Search

Background and Objective: Hemophilia A is the most common type of this disease. For the treatment of hemophiliacs, the injectable concentrate of human factor VIII, derived from plasma and recombinant, is available in the market. Due to the risk of contracting infectious diseases due to blood transfusions or donated blood products, including plasma-driven factor VIII, recombinant factor VIII can be used, which is safe and has a higher purity than the blood-derived sample, while showing a longer half-life in the blood of consuming patients. The aim of this study is to evaluate the expression and synthesis of recombinant human factor VIII using a cell-free eukaryotic expression system as a new strategy for the synthesis of recombinant drugs. Methods: In this experimental study, pcDNA3 vector containing the gene fragment without B-domain of blood coagulation factor VIII was transformed into susceptible DH5a bacteria. After the multiplication of the bacteria and the increase of the target vector, the amplified vector was extracted at this stage and its quality and concentration were determined. Then CHO-K1 eukaryotic cells were cultured and cell extract was prepared from the density of more than ten million of their cells. The extracted vector was combined with cell extract and expression solution, and the expression process was carried out at 30°C and 37°C. The level of expression was evaluated by ELISA and dot blot techniques. Findings: Based on the ELISA technique adopted in this study, the expression level of recombinant human blood coagulation factor VIII in the base sample at a temperature of 30°C, sample 2 at a temperature of 30°C, base sample at a temperature of 37°C, and sample 4 of negative control at a temperature of 30°C were respectively reported as 275, 276, 273 and 0 ng/ml in a period of 20 hours. In the dot blot technique, the mentioned quantitative results were qualitatively confirmed by the color intensity of the spots. Conclusion: The results showed that the cell-free expression method can be used for the synthesis of recombinant factor VIII. Under the conditions used in this research for cell-free expression of recombinant factor VIII, volume ratio of 50/50 of CHO-K1 cell extract was suitable, and the basic expression solution at 30°C showed the highest concentration of the product. Due to the use of CHOK1 eukaryotic cell extract, the synthesized recombinant human factor VIII has post-translational modifications, which are very important for the functionality of this protein in the human body. [ABSTRACT FROM AUTHOR]

Background: Klebsiella pneumoniae is a clinically relevant pathogen that has raised considerable public health concerns. This study aims to determine the presence of beta-lactamase genes and perform molecular genotyping of multidrug-resistant (MDR) K. pneumoniae clinical isolates., Methods: Clinical isolates of MDR K. pneumoniae were collected from educational hospitals affiliated with Babol University of Medical Sciences. The isolates of K. pneumoniae were identified through standard microbial and biochemical tests. Antibiotic resistance was assessed using disk diffusion, modified Hodge test (MHT), combined disk, and polymerase chain reaction (PCR) methods. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was performed for molecular typing., Results: A total of 42 MDR K. pneumoniae isolates were obtained from various clinical specimens. The highest antibiotic resistance was observed for ampicillin (100%), while the lowest resistance was noted for amikacin (19.04%). The MHT indicated that 38.09% of K. pneumoniae isolates produced carbapenemase enzymes. Metallo-beta-lactamase (MBL) production was found in 54.76% of isolates. Molecular detection of beta-lactamase genes revealed the presence of bla NDM (21.42%), bla KPC (42.85%), bla TEM (76.19%), bla SHV (47.16%), and bla CTX-M (80.95%) genes. ERIC-PCR molecular typing identified seven distinct genetic patterns among the isolates., Conclusions: This investigation demonstrates the high resistance levels of K. pneumoniae strains. The beta-lactamase genes with the highest and lowest frequencies correspond to bla CTX-M and bla NDM genes, respectively. ERIC-PCR dendrograms suggest a common origin for K. pneumoniae clinical isolates and the propagation of similar clones within hospital wards. These findings indicate that K. pneumoniae isolates are highly virulent, necessitating the development of more effective resistance-fighting techniques and gene transfer research., Clinical Trial Number: Not applicable., Competing Interests: Declarations. Ethics approval and consent to participate: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Islamic Azad University - Ayatollah Amoli Branch; Amol, Iran (2022-09-11/ IR.IAU.AMOL.REC.1401.087). Informed consent was obtained from all individual participants included in the study. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Antibiotic resistance is a significant global health concern, leading to increased morbidity, mortality, and health care costs. Integrons are genetic elements that could acquire and express gene cassettes, including those that confer antibiotic resistance. This comprehensive study focused on the distribution of integrons and their gene cassettes in clinical isolates. This study explored the structure and classification of integrons with particular emphasis on Class I, II, III, and IV integrons. It also discussed the role of integrons in antibiotic resistance. The findings of this study contribute to a better understanding of the mechanisms underlying antibiotic resistance and provide valuable insights for developing strategies to combat this public health crisis.

