Ovarian cancer (OC) is one of the most common and lethal malignant tumors worldwide and the prognosis of OC remains unsatisfactory. Transcription factors (TFs) are demonstrated to be associated with the clinical outcome of many types of cancers, yet their roles in the prognostic prediction and gene regulatory network in patients with OC need to be further investigated.TFs from GEO datasets were collected and analyzed. Differential expression analysis, WGCNA and Cox-LASSO regression model were used to identify the hub-TFs and a prognostic signature based on these TFs was constructed and validated. Moreover, tumor-infiltrating immune cells were analyzed, and a nomogram containing age, histology, FIGO_stage and TFs-based signature were established. Potential biological functions, pathways and the gene regulatory network of TFs in signature was also explored.In this study, 6 TFs significantly associated with the prognosis of OC were identified. These TFs were used to build up a TFs-based signature for predicting the survival of patients with OC. Patients with OC in training and testing datasets were divided into high-risk and low-risk groups, according to the median value of risk scores determined by the signature. The two groups were further used to validate the performance of the signature, and the results showed the TFs-based signature had effective prediction ability. Immune infiltrating analysis was conducted and abundance of B cells naïve, T cells CD4 memory resting, Macrophages M2 and Mast cells activated were significantly higher in high-risk group. A nomogram based on the signature was established and illustrated good predictive efficiencies for 1, 2, and 3-year overall survival. Furthermore, the construction of the TFs-target gene regulatory network revealed the potential mechanisms of TFs in OC.To our best knowledge, it is for the first time to develop a prognostic signature based on TFs in OC. The TFs-based signature is proven to be effective in predicting the survival of patients with OC. Our study may facilitate the clinical decision-making for patients with OC and help to elucidate the underlying mechanism of TFs in OC.The online version contains supplementary material available at 10.1186/s13048-021-00938-2. Ovarian cancer (OC) is one of the most common and lethal malignant tumors, threatening global female health . DespiteTranscription factors (TFs) are DNA-binding proteins that play essential roles in controlling genes expression . Many TFDue to the urgent demand for a reliable, prompt and accurate prognostic stratification to help clinical decision-making of cancers, many risk score models were identified for predicting the clinical outcome of patients with OC. However, those signatures were mostly focus on genes, miRNAs or lncRNAs , 9. TFs,(Scheme ). Firstly, differentially expressed TFs (DE-TFs) was obtained through the intersection of the DEGs and several TFs databases. Then DE-TFs co-expression network was constructed using WGCNA, and hub-TFs were identified in significantly OC-related modules by Cytoscape. Cox-LASSO regression was performed to finally generate a TFs-based signature containing 6 TFs. According to the risk scores in the signature, patients with OC were divided into high-risk group and low-risk group. The validity of the TFs-based signature was assessed between two groups both in training and testing datasets. The TFs-based signature was further combined with several clinicopathological factors to establish a nomogram that could effectively predict survival probability. In addition, the relative abundance of 22 types tumor infiltrating immune cells in two groups were analyzed using CIBERSORT. Moreover, a TFs-gene target regulatory network was constructed by TRRUST to give insight into the underlying mechanism of the TFs in OC. To our best knowledge, this is the first risk score model based on TFs to predict the survival of patients with OC. The TFs-based signature and nomogram we constructed could be effective in prognosis prediction and have potential to assist clinical decision-making. Our study also identified the hub-TFs and shed lights on the roles of TFs and related gene regulatory network in the occurrence and progression of OC.In the present study, we aimed to construct a prognostic signature for predicting the survival of patients with OC using TFs data mainly from GEO database Scheme . Firstlyhttp://jaspar.genereg.net/), TRANSFAC (http://gene-regulation.com/pub/databases.html), CISBP  and TRRUST (https://www.grnpedia.org/trrust/) were collected and processed, and the duplicate TFs in each database were removed. The combined TFs database was used for extracting the differentially expressed TFs from differential gene expression profiles of OC.TFs in 4 commonly used TFs databases in bioinformatic analysis, JASPAR .The microarray data of OC in the GSE26712 and GSE140082 datasets were downloaded from GEO database Table . We alsoWeighted Correlation Network Analysis (WGCNA) is a systems biology method that could identify the modules of highly correlated genes and find candidate biomarker genes as well as potential therapeutic targets, according to the links between gene sets and phenotypes. In this study, a co-expression network was constructed using WGCNA package in R based on the DE-TFs. The optimistic soft-thresholding power was screened in order to obtain a TFs co-expression network in accord with scale-free network model. Topological overlap matrix (TOM) was constructed and the hierarchical clustering tree was built based on the dissimilarity matrix (1-TOM) and the dynamic tree cut was used for identifying modules that were significantly associated with the tumorigenesis and development of the OC.The TFs expression profiles data in the significantly OC-related modules were then analyzed by Cytoscape and the connectivity of each TF was measured using CytoHubba, an add-in in Cytoscape. The TFs in the network were ranked by node degrees as hub-TFs and selected for further analysis.To explore the functions and signaling pathways of TFs in the modules that was significantly related with OC screened from WGCNA, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using clusterProfiler package in R.P < 0.05). Then the least absolute shrinkage and selection operator (LASSO) regression model was carried out to eliminate the redundant factors and to find the most significant survival-associated TFs. Finally, stepwise multivariate Cox proportional hazards model was employed to construct an optimized risk score model for predicting the clinical outcome of the patients with OC, which was defined as the TFs-based signature. The risk score was calculated according to following formula: 0(t), n, βi and Ki represent the baseline hazard function, number of most significant survival-associated TFs, the coefficient, and level of gene expression, respectively.GSE26712 was used as the training set to generate the model in this work. Firstly, univariate Cox regression analysis was performed to identify hub-TFs that was significantly associated with patient outcomes  and clinical outcome of patients between two groups were compared by plotting Kaplan-Merier survival curves using survival package in R. Then, GSE140082 was used as testing dataset to further evaluate the validity of the TFs-based signature. The TFs-based signature was also verified by plotting receiver operating characteristic (ROC) curve using survivalROC package in R. Moreover, a heatmap were depicted by clustering TFs expression, risk groups and clinical status of the patients to exhibit performance of the TFs-based signature.https://cibersort.stanford.edu). The profiles of 22 tumor-infiltrating immune cells in the high-risk group and low-risk group were depicted and wilcox test was used to calculate the statistical significance between the two groups.To explore the associations between tumor immune infiltration and the risk groups determined by the TFs-based signature, the CIBERSORT algorithm was introduced to infer the relative abundance of 22 types of tumor-infiltrating immune cells using microarray data of patients with OC .To explore potential functions of TFs in the prognostic signature and identify genes they may target, a TFs-target gene regulatory network was constructed utilizing TRRUST database and was visualized by Cytoscape. (adj. P < 0.05 and |logFC| > 0.05). Genes with |logFC| > 1 were presented as a volcano plot , TRANSFAC and CISBP (1639 TFs) and TRRUST (795 TFs) databases. After removing the duplicate TFs, the combined databases contained 1911 TFs. The data in GSE26712 was downloaded and the differentially expressed Genes (DEGs) were screened between OC tissues and normal tissues ). DE-TFs transcripts data in these 4 modules were further imported into Cytoscape for the recognition of hub-TFs. Finally, 70 DE-TFs with the highest gene node degree, defined as hub-TFs, were obtained and used for further analysis .To further obtain gene modules that strongly correlate with the cancers from the DE-TFs, the WGCNA was performed. The data meets the the scale-free condition and the optimal conditions was determined at β = 5. Then, we constructed the WGCNA network based on this condition to identify TFs data that is significantly correlated to the cancers from the DE-TFs. Next, 9 modules were constructed and 4 modules that have strong relevance were identified . The result showed that the highest enriched GO terms in molecular function (MF), biological process (BP), and cellular component (CC) were DNA-binding transcription activator activity (P < 0.001), covalent chromatin modification (P < 0.001), and transcription factor complex (P < 0.001), respectively. The significantly enriched KEGG pathways included many cancer-related pathways, such as transcriptional mis-regulation in cancer (P < 0.001), cell cycle (P < 0.001), and viral carcinogenesis (P < 0.01). The results showed hub-TFs were mainly involved in various transcriptional process. Besides, hub-TFs were involved in pathways like cell cycle, cell senescence and histone modification. A large number of hub-TFs also participated in the regulation of many types of cancers, such as prostate cancer.Functions and signal pathways of hub-TFs were conducted using GO functional enrichment analysis and KEGG pathway analysis Fig. . The res. These 6 hub-TFs and their coefficients were showed in Table In order to construct a risk score assessment for predicting ovarian cancer prognosis based on above hub-TFs, we used the univariate Cox regression analysis