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Brain–computer interfacing has been applied in a range of domains including rehabilitation, neuro-prosthetics, and neurofeedback. Neuroimaging techniques provide insight into the structural and functional aspects of the brain. There is a need to identify, map and understand the various structural areas of the brain together with their functionally active roles for the accurate and efficient design of a brain–computer interface. In this review, the functionally active areas of the brain are reviewed by analyzing the research available in the literature on brain–computer interfacing in conjunction with neuroimaging experiments. This review first provides an overview of various approaches of brain–computer interfacing and basic components in the BCI system and then discuss active functional areas of the brain being utilized in non-invasive brain–computer interfacing performed with hemodynamic signals and electrophysiological recording-based signals. This paper also discusses various challenges and limitations in BCI becoming accessible to a novice user, including security issues in the BCI system, effective ways to overcome those issues, and design implementations. Ministry of Defence (MINDEF) Nanyang Technological University Published version The authors acknowledge the support from Lee Kong Chian School of Medicine and MINDEF (NTU Project number: 04IDS001128A630). PP and BG also acknowledge the support from the Cognitive Neuro Imaging Centre (CONIC) at NTU, Singapore.

Introduction: It has been 17 years since the severe acute respiratory syndrome outbreak and Singapore is facing yet another daunting pandemic – the novel coronavirus (COVID-19). To date, there are 57,607 cases and 27 casualties. This deadly pandemic requires significant changes especially in the field of awake surgeries for intra-axial tumors that routinely involve long clinic consults, significant interactions between patient and multiple other team members pre, intra, and postoperatively. Materials and Methods: A retrospective review of all awake cases done during the COVID-19 pandemic from February to June 2020 was done. In this article, we outline the rigorous measures adopted during the COVID-19 pandemic that has allowed us to proceed with awake surgeries and intraoperative mapping at our institution. Results and Discussion: We have divided the protocol into various phases of care of patients planned for an awake craniotomy. Preoperatively, teleconsults have been used where possible thereby limiting multiple hospital visits and interaction. Intraoperatively, safety nets have been established during asleep-awake-asleep phases of awake craniotomy for all the team members. Postoperatively, early discharge and teleconsult are being employed for rehabilitation and follow-ups. Conclusions: Multiple studies have shown that with intraoperative mapping, we can improve neurological outcomes. As the future of the pandemic remains unknown, the authors believe that surgical treatment should not be delayed for intracranial tumors. Awake craniotomies and intraoperative mapping can be safely carried out by adopting the described protocols with combination of multiple checkpoints and usage of telecommunication.

Additional file 1.

Additional file 16. CD::UPRT::GFP_AD-MSCs were cocultured with U251MG™Z or U87MG™Z at the ratio of 1:10, in the presence or absence of 5FC. After seven days of incubation, images of the culture were captured. Representative images are presented.

Additional file 6. Supplementation of Enhancer to improve transfection in AD-MSCs. One day post seeding of 50,000 AD-MSCs, cells were transfected by 150-500 ng of PF463-CMV-GFP complexed with PEI at 1 μg pDNA to 3 μL PEI, in the presence or absence of Enhancer. a Two days post-transfection, the fluorescent images were captured at 4x magnification. Representative images are presented. b After which, cells were harvested for FACS analysis. Untransfected MSC served as negative control for gating. Data represents mean ± SD, n = 3 (Bar graph).

Additional file 2. Flow cytometry profiles of the cell cycle of the TMZ sensitive and resistant cell lines in the presence and absence of TMZ treatment. The number of cells acquired during flow cytometry measurement is 5000 per sample. The analysis of the cell cycle is presented in Fig. 1c and d.

