Molecular Cloning of Bone Morphogenetic Protein-2 (BMP- 2) in pGEM-b1 Vector and Transformation into E.coli Rosetta Strain

Background: Bone Morphogenetic Protein 2 (BMP- 2) belongs to the TGF-β superfamily of proteins and plays an important role in the development of bone and cartilage. BMP- 2 is also associated with maintenance and repair of damaged bone. Recombinant human bone morphogenetic protein 2 (rhBMP- 2) is now produced by genetic engineering techniques and used in treatment of thin bone fractures in the jaw and spine. In this study we aimed to extract and amplify the BMP- 2 gene from human osteoblast cell line MG-63, insert the amplified BMP- 2 gene into pGEM-b1 cloning vector, and then transform the recombinant vector into the E.coli strain of Rosetta. This technique can be used in future research and BMP- 2 expression.Methods: After culturing MG-63 cells, approximately 5 million viable cells were used for extraction of Total RNA. The extracted RNA was used for cDNA synthesis in RT-PCR reaction. Then, the BMP- 2 gene was amplified by specific primers and the PCR product was cloned in the pGEM-b1 vector. Chemically competent E.coli cells were prepared using CaCl2 0.1 M and transformed with recombinant pGEM-b1 vector under heat shock. The transformed E.coli Rosetta bacteria were inoculated on LB agar medium containing Ampicillin. Bacterial colony containing recombinant vector was isolated and used for plasmid extraction. The extracted plasmid was used for specific PCR to confirm the presence of BMP- 2 gene in pGEM- b1 vector.Results: After transformation, the E.coli Rosetta had the ability to become resistant to ampicillin and could grow on ampicillin- containing medium. While non-transformed E.coli Rosetta could not grow on LB agar containing ampicillin. The 1100 bp fragment was obtained from PCR amplification with specific primers, indicating that the BMP- 2 gene was inserted into pGEM-b1 vector.Conclusions: The pGEM-b1 vector and E.coli Rosetta strain were not used for BMP- 2 cloning in previous investigations. Therefore, this method may be a useful approach to reduce the challenges ahead of the optimization of BMP- 2 production.

Molecular Cloning of Bone Morphogenetic Protein-2 (BMP- 2) in pGEM-b1 Vector and Transformation into E.coli Rosetta Strain

Background: Bone Morphogenetic Protein 2 (BMP- 2) belongs to the TGF-β superfamily of proteins and plays an important role in the development of bone and cartilage. BMP- 2 is also associated with maintenance and repair of damaged bone. Recombinant human bone morphogenetic protein 2 (rhBMP- 2) is now produced by genetic engineering techniques and used in treatment of thin bone fractures in the jaw and spine. In this study we aimed to extract and amplify the BMP- 2 gene from human osteoblast cell line MG-63, insert the amplified BMP- 2 gene into pGEM-b1 cloning vector, and then transform the recombinant vector into the E.coli strain of Rosetta. This technique can be used in future research and BMP- 2 expression.Methods: After culturing MG-63 cells, approximately 5 million viable cells were used for extraction of Total RNA. The extracted RNA was used for cDNA synthesis in RT-PCR reaction. Then, the BMP- 2 gene was amplified by specific primers and the PCR product was cloned in the pGEM-b1 vector. Chemically competent E.coli cells were prepared using CaCl2 0.1 M and transformed with recombinant pGEM-b1 vector under heat shock. The transformed E.coli Rosetta bacteria were inoculated on LB agar medium containing Ampicillin. Bacterial colony containing recombinant vector was isolated and used for plasmid extraction. The extracted plasmid was used for specific PCR to confirm the presence of BMP- 2 gene in pGEM- b1 vector.Results: After transformation, the E.coli Rosetta had the ability to become resistant to ampicillin and could grow on ampicillin- containing medium. While non-transformed E.coli Rosetta could not grow on LB agar containing ampicillin. The 1100 bp fragment was obtained from PCR amplification with specific primers, indicating that the BMP- 2 gene was inserted into pGEM-b1 vector.Conclusions: The pGEM-b1 vector and E.coli Rosetta strain were not used for BMP- 2 cloning in previous investigations. Therefore, this method may be a useful approach to reduce the challenges ahead of the optimization of BMP- 2 production.

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Isfahan School of urban design: a morphological perspective

Urban morphology of cities in the Middle East and Central Asia has been of great interests to academics from various disciplines. Through centuries of development Iranian (Persian) art and architecture contributed to what is called now Islamic cities. Isfahan school of urban design has particularly played a significant role in creating to be highly influential in development of Islamic cities in the region. We discuss how establishment of this new school of thought on Islamic cities in 16th century (concurrent with Renaissance) influenced the way we think about cities in the Middle East and Central Asia today. Using content analysis and direct observation as our research methodology we investigate urban fabric of Isfahan- the capital of the Isfahan region in Iran and once its most largest and glorious city in order to extract the conceptual and practical lessons about traditional urban morphology and advocate their importance and relevance for the contemporary urban development practices in Islamic cities

Comparative Molecular and Microbiologic Diagnosis of Vaginal Colonization by Group B Streptococcus in Pregnant Women during Labor

Objective(s)Rapid tests for detection of Streptococcus agalactiae or Group B Streptococci (GBS) at the onset of labor are needed to permit early intrapartum antibiotic prophylaxis. This study aimed to evaluate the PCR assays targeting the 16S ribosomal RNA gene (16S rDNA) for detection of the GBS in comparison with a specific culture method. Materials and MethodsTwo swabs were used to obtain vaginal specimens from the 330 pregnant women attended delivery room at Hedayat hospital, Tehran, Iran. One swab was analyzed by direct plating onto selective GBS agar medium (ISLAM) and the other swab was used for a PCR assay, which amplified the 16S rDNA of S. agalactiae. Comparative study between the selective culture and the PCR assay was done among the 330 tested women.ResultsThe GBS colonization rate based on the culture results was 20.6% (68/330). Both culture and PCR methods were positive for 56 and negative for 253 women. The culture method was positive and PCR was negative in 12 women. The culture was negative and the PCR positive for 9 women. Sensitivity of the PCR assay was 82.3% and specificity was 96.5%. The positive predictive value was 86.15% and negative predictive value was 95.4%. ConclusionISLAM diagnostic procedure and PCR are rapid and reliable analyzing methods, which might be useful for accurate diagnosis of GBS colonization in pregnant women at the time of delivery