in each hub-TF first, which identified 14 hub-TFs that were significantly correlated with clinical outcome of OC patients. Then, the model was further optimized through LASSO regression and multivariate Cox regression. Eventually, a prognostic signature was constructed containing 6 hub-TFs, including ZNF304, HSF1, SNAI2, MLXIP, ZNF518A and RFXANK . The Kaplan-Merier survival curve revealed that high-risk patients had a significant worse survival in both training set (P = 8.007E-6) and testing set (P = 0.0362). In addition, the time-dependent ROC showed that the 2-year AUC of training cohort and validation cohort was 0.746 and 0.613, respectively. Moreover, the TFs expression profile of OC patients between high-risk and low-risk groups in training set was presented as a heatmap  and testing set (GSE140082) are shown in Fig. 5 demonstrate that the abundance of B cells naïve, T cells CD4 memory resting, Macrophages M2 and Mast cells activated were significantly higher in high-risk group (P < 0.05). On the contrary, Macrophages M0 were significantly higher in low-risk group (P < 0.001)  Table , which s is one of the most common and lethal malignant tumors worldwide, with the second highest incidence rate and the highest mortality rate in all types of gynecological tumors. Despite the progress in imaging technologies, cytoreductive surgery and chemotherapy, the prognosis of OC remains poor, with less than half of 5-year survival rate. Early diagnosis of patients with OC is difficult owing to the absence of cancer-specific symptoms and effective screening tools. Patients with OC also suffered from relapse, high invasion and rapid drug resistance. Therefore, the improvement of individualized treatment is urgently needed. To this end, reliable, fast and accurate prognostic biomarkers of OC is critical for improving clinical stratification and determining personalize treatment options. Molecular markers, like genes, proteins, miRNAs and lncRNAs, which contribute to the pathogenesis of OC, are widely acknowledged as ideal candidates to predict the prognosis in OC patients and give clues to the underlying mechanism of OC. Due to the decrease of sequencing cost, the amount of data has rapidly increased in those years, which laid a solid foundation for the construction of a more accurate molecular-based prognostic model for predicting survival of patients with OC , 14. It Identification of TFs that play significant roles in tumorigenesis and progression of OC is the prerequisite for constructing a reliable and accurate risk score model. In order to minimize the potential missing of TFs, we identified DEGs in OC dataset on the one hand, and we acquired the combined TFs database on the other hand. Then, the first identification was completed by selecting the intersection between DEGs and TFs database and defined as DE-TFs. In order to obtain the TFs that are significantly associated with the OC among the DE-TFs, the WGCNA was used. The results showed that modules were successfully constructed, which strongly correlate with cancers. Then, the hub-TFs were selected from DE-TFs in cancer-related gene modules and the second identification was completed. We further screened the hub-TFs that are associated with the prognosis of OC using univariate Cox regression analysis. To construct the optimized risk score model, LASSO and stepwise multivariate Cox regression analysis were used to reduce the numbers of TFs. Finally, a TFs-based signature was generated after above three identifications. In previous studies, Zhao et al. screened 5 lncRNAs using WGCNA and Cox regression to construct a risk score system for prognosis assessment of OC . Bao et Our study established a TFs-based signature containing 6 hub-TFs, ZNF304, HSF1, SNAI2, MLXIP, ZNF518A and RFXANK. ZNF304 (Zinc Finger Protein 304) plays a role in gene silencing, including several tumor suppressor genes, and is overexpressed in several types of cancers. It has been reported that ZNF304 regulated β-1 integrin expression, promotes ovarian cancer cell survival and protects against anoikis in OC . HSF1 (HP < 0.001) and testing set (P = 0.0362), with the 2-year AUC of 0.746 and 0.613, respectively. In the previous studies, several prognostic signatures for predicting survival in OC were built based on other molecular biomarkers, for instance, multi-gene signature including immune-related and energy metabolism-related gene signature, lncRNA signature and signaling pathway signature. Ding et al. constructed a signature containing nine genes related to tumor microenvironment with the 3-year AUC of 0.684 and 0.606 in the training cohort and testing cohort, respectively [Next, the TFs-based signature generated by our study was validated in both internal training dataset GSE26712) and external dataset (GSE140082). Patients with OC are divided into high-risk group and low-risk group according to the level of risk score. Then, to validate the performance of the signature, Kaplan-Merier survival analysis and time-dependent ROC were conducted between the two groups. The results showed that high-risk patients had a significantly worse survival in both training set (712 and eectively . CompareP < 0.05). On the contrary, higher abundance of Macrophages M0 were related to favorable prognosis (P < 0.05). Previous studies showed lymphocytes infiltration was associated with prognosis of patients with OC [The tumor immune microenvironment was identified to play a significant role in the progression and metastasis of OC, especially during the development of chemoresistance, which may present potential prognostic factors and therapeutic targets for ovarian cancer , 28. The with OC . It was  with OC . M2-type with OC . Limitin with OC . Our finLimitations in our study including the following: 1) although Kaplan-Merier survival curve illustrated a significantly poorer outcome in patients with OC in the high-risk group according to the TFs-based signature, it is necessary to improve the performance of AUC in the testing datasets, like 1-year and 5-year. Therefore, our TFs-based signature needed to be further validated and updated to elevate the prognostic ability. The TFs-based signature could also be improved when there are more appropriate datasets in the future. 2) despite the construction of the TFs-target gene regulatory network, the molecular mechanism behind the network need to be further elucidated in in vitro and in vivo experiments. Moreover, in order to further help the clinical stratification and personalize treatment options of patients with OC, it is reasonable to combine our TFs-based signature with more factors, such as lncRNAs, and clinicopathological parameters to continue developing the prognostic risk score model of OC.Our study established the first TFs-based signature that might be effective in the prediction of prognosis in patients with OC. The signature contains 6 hub-TFs, which are identified mainly using differential expression analysis, WGCNA and Cox-LASSO regression analysis. The TFs-based signature was validated. A nomogram based on the signature was constructed and demonstrated good performance in predicting survival of patients with OC. Furthermore, immune infiltrating analysis showed that TFs included in the signature may play a role in the tumor immune microenvironment. In addition, the TFs-target gene regulatory network suggested that these TFs participate in many cancer-related pathways. Our study could provide helpful information for the clinical stratification and personalized treatment options in patients with OC. It also gives insights into the potential roles of TFs in the molecular mechanism of OC.Additional file 1: Table S1 Characteristics of patients with ovarian cancer in GSE140082


Small-scale magnetic robots are remotely actuated and controlled by an externally applied magnetic field. These robots have a characteristic size ranging from several millimetres down to a few nanometres. They are often untethered in order to access constrained and hard-to-reach space buried deep in human body. Thus, they promise to bring revolutionary improvement to minimally invasive diagnostics and therapeutics. However, existing research is still mostly limited to scenarios in over-simplified laboratory environment with unrealistic working conditions. Further advancement of this field demands researchers to consider complex unstructured biological workspace. In order to deliver its promised potentials, next-generation small-scale magnetic robotic systems need to address the constraints and meet the demands of real-world clinical tasks. In particular, integrating medical imaging modalities into the robotic systems is a critical step in their evolution from laboratory toys towards potential life-savers. This review discusses the recent efforts made in this direction to push small-scale magnetic robots towards genuine biomedical applications. This review examines the accomplishment achieved so far and sheds light on the open challenges. It is hoped that this review can offer a perspective on how next-generation robotic systems can not only effectively integrate medical imaging methods, but also take full advantage of the imaging equipments to enable additional functionalities. In the past several decades, small-scale robotics has been attracting an increasing amount of attention from researchers, entrepreneurs, and healthcare professionals. These miniature devices have the potential to access hard-to-reach regions in human bodies and perform unprecedented medical procedures in a minimally invasive manner ,5,6,7,8.Small-scale magnetic robotics emerged as an interdisciplinary research field several decades ago and quickly gained momentum in advancement ,18,19,20It is beneficial to clarify the robot definition in the context of small-scale robotics. As individual device gets smaller, it becomes simpler in materials and structures. And more capabilities that were used to be accomplished by onboard components are now fulfilled by off-board peripherals and supporting equipments. From a few millimetres down to a few nanometres, these devices serve more like one ,24,25,26With a short history of only several decades, studies in small-scale magnetic robotics have hitherto limited themselves to simplified laboratory environments and proof-of-concept demonstrations. In these pioneering investigations, direct line-of-sight was maintained to easily track the robots within different workspace. Optical cameras and sometimes even naked eyes were employed as the primary