were followed up for at least 120 months. The Kaplan-Meier method calculated the survival rate using SPSS version 23 software. Result: The 5 and 10 years survival rates of neoadjuvant and adjuvant groups were 87 ± 0.04, 80 ± 0.07% and 87 ± 0.02, 83 ± 0.03%, respectively. Statistical analysis results with the mentioned treatment strategies did not show any significant difference in overall survival. Conclusion: The result of this study on LABC patients demonstrated that compared to surgery first following adjuvant chemotherapy, the neoadjuvant chemotherapy has several benefits, including downstaging and more BCS, with no statistically significant difference in the overall survival rate of the patients.

Application of mesenchymal stem cells in corneal regeneration.

Due to delicate its structure, the cornea is susceptible to physical, chemical, and genetic damages. Corneal transplantation is the main treatment for serious corneal damage, but it faces significant challenges, including donor shortages and severe complications. In recent years, cell therapy is suggested as a novel alternative method for corneal regeneration. Regarding the unique characteristics of Mesenchymal stem cells including the potential to differentiate into discrete cell types, secretion of growth factors, mobilization potency, and availability from different sources; special attention has been paid to these cells in corneal engineering. Differentiation of MSCs into specialized corneal cells such as keratocytes, epithelial and endothelial cells is reported. Potential for Treatment of keratitis, reducing inflammation, and inhibition of neovascularization by MSCs, introducing them as novel agents for corneal repairing. In this review, various types of MSCs used to treat corneal injuries as well as their potential for restoring different corneal layers was investigated.