Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Extended adipofascial wrap around radial forearm flap for hard palate reconstruction.

BACKGROUND: While using radial forearm free flap in palate reconstruction, the pedicle lies in the nasal floor, constantly exposed to the nasal secretions and turbulent air current. To overcome this problem, we have designed a procedure which utilises the adipofascial extension to wrap the pedicle and nasal side of the flap. MATERIALS AND METHODS: The study was done during 2017 and 2018, 2 years' period. Totally 13 consecutive patients with defect in the palate status post-oncological resection and those in whom local flaps were not enough to cover the defect were included into the study. These patients were divided into two groups. First group in whom adipofascial extension was not used to cover the pedicle and second group in whom adipofascial extension was used to cover the pedicle. The incidence of nasal crusting, secondary haemorrage, blow out and flap necrosis were analysed and compared. RESULTS: In Group 1, we had 2 among 6 (33%) patients with secondary haemorrage. One patient had partial flap loss. On exploring, we noticed thrombosis of cephalic vein. We did not had any incidence of blow out of the pedicle. In Group 2, none of the patients had any secondary haemorrage. All flaps healed well. On doing nasal endoscopy at 6 months of follow-up, all flaps showed complete mucosalisation at the nasal side. CONCLUSION: Use of adipofascial extension while planning a radial forearm free flap to cover the nasal side of the flap and pedicle in the nasal floor helps to reduce the nasal crusting and secondary haemorrhage.

Use of selfie sticks and iPhones to record operative photos and videos in plastic surgery.

Use of smartphone has become ubiquitous. With smartphone cameras becoming powerful, they are replacing digital cameras and digital SLRs as primary instruments to take photos and record videos. It is natural even for plastic surgeons that smartphones are handy to take still photographs and even record high-definition or 4K videos. Another invention which has become popular with smartphone photography is a selfie stick. We explain the possibility and methodology of using an iPhone and selfie stick to take operative photographs and high-quality videos.

A novel and innovative technique of using a disposable syringe and mesh for harvesting fat for structural fat grafting.

BACKGROUND: Successful restoration of structure and function using autologous free fat grafts has remained elusive. Review of literature shows that various harvesting and preparation techniques have been suggested. The goal of these techniques is to obtain greater adipocyte cell survival and consequently more reliable clinical results. MATERIALS AND METHODS: In our technique, a piece of mesh is kept at one end of the lipoaspiration syringe, which is then connected to the Suction pump. As one syringe fills, it is replaced by another one until the required amount of fat is obtained. RESULTS: By using a polypropylene mesh in our technique, we can separate the transfusate from the harvested fat graft during harvesting itself. The fat graft thus obtained is dense and concentrated, with fewer impurities. CONCLUSION: Hence, we recommend our technique as a reliable method for extracting sterile emulsified fat in an economical way.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog.

BACKGROUND: The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells. METHODS: Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro- and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response. RESULTS: We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition (EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NFκB promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NFκB pathway whereas Bmi1 knockdown reduced the expression of NFκB target genes, suggesting that Bmi1 might regulate Nanog expression through the NFκB pathway. CONCLUSIONS: Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NFκB pathway.