or only imaging modalities, see Not willing to forsake the aforementioned convenience, researchers have been trying to maintain such a direct line-of-sight in as many cases as possible. Many experiments, especially the preliminary ones, of small-scale magnetic robots are performed in air ,36,37,38 surface . The she surface . Positio surface . Huang e surface .Even when medical phantoms or animal models were used for realistic biomedical working environments, open ,60,61,62sruption . Biofilmsruption . An optiAlthough the optical imaging approach is widely adopted, only using this technique compromises the clinical relevance of the corresponding small-scale robotic systems. Because direct line-of-sight is unavailable for most clinical scenarios, especially for minimally invasive diagnostic and therapeutic tasks that motivate small-scale robotics in the first place. As a result, researchers have started to restrain the usage of optical imaging to only early stage proof-of-concept demonstrations. Instead, they actively seek to deploy more realistic and clinically relevant imaging modalities in next-generation small-scale magnetic robotic systems.Imaging biology tissues and tracking markers are pivotal capabilities in clinical environments. They provide healthcare practitioners with static and dynamic information beyond direct visual observations to assist in disease prevention, diagnosis, and therapy . A wide Though these imaging modalities are common in clinical workspace, they require dedicated and specialized equipments which are often expensive to purchase, setup, operate, and maintain within research laboratories hosted by universities and institutes. The resultant medical images are different with the optical ones that people are used to. And professional training is required for proper interpretations and information extraction from medical images. In addition, as all these imaging modalities were developed prior to the emergence of small-scale magnetic robotics, they are not inherently compatible with these novel robotic systems. Comprehensive evaluations and validations of compatibility and safety are needed before integrating them together. The steep upfront monetary investment leads to the scarcity of these imaging modalities in research communities. For the privileged researchers who have access to these modalities, the operational cost, expertise requirement, and prerequisite compatibility and safety studies discourage them from adopting these modalities in their investigations of small-scale magnetic robots. Although clinical imaging modalities have the aforementioned inhibitive features, researchers still have started to make efforts towards the integration of them in the next-generation of small-scale magnetic robotic systems. Because these imaging modalities constitute an indispensable portion of future robotic systems applied in real-world biomedical applications, where direct line-of-sight is unavailable.This section reviews some exemplar studies that take advantages of medical imaging modalities to enhance the relevance of the developed small-scale magnetic robotic systems with real-world biomedical applications. The scope of this section is intentionally limited to only the studies involving MRI, US, X-ray, or fluorescence imaging.MRI is a versatile imaging technique to generate images of human bodies, especially soft tissues. It uses a strong uniform axial magnetic field, magnetic field gradients, and radio waves. The strong axial magnetic field has a magnitude of several teslas. And it poses challenging constraints on the use of ferromagnetic materials as well as conductive cables. During MRI imaging, patients are confined to a long and narrow central chamber with loud noise that could range from 65 to 130 decibels, limiting the appropriate time period of operation. Thus, small-scale magnetic robots have to work in a strong constant magnetic field, with all necessary peripherals fitting inside the limited space and operation finished by a relatively short period of time. But at the same time, MRI machines have the potential to carry out imaging and actuation of small-scale magnetic robots without requiring additional setups, assuming the magnetic robots could take advantages of the magnetic field generated by the MRI machines.Efforts have been made to use MRI machines to obtain real-time position information of small-scale magnetic robots. In 2008, Felfoul et al. developed a 3D magnetic resonance (MR)-tracking method specifically for small-scale robots with a ferromagnetic core . In vitrIn 2017, Yan et al. reported biohybrid small-scale robots with helical shapes for MRI-guided therapy . In vivoSince MRI machines generate both uniform magnetic field as well as magnetic field gradient, it is preferable to develop small-scale magnetic robots that can not only be imaged but also be actuated and controlled by MRI machines, obviating the need for additional magnetic field generation setups. Though at any instance, only one of the functions, i.e., either sensing or actuation, can be executed by the same MRI machine. In 2015, Lalande et al. took advantages of the presence of a strong magnetic field in a MRI machine and successfully demonstrated in vivo magnetic steering of a ferromagnetic guidewire in a MRI system equipped with additional gradient coils for selective catheterization of renal arteries on rabbits . Also toBesides experimental explorations, theoretical and computational research has also been performed to construct a plan and control strategies as well as simulation environment for microrobots using a MRI machine ,82. In 2The configurations and working conditions of MRI machines pose severe constraints on the deployment of small-scale magnetic robots. But at the same time, the increasing accessibility and popularity of MRI machines in healthcare keeps attracting researchers. In addition, MRI is a widely applied and thoroughly investigated medical imaging modality. It poses no safety concerns for patients assuming standard precautions and operational procedures are followed. As a result, it is a particularly intriguing promise of fusing imaging and controlling systems into one MRI setup for better reliability and less complexity. Imaging requires a strong axial magnetic field and short pulses of radio frequency (RF) fields. While actuation requires quasi-static magnetic fields at various directions with smaller magnitude compared with the axial magnetic field for imaging. As the magnetic fields used for imaging and actuation are different, it exists a possibility of developing a multiplex of magnetic field sequence to conduct both tasks. One challenge is that the strong axial magnetic field required for imaging is always present in the workspace and override the effects of any quasi-static magnetic fields used for actuation. Next-generation robotic systems are being designed to utilize an MRI machine to its full potential. US imaging is widely used in clinical scenarios to visualize muscles, tendons, and other internal organs of human body . It stanIn 2018, Hu et al. demonstrated that a millimetre-scale magnetic sheet robot was wirelessly modulated to move inside a concealed areas of an ex vivo chicken tissue . Figure In 2020, Wang et al. reported a reversible anchoring robot that worked within a tubular structures under the guidance of real-time US images . The sofUS has become one of the most accessible and popular medical imaging modality for researchers in small-scale magnetic robotics. It has also been used to provide acoustic energy as the primary or additional input for small-scale robots. For example, Youssefi et al. reported a dual micromanipulation system for small-scale robots to achieve 3D, contactless, and semi-autonomous micromanipulation in 2019 . Robots mulation . Thus, tX-ray imaging modality employs ionizing radiation to generate images of human bodies. When (a part of) human body is sandwiched between a radiation source and sensor, different biological substances of the body block the radiation to various extents, resulting in different signal strength received by the sensor. X-ray is widely used in clinical workspace and includes different specific techniques and procedures tailored for different purposes. Examples include radiography, computed tomography (CT), and X-ray fluoroscopy. To roughly differentiate these three techniques from each other in terms of their usage, radiography captures single still images, CT provides a reconstruction using many cross-sectional images, while X-ray fluoroscopy produces a continuous X-ray image. But recent advancement of these technologies has blurred their distinctions, and the overlap of their respective functionalities grows larger. Although X-ray imaging modalities are widely deployed and frequently utilized in clinical environments, the exposure to radiation leads to potential risks, such as injuries to the skin and underlying tissues (“burns”) shortly after the procedure and radiation-induced cancers occurring some time later. To minimize these risks, X-ray imaging is always performed with the lowest acceptable exposure for the shortest time necessary.In 2018, Nguyen et al. proposed a real-time position and spatial orientation tracking method for a millimetre-scale bullet-shaped robot using a principle component analysis and an X-ray reconstruction . The devX-ray is a convenient choice to image small-scale magnetic robots for in vitro, ex vivo, and in vivo biological experiments performed. However, as it involves ionizing radiation by its nature, the safety of its wide deployment in next-generation robotic systems warrants further investigations. Established medical guideline such as “As Low as Reasonably Achievable” (ALARA) requires careful evaluations been performed to compare the benefits with the risks associated with X-ray imaging. Although they may sound similar, fluorescent imaging is different with X-ray fluoroscopy. Fluorescence imaging utilizes fluorophores and a fluorescence-detecting imaging system, avoiding the exposure to radiation and thus ameliorating the safety concern . FluoropIn 2015, Servant et al. employed real-time fluorescence imaging to observe a swarm of micron-scale functionalized artificial bacterial flagella (f-ABFs) . The f-AIn 2019, Wang et al. reported magnetic helical microstructures that were controlled to swim along pre-defined trajectories under the control of rotational magnetic fields for single-cell targeting . ExperimFluorescence imaging is popular in small-scale