Gingival-derived mesenchymal stem cells (GMSCs) stand for a unique source of mesenchymal stem cells (MSCs) isolated from a neural crest origin with potential application in regenerative medicine. However, there are some limitations to the usage of these cells in clinical cell therapy such as reduced cell number and undesirable differentiation of the cell throughout frequent passages. Nowadays, studies have applied manipulation strategies to improve MSCs' effectiveness in clinical therapy. Among all of the materials used for this purpose, there is a growing trend for the use of biomaterials such as probiotic extracts or their conditioned media due to their lower toxicity. In the present study, we utilized extracts from Lactobacillus reuteri and Lactobacillus rhamnosus to assess their potential to enhance the function of GMSCs. We compared the effectiveness of these bacterial extracts to determine their relative efficacy. Bacterial extracts of two lactic acid bacteria were prepared using an ultrasonic homogenizing device. The impact of these bacterial extracts on GMSCs was evaluated through Alizarin Red and Oil Red O staining, cell counting by Trypan Blue staining, and real-time polymerase chain reaction. The findings of our study indicate that the administration of 50 μg/mL L. rhamnosus extract resulted in a greater enhancement of stemness marker expression, osteogenic differentiation, and proliferation of GMSCs compared with an equivalent concentration of L. reuteri extract. Neither of these bacterial extracts revealed any effect on the differentiation of the GMSCs into the adipogenic lineage. These findings suggest that L. rhamnosus extract could be more effective at promoting GMSCs' efficacy in tissue engineering and regenerative medicine.

The vagina hosts a community of microorganisms known as the vaginal microbiota. This community is relatively stable and straightforward, with Lactobacillus species being the most dominant members. The vaginal microbiota has various functions that are essential for maintaining human health and balance. For example, it can metabolise dietary nutrients, produce growth factors, communicate with other bacteria, modulate the immune system, and prevent the invasion of harmful pathogens. When the vaginal microbiota is disrupted, it can lead to diseases and infections. The observed disturbance is distinguished by a reduction in the prevalence of Lactobacillus and a concurrent rise in the number of other bacterial species that exhibit a higher tolerance to low oxygen levels. Gynecologic cancers are a group of cancers that affect the female reproductive organs and tissues, such as the ovaries, uterus, cervix, vagina, vulva, and endometrium. These cancers are a major global health problem for women. Understanding the complex interactions between the host and the vaginal microorganisms may provide new insights into the prevention and treatment of gynecologic cancers. This could improve the quality of life and health outcomes for women., (© 2024. The Author(s).)

Background: Coagulation factor VIII (FVIII) is applied for spontaneous hemorrhaging inhibition and excessive bleeding after trauma in patients with hemophilia A. High-quality human recombinant factor VIII (rFVIII) has been produced relatively in large quantities in cultured mammalian cells. NS0 is one of the most common mammalian cell lines for therapeutic protein production. Production of rFVIII has increased due to low FVIII expression levels and rising demand for hemophilia A prophylactic treatment. Several methods have been developed to prevent cell cycle progression in mammalian cells for increased recombinant protein yields., Objective: The aim of the study was to investigate the level of recombinant BDD-FVIII expression in NS0 mouse myeloma cells. Additionally, the study aimed to determine the effects of chemical drugs, Mitomycin C, Lovastatin, and Metformin on the secretion of FVIII through cell cycle arrest., Materials and Methods: We cultured NS0 cells and transfected them with the 2 μg pcDNA3-hBDDFVIII plasmid by Lipofectamine 3000. The cells were treated with 10 μg.mL -1 Mitomycin C, 20 μM Lovastatin, and 20 mM Metformin separately. After 24 and 48 hours, the samples were collected and, protein expression was analyzed using RT-PCR, Dot blot, and ELISA., Results: A higher protein expression level was observed in treated cells 24h and 48h after treatment with all three drugs. According to real-time PCR, Metformin treatment resulted in the highest expression level within 24 h (P=0.0026), followed by Mitomycin C treatment within 48 h (P=0.0030)., Conclusion: The NS0 cell line can be regarded as a suitable host for FVIII production. FVIII protein expression level was increased by using Lovastatin, Metformin, and Mitomycin C drugs. Further investigations are suggested, and the potential application of these drugs to increase recombinant protein yield can be used to produce therapeutic proteins in the industry., Competing Interests: The authors declare no conflict of interest, (Copyright: © 2021 The Author(s); Published by Iranian Journal of Biotechnology.)

Interleukin-2 (IL-2) is a cytokine that acts in dual and paradoxical ways in the immunotherapy of cancers and autoimmune diseases. Numerous clinical trial studies have shown that the use of different doses of this cytokine in various autoimmune diseases, transplantations, and cancers has resulted in therapeutic success. However, side effects of varying severity have been observed in patients. In recent years, to prevent these side effects, IL-2 has been engineered to bind more specifically to its receptors on the cell surface, decreasing IL-2 toxicities in patients. In this review article, we focus on some recent clinical trial studies and analyze them to determine the appropriate dose of IL-2 drug with the least toxicities. In addition, we discuss the engineering performed on IL-2, which shows that engineered IL-2 increases the specificity function of IL-2 and decreases its adverse effects.