Additional file 7. Scaling out MSC modification process on flat-bed culture. AD-MSCs were seeded in 6-well plate, T25, T75 and T175 flasks at 20,000/cm2. One day post seeding, cells were transfected with 150 ng PF463-CMV-GFP/ cm2 complexed at 1 μg pDNA to 3 μL PEI. Two-day post transfection, cells were harvested for analysis. Total number of cells harvested from each culture vessels were determined by the automated cell counter NC-3000. After which, the % of GFP+ cells measured with flow cytometry. a The scatter plot represents absolute number of GFP+ cells collected from each culture vessel; deduced from the readout of cell counting and flow cytometry of the GFP expression. Data of biological triplicates are expressed as mean + SD. b Representative images present the flow cytometry profile of unmodified and modified AD-MSCs harvested from T175 flasks.

Additional file 5. Determination of the transfection efficiencies of various commercial carriers in AD-MSCs. One day post seeding of 50,000 AD-MSCs, cells were transfected with different gene carriers at various GFP encoded plasmid (PF463-CMV-GFP, PlasmidFactory) and gene carrier amounts. AD-MSCs were transfected with a Lipofectamine 3000, b Polyfect, c Transficient, and d Turbofect according to the manufacturer’s instructions. e For cells transfected with PEI, the protocol is detailed in methods and materials section. Two-day post transfection, AD-MSCs were harvested. Transfection efficiency and cell viability (Propidium Iodide exclusion assay) were determined using NucleoCounter®NC-3000™. For each gene carrier, the left and right graph bar presents % of GFP+ cells and % of cell viability (% of PI- population), respectively. Data represents mean ± SD, n = 3.

Additional file 3. Gene expression of genes related in 5FU resistance pathway. Fold change of expression for DPYD, TYMS and UMPS in a U-251MG and b U-87MG TMRZ cell lines were calculated in relative to their respective parental cell lines. Similarly, changes in the expression of ABCC5 transporter were calculated accordingly for c U-251MG and d U-87MG TMZR cell lines. All samples were analysed in triplicates. The fold change in the expressions of the gene of interest was calculated after normalization to the house keeping gene GAPDH. Line graph shows the average fold change in gene expression, mean + SD (n = 3). Significant differences between parental and TMZR cells were calculated using unpaired, two-tailed Student’s t-test. p-value

Additional file 8. Scaling up MSC modification process on microcarrier culture. AD-MSCs were seeded at 28,000/cm2 on Cytodex® 3 microcarriers (total surface area of 1.9 cm2) in 24-well non-adherent plates, with agitation speed of 50 rpm for 24 h before transfection. Similar to flat-bed transfection, the polymer and DNA complex (at 200-300 ng of pDNA) were added to the cell culture using a dropwise manner after 15 min incubation. Cells were transfected in the presence or absence of the enhancer. Two-day post transfection, a the fluorescent images were captured at 10x magnification. Representative images are shown. b After which, cells were harvested through trypsinization. To separate the cells from microcarrier, the suspension cells were filtered through 70 μm cell strainer. The transfection efficiencies were determined through flow cytometry analysis. Cell viability based on PI exclusion was determined with NC-3000 automated cell counter. The combination chart presents % of GFP+ cells (graph bar) and cell viability (mark type) Data of biological triplicates are expressed as mean + SD. c For microcarriers with total surface area of 1.9 cm2 and 9.5 cm2, cells were transfected in 24-well non-adherent plates. To further scale up the transfection process, AD-MSCs were seeded on Cytodex® 3 (total surface area of 47.5 cm2) at 28,000 cells/cm2 in 125 mL Erlenmeyer flasks. One day later, cells were transfected in the presence of Enhancer. The amount of DNA was fixed at 150 ng/cm2 of microcarriers. Two-day post transfection, transfection efficiency was analysed. The scatter plot represents absolute number of GFP+ cells collected from each culture vessel; deduced from the readout of cell counting and flow cytometry analysis of the GFP expression. Data are expressed as mean + SD, n = 3.

Additional file 9. Representative images and FACS profile of AD-MSCs transfected with 300 ng of CD::UPRT::GFP plasmid, in the presence or absence of the Enhancer.