magnetic robotic systems. Different with other imaging modalities, it lacks the capability to reveal hidden features of the biological environment surrounding the robots. Thus, it cannot help the robots navigate within complex and tortuous environments buried deep in the body. Instead, it is often used to reveal the position of one  or a swaThe past several decades witnessed the emergence and rapid advancement of small-scale magnetic robotic systems. Pioneering explorations carried out by researchers across the globe have presented auspicious preliminary results. Researchers, engineers, and entrepreneurs are joining efforts to push this field forwards and determined to deliver its full potential. These robots have started to evolve from laboratory toys towards the promised life-savers in biomedical tasks, especially in the peri- and post-pandemic eras. To realize a such transition, next-generation small-scale magnetic robots demand more than proof-of-concept demonstrations in simplified ideal environments. Instead, application-oriented robots need to be developed for unstructured biological workspace under real-time guidance of medical imaging modalities. The success of this transition will warrant a long-term prosperity of this field and lead to far-reaching societal and economic impact.Imaging small-scale robots using medical imaging modalities is challenging. Concerted and also independent efforts are being made in the research community to explore various approaches and strategies. However, medical imaging modalities still mostly serve as a secondary or optional component. The next-generation small-scale magnetic robots should at least be compatible with medical imaging modalities to work without direct line-of-sight. In an ideal case, they should also utilize the existing imaging equipments, such as an MRI machine and a US probe, to extend their functionalities catalogue. Recently, healthcare professionals are starting to get involved in the development and deployment of these robots. It is hoped that this collaborative initiative between scientists and doctors will grow stronger with each party bringing unique expertises and perspectives to the table.In view of the active research activities in the field of small-scale magnetic robotics, this review aims to provide a sense of the context, the landscape, and the state-of-the-art for fellow researchers and engineers. It discusses some exemplar pioneering work that integrated medical imaging modalities into small-scale magnetic robotic systems to various extents. Focus is given to the studies involving MRI, US, X-ray, and fluorescence imaging, due to their popularity and accessibility in existing clinical environments. Although they are not covered in this review, other medical imaging modalities have also been explored to image and track small-scale magnetic robots ,104. For


The sensitivity of cytology after fine needle aspiration (FNA-cytology) in detecting medullary thyroid carcinoma (MTC) is low. To overcome this problem, measuring calcitonin (CT) in washout fluid of FNA (FNA-CT) has been largely diffused and showed good performance. However, no evidence-based study exists comparing systematically the sensitivity of FNA-cytology and FNA-CT. This study aimed to systematically review the literature and collect data allowing a head-to-head comparison meta-analysis between FNA-cytology and FNA-CT in detecting MTC lesions.The online databases of PubMed/MEDLINE and Scopus were searched until June 2021. Original articles reporting the use of both FNA-cytology and FNA-CT in the same series of histologically proven MTC lesions were included They were extracted general features of each study, number of MTC lesions (nodule and neck lymph nodes), and true positive and false negatives of both FNA-cytology and FNA-CT.Six studies were included. The sensitivity of FNA-cytology varied from 20% to 86% with a pooled value of 54% (95% CI 35–73%) and significant heterogeneity. The sensitivity of FNA-CT was higher than 95% in almost all studies with a pooled value of 98% (95% CI 96–100%) without heterogeneity. The sensitivity of FNA-CT was significantly higher than that of FNA-cytology.FNA-CT is significantly more sensitive than FNA-cytology in detecting MTC. Accordingly, FNA-CT represents the standard method to use in patients with suspicious MTC lesions, combined with cytology. Medullary thyroid carcinoma (MTC) is an infrequent thyroid malignancy originating from C-cells and occurring as a familial disorder in about one in five cases , 2. The In addition to the above technical issues, the need for systematic reviews of diagnostic accuracy has gained interest in the modern cytopathology . In factThe present systematic review was conceived to collect data allowing a head-to-head comparison meta-analysis between FNA-cytology and FNA-CT in detecting MTC lesions. Then, an online search strategy was designed to find original papers reporting the performance of both methods in the same patients’ series.The systematic review was conducted according to the PRISMA-DTA statement .The online databases of PubMed/MEDLINE and Scopus were searched. Initially, the search was based on the combinations of the following terms . Later, a specific algorithm,  AND (biopsy OR FNA OR FNAC) AND (washout OR fluid), was used. No beginning date limit was used. No language restriction was adopted. The last search was done on June 28th, 2021. To identify additional studies, the references list of the retrieved articles was screened.In accordance to the study’s aim, the ideal study to be included was that reporting the use of both FNA-cytology and FNA-CT in the same series of nodules or neck lymph nodes. In fact, only this kind of study could allow us to calculate the pooled sensitivity of both FNAs and compare them in a head-to-head fashion. After screening the online databases, the main exclusion criteria were: (a) articles not within the field of interest of the study; (b) review articles, editorials, letters, comments; (c) articles that did not provide clear study characteristics or reporting overlapping patient data; (d) studies including <10 MTCs. Two researchers  independently reviewed title and abstract of the retrieved articles, applied the above selection criteria, and reviewed the full-text of the remaining articles to determine their inclusion. Disagreements between them were resolved in a mutual consensus.For each included study, the following data were extracted: (a) authors and country of origin; (b) journal; (c) year of publication; (d) number of patients evaluated; (e) number of histologically proved MTC lesions (nodule and neck lymph nodes); (f) number of MTC lesions with suspicious or positive report at FNA-cytology; (g) number of MTC lesions with positive FNA-CT; (h) number of MTC lesions with negative FNA results. Then, true positives (TP) and false negatives (FN) of both FNA-cytology and FNA-CT were obtained. In addition, other technical data about the CT measurement were extracted for each study.The risk of bias of the studies included was assessed independently by two reviewers , according to QUADAS-2 . The ris2 index and it was significant with value >50%. Statistical analyses were performed by using OpenMeta[Analyst] .The sensitivity of FNA-cytology and FNA-CT in detecting MTC lesions was calculated from each article on per lesion-based analysis by using this formula: Sensitivity = TP / (TP + FN). FNA-cytology was considered as TP when an MTC lesion was defined as malignant or when it was defined suspicious for MTC by authors of the studies. FNA-CT was considered as TP according to the criterion adopted by the authors of the studies. A random effects model was used. Pooled data were presented with 95% confidence intervals (95% CI). To compare the sensitivity between FNA-cytology and FNA-CT, a significant difference could be detected if the 95% CIs of the two FNAs were not overlapping. Heterogeneity was assessed by IAccording to our searching algorithm, a number of 204 records was retrieved. After excluding duplicates and applying the above selection criteria, 6 articles –16 were From the six studies included in the systematic review, four were published by Italian authors and the remaining two were published by researchers from Portugal and China. One study presented a multi-center series while the other studies were single-center. All studies included patients with suspicious MTC or recurrent MTC based on high serum CT levels. The overall number of patients enrolled in the studies was 278 and the lesions that undergone FNA-cytology and FNA-CT was 399. Finally, a total of 173 histologically proven MTC lesions were available. The sensitivity of both FNA-cytology and FNA-CT was reported in all six studies. In addition, some of them , 15 alsoThe risk of bias was assessed based on four study characteristics Table . The risThe sensitivity of FNA-cytology recorded in the six studies varied from 20% to 86%. The pooled sensitivity of FNA-cytology obtained by the meta-analysis was 54% with 95% CI from 35 to 73% and significant heterogeneity .As usual, some potential limitations may affect the present meta-analysis. First, no large-sample studies were found and this influenced the pooled sample size. This obviously depends on the infrequent incidence of MTC. The herein recorded difference in sensitivity between FNA-CT and FNA-cytology was significant  and this data suggests to perform, as a further proof, prospective multicenter studies with direct comparison between FNA-CT and FNA-cytology. Second, some studies were retrospective. This could influence the findings of FNA-cytology  and explain the heterogeneity observed only in FNA-cytology. Third, the series of patients should be not consecutive and a potential selection bias should be taken into account. Fourth, we must be aware that a different use of serum CT testing could be adopted by different institutions. This might have influenced in clinical practice the selection of patients undergoing FNA with or without measurement of CT in washout fluids. However, the major strength of the present data is that FNA-CT showed excellent sensitivity with no discordant data among studies  confirms that FNA-cytology can only detect approximately a half of histologically proven MTC lesions, and (2) demonstrates that FNA-CT is significantly more sensitive than FNA-cytology. Accordingly, FNA-CT is the standard method to use in patients with suspicious MTC lesions and it has to be combined with FNA-cytology in all cases.