Toxoplasma gondii is an intracellular protozoan parasite that causes neuroinflammation in the brain and a constant need for peripheral leukocyte migration. Matrix metalloproteinase 9 (MMP-9) can play a major role in this neuroinflammation and be implicated in some neurological disorders, such as migraines. Therefore, the genetic polymorphism evaluation of MMP-9 in migraine patients with T. gondii infection was performed. One hundred fourteen migraine patients and 114 healthy controls were evaluated for the presence of anti-Toxoplasma IgG antibodies. Seventy-two migraine patients and 40 healthy controls were randomly selected for assessment of the MMP 9-1562C/T genetic polymorphism. In the preliminary examination, 61 (53.5%) migraine patients and 43 (37.3%) healthy controls were positive for IgG antibodies, with a significant association between T. gondii seropositivity and migraine (OR = 1.90; 95% CI = 1.21-3.223; P = 0.012). Genetic distribution for the polymorphism was not in Hardy-Weinberg equilibrium in cases but showed no significant variation in control groups (P = 0.03 and P = 0.180, respectively). A significant preponderance of the CT + TT genotype was found in migraine subjects compared to controls (P = 0.042) (OR, 1.77, CI, 1.013-2.229). The homozygote muted allele TT had a higher rate in migraine patients (6.9%). There were significant differences in CT + TT genotype between T. gondii positive and negative migraine patients (P = 0.024), but T allele frequencies had no significant variation (OR 1.7 CI, 1.084-2.44 and 0.42 CI, 0.044-3.97, respectively). In conclusion, the results may provide the first evidence for the involvement of the MMP-9 gene polymorphism in the mechanism of migraine pathology following Toxoplasma infection., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Helicobacter pylori as a common gastrointestinal (GI) pathogen must possess certain virulence characteristics to colonize the stomach, evade host immune responses, and subsequently induce GI diseases. This research aimed to investigate the expression level of two important genes, the sialic acid-binding adherence ( SabA ) and the blood group antigen-binding adhesion ( BabA ) in H. pylori strains isolated from adult patients living in the northern part of Iran, and their association with peptic ulcer disease (PUD) and gastric cancer (GC). This cross-sectional study was carried out on adult patients referring to the GI clinic of the hospitals affiliated to Babol University of Medical Sciences, Iran. New cases diagnosed with gastritis, peptic ulcer or gastric cancer were included. Endoscopic-guided gastric biopsies were examined and H. pylori positive colonies were analyzed to determine the expression of babA and sabA genes, utilizing specific primers and the SYBR Green dye. Among 175 patients with mean age of 51.6±15.6 years, 101 (57.7%) of the individuals tested positive for H. pylori infection. Statistical analysis revealed a significant correlation between sabA (P=0.003) and babA (P=0.002) gene expression and development of PUD and GC. Smoking (P=0.052), gender (P=0.004) and positive babA gene expression (P=0.009) had the greatest association with occurrence of PUD or GC in H. pylori positive patients.  In summary, the presence of the sabA gene in people infected with H. pylori increased the risk of GC compared to gastritis, while, the presence of the babA gene was significantly increased in gastric ulcer patients. Considering the diversity of H. pylori isolates and the varying results observed in different geographical regions, further comprehensive studies are required to evaluate the function of these genes in H. pylori pathogenesis and their relationship with clinical outcomes., (© The Author(s).)

Background: A combination of bioceramics and polymeric materials has attracted the research community's interest in bone tissue engineering. These composites are essential to support cell attachment, proliferation, and osteogenesis differentiation, which are vital as a classic strategy in bone tissue engineering. In this study, NiFe2O4/ZnO-coated poly L-Lactide (PLLA) was employed as a scaffold to evaluate the osteogenic differentiation capability of human adipose tissue derived mesenchymal stem cells (hAMSCs). Material and methods: The electrospun PLLA nanofibers were fabricated, coated with nanocomposite (NiFe2O4/ZnO), and evaluated by the water contact angle (WCA), tensile test, attenuated total reflectance fourier-transform infrared (ATR-FTIR) and scanning electron microscopy (SEM). Then, the osteogenic differentiation potential of hAMSCs was assessed using NiFe2O4/ZnO-coated PLLA compared to tissue culture plastic (TCP) and a simple scaffold (PLLA) in vitro conditions. Results: The adhesion, proliferation, and differentiation of hAMSCs were supported by the mechanical and biological properties of the NiFe2O4/ZnO-coated PLLA scaffold, according to SEM and 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining patterns. During bone differentiation, Alkaline phosphatase (ALP) enzyme activity, biomineralization, calcium content, and osteogenic gene expression (ALP, Osteonectin, Osteocalcin, Collagen type I, and Runx2) were higher on NiFe2O4/ZnO-coated PLLA scaffold than on PLLA scaffold and TCP. Conclusion: Based on our results, the osteogenic differentiation of hAMSCs on the improved biological scaffold (PLLA coated with NiFe2O4/ZnO) could accelerate due to the stimulating effect of this nanocomposite., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shariati, Seyedjafari, Mahdavi, Maali and Ferdosi-Shahandashti.)