Little is known about associations between performance-based measurements and self-reported scales, nor about ceiling effects or sensitivity to change to evaluate effects in the target population for self-managed exercise interventions. This study aimed to explore the feasibility of using performance-based outcomes for gait speed, functional leg strength and balance, and self-reported outcomes of falls-efficacy and functional ability in two self-managed falls prevention exercise interventions for community dwelling older adults.n = 67) exercised with one of two self-managed falls prevention exercise programmes, a digital programme (DP) or a paper booklet (PB) in a 4-month participant preference trial. Pre- and post-assessments, by blinded assessors, included Short Physical Performance Battery (SPPB) and 30s Chair stand test (30s CST). Participants completed self-reported questionnaires: Activities-specific and Balance Confidence scale (ABC), Iconographical Falls Efficacy Scale (Icon-FES), Late-Life Function and Disability Instrument Function Component (LLFDI-FC). In addition, improvement in balance and leg strength was also self-rated at post-assessment. Participants’ mean age was 76 ± 4 years and 72% were women.Independent living, community-dwelling older adults . At pre-assessment all performance-based tests correlated significantly with the self-reported scales, however, no such significant correlations were seen with change-scores. Improvement of performance-based functional leg strength with substantial effect sizes and significant correlations with self-reported exercise time was shown. There were no differences in outcomes between the exercise programmes except that DP users reported improved change of leg strength to a higher degree than PB users.The LLFDI-FC and sit-to-stand tests were feasible and sensitive to change in this specific population. The balance sub-component of SPPB and self-reported measures ABC and Icon-FES indicated ceiling effects and might not be suitable as outcome measures for use in a high functioning older population. Development and evaluation of new outcome measures are needed for self-managed fall-preventive interventions with high functioning community-dwelling older adults.The online version contains supplementary material available at 10.1186/s12877-022-02851-9. Falls prevention is important as the ageing population increases, and the occurrence of falls rise . ExercisOutcome measures that are sensitive enough to detect important differences in physical capacity are crucial to evaluate the effects of exercise interventions. Older adults are a heterogenous group and it is therefore important to evaluate psychometric properties for the intended target group. For high functioning community-dwelling older adults, commonly used outcome measures might have ceiling effects, which can limit evaluations of exercise effects –14. WhenThe overall aim of this study was to explore the feasibility of using performance-based outcomes of gait speed, functional leg strength and balance, and self-reported outcomes of falls-efficacy and functional ability in two self-managed falls prevention exercise interventions. More specifically to investigate the feasibility of the outcome measures regarding ceiling effects and sensitivity to change, and to explore associations between performance-based measurements and self-reported scales. We also explored measured effects and differences depending on format of the programme .The intervention was a participant preference trial in which the participants choose to self-manage their exercise with either a digital programme (DP) or a paper booklet (PB). Each participant started the intervention by attending an introduction meeting, including individual pre-assessments and a short presentation of the exercise programme of their choice. The intervention included four months of fully self-managed exercise, guided by the chosen programme, and the intervention ended with a post-assessment meeting. The study complies with the Declaration of Helsinki and received approval from The Regional Ethical Review Board in Umeå (Dnr 2016/106–31). All participants were given written and verbal information and gave written informed consent. The study was registered on ClinTrials.gov 28/09/2016, with ID: NCT02916849.Recruitment was performed in senior citizen organizations and at one health care centre. At the senior organizations the participants were recruited by members of the research team and at the health care centre by health care professionals. Inclusion criteria were: ≥70 years old, living independently in the community, able to rise from a chair and stand without support, experiences of deterioration in balance and/or need to be more careful not to lose balance and/or have experienced a fall the past year. Exclusion criteria were: doing physical exercise more than three hours per week, self-reported progressive disease that was likely to influence mobility and cognitive difficulties. Status of cognitive condition was judged during the screening interview, if the person was able to answer questions satisfactorily and able to converse about matters regarding the study, they were considered suitable to take part in the study. The study participants were 72% women and the mean age was 76 years.Both programmes were fully self-managed, meaning the programme was used without professional guidance and it could be tailored and progressed by the participant as often as desired. The PB was based on the Otago Exercise Programme , the DP All participants attended an introduction meeting for approximately two hours including a short lecture on falls and falls prevention followed by physical assessments, completion of self-administered questionnaires, and an introduction of either the DP or PB. As self-management was the focus of the intervention, interactions with participants were limited during the study. A contact number was provided in case of any emerging technical problems during the intervention and all participants received a telephone call a few weeks after study start to identify any potential problems with the programmes. For some of the participants in the DP group more interaction was scheduled, six participants had an observation in their own home midway in the intervention, and twelve participants were in a sub-group who were offered monthly peer-mentor meetings. Further detailed description of the interventions can be found in the Supplementary Table S, TemplatAt the introduction meeting, sociodemographic characteristics, access to technology, self-reported health, function, and mobility, as well as history of falls in the past year, was collected through a self-report questionnaire. One of the researchers was present in the room to answer questions when participants filled in the questionnaires. A physiotherapist blinded to group allocation, and previous results, performed all measurements of physical functioning at the pre- and post-assessment.Functional performance was measured using the Short Physical Performance Battery (SPPB) . The SPPR = 0.89; 95% CI = 0.79–0.93) and concurrent validity with 1 RM leg-press among community-dwelling older adults  was used to assess functional leg strength and endurance . During .64–0.85 .Different definitions and tests have been used to describe and report fear of falling, where loss of confidence in balance abilities to function safely may cause fear and avoidance of activities .Self-reported balance confidence was measured using a Swedish version of the Activities-specific Balance Confidence Scale (ABC) showing internal consistency Cronbach’s α = 0,95–0,97, and test-retest ICC 0.82, 95% CI = 0.72–0.88 . Verbal Fear of falling was measured by the 30-item Iconographical Falls Efficacy Scale (Icon-FES) a picture-based questionnaire , translaThe Late-Life Function and Disability Instrument (LLFDI) measures functional limitations and disability in community-dwelling older adults , 35. In Self-rated improvements in leg strength and balance were assessed by two single-item questions with a 5-point Likert scale . The questions were developed by the research group for the purpose of this study.https://www.jamovi.org, The jamovi project (2020).Participant characteristics are reported using means with standard deviations or as proportions when applicable. In this feasibility study both groups received an exercise intervention (DP and PB). To improve power in analyses of the outcome measures data were pooled from both groups. Differences in effects between groups were explored although the study was not powered for this purpose. Pre-post values are summarized using non-parametric measures of central tendency and dispersion (median and interquartile range). Eventual occurrence of ceiling effects were explored by examining pre-assessment median values and the scores for the third or first quartile depending on whether a high or low score was considered superior. For outcomes with no ceiling effects, and available limit values for Minimal Clinically Important Difference (MCID), the proportions of participants who improved or deteriorated were calculated. For SPPB the MCID applied was 1 point , for gaip = 0.009). A few participants who withdrew still filled in self-reported scales at the end of the study. More information on attrition and adherence from this feasibility study has been reported elsewhere [In total, 67 participants were enrolled in the study Table . Over 90lsewhere . The meaMedian values, interquartile ranges and effect sizes are presented in Table In terms of sensitivity to change, about half of the participants improved according to limits of MCID Table  for the At pre-assessment, the performance-based tests SPPB, gait speed, balance, and 30s CST all had significant low to moderate correlations with self-reported scales, with the exception of poorer correlations between balance and Icon-FES Table . For theChange scores, between pre- and post-assessment, showed no significant correlations between performance-based measurements and self-reported scales Table . There wrrb = − 0.65, 30s CST rrb = 0.76) following four months of either self-managed exercise intervention. The effect size for total SPPB score was smaller (rrb = 0.34). Small effect sizes were seen for gait speed, balance, fall-efficacy and self-reported functional ability (rrb < 0.21). Participants in both groups self-reported improved balance  post intervention. Participants in the DP group were significantly more inclined than participants in the PB to agree that their leg strength had increased. No other significant differences between the two programmes were found.Outcome measures at pre- and post-assessments for the whole sample and per group are presented in Table p = 0.006) and 5TSTS . Although total SPPB did not improve significantly, the outcome was correlated to reported exercise time . No significant associations were seen between exercise time and outcomes for gait speed (rho − 0.063), balance (rho 0.159), ABC (rho 0.078), Icon-FES (rho − 0.079) LLFDI-FC (rho 0.04), or with self-rated improvements in leg strength (rho 0.202) or balance (rho 0.264).For the whole sample, improvements in functional leg strength correlated significantly with self-reported exercise time, 30s CST  or fear of falling (Icon-FES). In these tests, a large proportion of the participants obtained either close to the best or the best scores at pre-assessment. These ceiling effects were not seen in the self-reported functional ability according to LLFDI-FC. The measurements of functional leg strength according to time for 5TSTS or number of 30s CST, have in themselves no maximum score, and are thus not subject to ceiling effects. Using these outcome measures, substantial effect sizes were seen for functional leg strength, which also correlated with exercise time (minutes accomplished). Associations between performance-based measurements and self-report scales were seen at pre-assessment but not for change over time.The ABC scale demonstrated ceiling effects, which is in accordance with previous studies –48. MyerThe SPPB was used to measure lower-extremity function. The psychometric properties of this assessment battery have previously been extensively evaluated and SPPB is strongly recommended as the first choice of outcome measure in older community-dwelling persons . The parIn contrast to performance-based measurements of balance the self-rated change in balance, with questions made for this study , indicatAt pre-assessment significant associations were seen between performance-based measurements and self-reported scales. Previous cross-sectional studies have also shown associations between balance confidence (ABC) and self-reported function (LLFDI-FC), with various balance, physical performance measurements and self-rated health , 46. HowNo differences were seen in the additional analyses between groups using the DP and the PB, except that DP participants were more inclined to agree that their leg strength improved post intervention. This is in line with our previous reported results showing that adherence to the programme were similar in both groups among participants who completed the intervention . The resThe majority of study participants were women 72%), a distribution normally seen in studies concerning falls prevention [%, a distThe use of MCID is based on previous studies where both length of the intervention and study population have to be considered. The reference values used in this study were obtained from studies that varied in length from nine weeks to one year follow-ups –43. All A limitation with the present study was that it was a feasibility study with a relatively short intervention, and was not powered to evaluate exercise effects between the two programmes. Although, the four-month self-managed falls prevention exercise study showed some positive effects on self-reported balance and measured leg strength in both groups combined. We also created new questions on self-reported change in functional ability as no evaluations of such questions were found in the literature. The PB was used as a comparative group to the new DP and the aim was explore the feasibility of the outcome measures.We did not apply any strict limits for what was considered a ceiling affect. Commonly ceiling effects are described when the majority of values obtained for a variable approach the upper limit of the scale. A criterion often applied is that ceiling effects are considered if more than 15% of respondents achieve the highest possible score . Such guA strength of this study was that data was collected before and after the intervention by the same small group of assessors. Experienced physiotherapists assessed performance-based measurements blinded to results of self-rated scales and to group allocation. For ABC, individual verbal instruction was provided as the original scales have extensive written instructions. The participants answered all self-report questionnaires independently, with a researcher close by in case of any questions. The LLFDI-FC also had individual verbal instructions, yet many participants found the parted page for function unclear, which required some explanation. Several participants had questions regarding some illustrations in Icon-FES. For example: How concerned are you about crossing the street on a pedestrian crossing against the lights? How concerned are you when cleaning the gutter? These were all things that many participants were warned to do and never did. Consequently, they had objections to answering these questions, resulting in a few contradicting answers in this questionnaire.The outcome measures used to assess participants changes following two self-managed falls prevention interventions showed limitations. The balance sub-component of the SPPB and the self-reported measures ABC and Icon-FES might not be suitable for assessments in all community-dwelling older adults due to risk of ceiling effects. The LLFDI-FC did not show any ceiling effects, and could be used as a self-administered instrument. The 5TSTS and 30s CST were feasible in this study including high-functioning older adults. Both leg strength assessments were able to detect improvements and also showed correlation with exercise time, suggesting adequate level of challenge to reflect the capacity/function of relatively high-functioning older adults. Associations were seen at pre-assessment for performance-based and self-reported scales, considering change-scores no significant correlations were seen. More research is needed to identify suitable instruments for assessment of balance performance, and balance confidence in self-managed fall-preventive interventions in this group of older adults. Additionally, it is desirable to explore the participants’ own experiences of improvements in balance and strength in further studies.Additional file 1: Supplementary Table (S1). Template for Intervention Description and Replication (TIDieR) checklist for Safe Step feasibility study.


Adolescent e-cigarette use has increased dramatically. Most e-cigarette liquids contain nicotine, which can harm the developing adolescent brain. Local studies examining the risk factors of adolescent e-cigarette use and interactions between its risk factors are limited. This study was aimed at determining the prevalence of current e-cigarette use and its associated factors among in-school adolescents in West Malaysia. We also examined the possible sociodemographic characteristic, lifestyle risk behaviour and parental factor interactions that affect the probability of current e-cigarette use.We conducted a cross-sectional study using data from the National Health and Morbidity Survey 2017. Respondents aged 13–18 years were included in the study. The data were analysed using STATA (v.15).The prevalence of current e-cigarette use was 9.1%. Male gender, older age, Malay ethnicity, schooling in urban area, current smoking, current alcohol use, current drug use, having parents that are not married and living together and parental tobacco product use were significantly associated with current e-cigarette use. There were significant interactions between: i) gender with age, ethnicity, current smoking, current alcohol use and current drug use, ii) ethnicity with current smoking and current alcohol use, iii) locality with ethnicity, current smoking and current alcohol use, iv) current drug use with age, ethnicity, current smoking and current alcohol use, v) parental marital status with gender, age and ethnicity, and vi) parental tobacco use with ethnicity and current smoking.Our findings identify significant associations between sociodemographic characteristics, lifestyle risk behaviours and parental factors with current e-cigarette use. They also provide new insight into the interactions between these factors that affect the probability of current e-cigarette use among West Malaysian adolescents. Efforts to tackle e-cigarette use in Malaysian adolescents should target sociodemographic characteristics, lifestyle risk behaviours and parental factors such as smoking cessation intervention for parents. According to the World Health Organization (WHO), adolescents are individuals aged 10–19 years . AdolescThere have been studies aimed at understanding the factors that influence adolescent e-cigarette use. Individual risk factors such as being male , older  and an uWith growing concern over adolescent e-cigarette use, more research is needed to better understand the associations between e-cigarette use with certain risk factors and whether certain groups of adolescents may be more likely to engage in concurrent use of substances. A recent study highlighted the moderating effect of age and sex in the association between e-cigarette use and other risk factors. For example, female e-cigarette users were more likely to use alcohol and drugs compared to males, while younger e-cigarette users were more likely to smoke and use drugs compared with older adolescents . FurtherStudies on e-cigarette use in Malaysia have focused on the adult population , 22, whiWest Malaysia consists of 11 states  and two federal territories , with an estimated population of 25.9 million in 2020 . This stThe NHMS 2017 was a cross-sectional study which used two-stage stratified cluster sampling design to select a representative sample of secondary in-school adolescents in Malaysia . The samd) (schools) to be sampled was determined; (iv) The number of respondents to be sampled from each cluster was determined ; (v) Obtain the sampling interval (SI) by dividing the total population by the number of clusters to be sampled; (vi) A random number between 1 and the SI was chosen (this is the random start (RS)); (vii) The following series were calculated: RS; RS + SI; RS + 2SI; …. RS + (d-1)*SI; (viii) The probability for each cluster being sampled: Prob 1 = (a x d) / b . Prob 1 is proportionate to the value of “a” (number of students in the clusters).West Malaysia was stratified into 11 states and two federal territories. The first stage of sampling was the random selection of secondary schools with probability proportionate to school enrolment size. Probability proportion to size is a sampling procedure under which the probability of a unit being sampled is proportional to the size of the ultimate unit, giving larger clusters a greater probability of selection and smaller clusters a lower probability of selection. Based on the required sample size for each stratum, the following steps were applied for each stratum: (i) A list of primary sampling units (schools) and their population size was developed; (ii) The cumulative sum of the population size was determined; (iii) The number of clusters (SI) was determined by dividing the total number of classes with the number of classes to be sampled; (iii) A random number for the start of selection (RS) was determined; (iv) Classes were selected accordingly based on the SI. A total of 176 secondary schools were selected from West Malaysia and subsequently four to 10 classes were selected from each selected school to participate in the NHMS 2017. The present study was a cross-sectional study involving in-school adolescents in West Malaysia . Respondents aged 13–18 years were included in the present study.The second stage of sampling was the selection of classes in the selected schools by using the systematic random sampling. As mentioned earlier, in order to ensure that all individuals in the population have the same probability of selection irrespective of the size of their cluster, the same number of individuals were sampled from each cluster. The following steps were done for each selected schools: (i) The average number of students in a class from form 1 to 5 was determined and the number of classes to be sampled was determined accordingly to obtain the required number of students from each school; (ii) The sampling interval  2012 . The queThe dependent variable in our study (current e-cigarette use) was defined as the use of e-cigarettes in the past 30 days .The independent variables were gender , age , locality , ethnicity , current smoking , current alcohol use , current drug use , parents’ marital status , parent(s) use of any tobacco product  and parental supervision in the past 30 days .Locality was categorized based on the school location , while tCurrent smoking was defined as the use of any smoked tobacco products in the past 30 days, including manufactured cigarettes, roll-your-own cigarettes, traditional hand-rolled cigarettes, shisha, cigar or pipe . CurrentParents’ marital status was categorized into ‘married and living together’ and ‘other’  , while pn) and percentages (%) were used for qualitative data, while the appropriate measure of central tendency was used for quantitative data. The prevalence of current e-cigarette use was estimated using the percentage (%) and its corresponding 95% confidence interval (CI). Chi-square analysis was performed to determine the association between current e-cigarette use with all the independent variables. The associations between current e-cigarette use with all the independent variables were tested with simple logistic regression to obtain the crude odds ratio (OR). Variables with p < 0.25 [The data were analysed using STATA (v.15). Descriptive statistics of frequencies  2017 , a crossA total of 22,228 respondents aged 13–18 years with the mean age of 14.99 years (SD = 1.42) were included in the study. The prevalence of current e-cigarette use among the respondents was 9.1% . Higher There were significant interactions between: i) gender with age, ethnicity, current smoking, current alcohol use and current drug use, ii) ethnicity with current smoking and current alcohol use, iii) locality with ethnicity, current smoking and current alcohol use, iv) current drug use with age, ethnicity, current smoking and current alcohol use, v) parental marital status with gender, age and ethnicity, and vi) parental tobacco use with ethnicity and current smoking. The gender-age interaction  shows thThe gender-current smoker interaction  shows thIn the ethnicity-current smoker interaction , currentFor the locality–ethnicity interaction , Malay rFor the current drug use-age interaction , there iFor the parental marital status-gender interaction , male adFor the parental tobacco use-ethnicity interaction , Malay aThe present study reports the most recent prevalence of current e-cigarette use among in-school adolescents in West Malaysia. We found that almost one-tenth of in-school adolescents in West Malaysia are current e-cigarette users. The prevalence is lower than that reported among adolescents in Canada (11.0%)  and IndoNevertheless, the prevalence of current e-cigarette use among in-school adolescents in Malaysia in the present study is higher compared to a report in 2012 (1.2%) , but simWe found that male respondents, older adolescents, Malays, respondents who were schooling in urban areas, current smokers, current alcohol users, current drug users, respondents whose parents were not married and living together and respondents whose parent(s) used any tobacco product were more likely to be current e-cigarette users. All these nine factors have interactions with other factors. The nineteen interactions between factors in this study are discussed as the probability of current e-cigarette use.The interaction of gender with age shows a greater probability of current e-cigarette use among older adolescents. Studies conducted in the United States  and MalaThe gender-ethnicity interaction shows a greater probability of current e-cigarette use among Malay adolescents, which is in line with that of another recent survey among Malaysian adolescents . The lowThe interaction of gender and current smoking shows a greater probability of current e-cigarette use among current smokers. This finding is consistent with that of other studies in Indonesia  and MalaThe gender-current alcohol use interaction and gender-current drug use interaction show that current alcohol users and current drug users had higher probability of current e-cigarette use. These findings are explainable as local  and overIn the ethnicity-current smoker interaction, current smokers had a higher probability of current e-cigarette use, while the effect of smoking status differences for current e-cigarette use was greater among Malay adolescents. In line with our findings, a study reported that both ethnicity  and smoking are associated with increased odds of using e-cigarettes among adolescents in Malaysia. The greater effect in the high-risk group is explainable as Malay adolescents had higher odds of current e-cigarette use compared to non-Malay adolescents  .The interaction of ethnicity and current alcohol use shows a higher probability of current e-cigarette use among current alcohol users. Apart from the fact that alcohol, tobacco and drug use tend to cluster among adolescents , 47, stuFor the locality–ethnicity interaction, Malay respondents had a higher probability for current e-cigarette use. Ethnicity differences had a greater effect for current e-cigarette use in the high-risk group (urban), which could be partly explained by easier access to internet, as websites serve as platform for information and sales of e-cigarettes . For theFor the locality–current alcohol use interaction, the probability of current e-cigarette use was higher for current alcohol users, while the effect of alcohol use differences for current e-cigarette use was greater in the low-risk group . This finding is explainable as rural dwellers in Malaysia are more likely to be heavy alcohol drinkers , while sThe interaction of drug use and ethnicity shows a greater effect for current e-cigarette use among non-current drug users. In contrast to our findings, local studies have reported that the use of substances was less likely to cluster in Malay compared to non-Malay adolescents , 53. WitFor the parental marital status-gender interaction, male adolescents had a higher probability of current e-cigarette use. Studies conducted in Hong Kong , Korea  and MalaThe interaction of parental marital status and age shows a higher probability of current e-cigarette use among older adolescent. The greater effect of age differences for current e-cigarette use among the low-risk group (married and living together) is mainly due to the markedly lower probability of current e-cigarette use in the low-risk group than in the high-risk group among younger adolescents. In line with our finding, a study conducted in the United States reported that adolescents who experience family instability are at higher risk of developing poor-quality peer networks, which contribute to problem behaviour. The association between family instability and problem behaviour was also found to be stronger for younger adolescents .The parental marital status-ethnicity interaction shows that Malay adolescents had higher probability of current e-cigarette use. The reason of such finding could be due to the highest prevalence of e-cigarette use among adults of Malay ethnicity compared to other ethnicities , as adolFor the parental tobacco use-ethnicity interaction, those whose parent/s used any tobacco product had higher probability of current e-cigarette use. Our findings substantiate the findings of studies that reported a significant association between parental tobacco product use and current e-cigarette use among adolescents , 59. ThiThere are limitations to our study. First, temporal causality cannot be determined, as our study utilized cross-sectional data. Second, data were self-reported, and there is the possibility of imprecise reporting due to recall bias. Despite certain methodological limitations, our study used a large representative sample of adolescents from West Malaysia, and therefore produces more generalisable findings.The present study found that almost one-tenth of Malaysian adolescents were current e-cigarette users. Current e-cigarette use is associated with sociodemographic characteristics, lifestyle risk behaviours and parental factors. Our findings provide new insight into the interactions between various combinations of sociodemographic characteristics , lifestyle risk behaviours  and parental factors  that affect the probability of current e-cigarette use among West Malaysian adolescents. Efforts to tackle e-cigarette use should target sociodemographic characteristics, lifestyle risk behaviours and parental factors such as smoking cessation intervention for parents. Adolescents who are current e-cigarette users were more likely to engage in other lifestyle risk behaviours, including smoking, alcohol and drug use. Thus, there is a need to monitor these risk behaviours simultaneously among adolescents. The need for smoking cessation intervention must be emphasized. These efforts should be complemented with findings from the sociodemographic characteristic, lifestyle risk behaviour and parental factor interactions. Policies, programmes and interventions must be strengthened and tailored towards the needs of adolescents who are at risk of e-cigarette use.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Beta-blockers affect glioma proliferation by inducing a brief reduction of cAMP and a temporary cell cycle arrest in vitro. Contrasting results were observed concerning glioma cell migration. The identified clinical study did not find an association between beta-blockers and survival in glioma patients. Although preclinical studies provide scarce evidence for the use of beta-blockers in glioma, they identified potential pathways for targeting glioma. Future studies are needed to clarify the effect of beta-blockers on clinical endpoints including survival outcomes in glioma patients to scrutinize the value of beta-blockers in glioma care.Given the median survival of 15 months after diagnosis, novel treatment strategies are needed for glioblastoma. Beta-blockers have been demonstrated to inhibit angiogenesis and tumor cell proliferation in various cancer types. The aim of this study was to systematically review the evidence on the effect of beta-blockers on glioma growth. A systematic literature search was performed in the PubMed, Embase, Google Scholar, Web of Science, and Cochrane Central to identify all relevant studies. Preclinical studies concerning the pharmacodynamic effects of beta-blockers on glioma growth and proliferation were included, as well as clinical studies that studied the effect of beta-blockers on patient outcomes according to PRISMA guidelines. Among the 980 citations, 10 preclinical studies and 1 clinical study were included after title/abstract and full-text screening. The following potential mechanisms were identified: reduction of glioma cell proliferation (The online version of this article (10.1007/s10143-020-01277-4) contains supplementary material, which is available to authorized users. Glioblastomas are the most common primary malignant brain tumors . With a Beta-blockers are competitive antagonists of the sympathetic effects of catecholamines on beta-adrenergic receptors , 8. TheyA systematic literature review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The EmbThe following data were extracted from the included preclinical studies: year of publication, name of the first author, type of cell line, beta-blocker used, optimum concentration, cyclic adenosine monophosphate (cAMP) formation, morphology, and percentage of beta-adrenergic receptor blocked as determined relative to all cells on the plate. From clinical studies, the following parameters were extracted: year of publication, name of the first author, glioma categorized by grade, beta-blocker used, number of patients, 1-year survival rate, and median overall survival in months.The search identified 980 unique studies. After screening of title and abstract followed by screening of the full text, we included 11 studies  that examined the effect of beta-blockers on glioma growth and patient outcomes  reduction of glioma cell proliferation , 28, 29,Eight studies investigated the effect of beta-blockers on glioma cell proliferation , 28, 29.In two in vitro studies, elevated cAMP levels were associated with increased glioma cell proliferation and stellate transformation , 24. SteErguven et al. explored the effect of beta-blockers on the cell cycle . In theip < 0.001).Two studies examined the effect of beta-blockers on angiogenesis and glioma cell migration , 27. PavAnnabi et al. did not observe a direct association with cell migration in vitro but reported an association between propranolol administration and matrix metalloproteinase nine (MMP-9) and Hu antigen R (HuR) . MMP-2 wOne study investigated the effect of beta-blockers on the sensitivity of glioma cells to cytotoxic drugs . In thisp < 0.05). At 72 h, this percentage of apoptotic cells was decreased to 2% (p < 0.01) [Erguven et al. also examined the effect of beta-blockers on programmed cell death . They sh < 0.01) . The und < 0.01)  Fig. 3)p < 0.05)p value 0.53) or progression-free survival .Johansen et al. studied the effect of beta-blockers on recurrent glioblastoma in a retrospective cohort of 218 patients . This stThis systematic review synthesizes all preclinical and clinical evidence on the effect of beta-blockers on glioma growth and patient outcomes. Preclinical studies have identified several potential mechanisms of action through which beta-blockers might have a potential effect on (i) glioma cell proliferation , 28, 29,Beta-blockers have been suggested to reduce the risk of prostate cancer , as wellThe current literature provides limited evidence to integrate beta-blockers into glioma treatment. However, the preclinical studies have identified various mechanisms through which the application of non-selective beta-blockers could potentially attenuate glioma cell proliferation.Microglia have been demonstrated to upregulate matrix-altering enzymes, especially MMP-2 and MMP-9, to facilitate glioma infiltration. Downregulation of the MMP-cascade results in less glioma invasion, angiogenesis, and growth . This syFinally, the observational study did not find a significant effect of beta-blockers on the survival rate of recurrent glioblastoma patients. In the multivariate analysis, neurocognitive deficits and steroids were included without reported association with overall survival. The aforementioned variables were included as a result of a study which reviewed variables influencing the quality of life and prognosis in glioblastoma patients . The sta. Studies that consider mediating proteins as surrogate endpoints rely on the strong assumption that these mediators are included in the causal pathway. Outcomes such as cell growth or migration are independent of such assumptions and thus would be able to support more robust conclusions. Additionally, this systematic review is subject to publication bias. Despite these limitations, we believe this review provides valuable insights into the potential utility of beta-blockers in glioma care and valuable target mechanisms for the development of anti-glioma drugs. To the best of our knowledge, this is the first systematic review synthesizing the current body of evidence on the effects of beta-blockers on glioma growth and patient outcomes, both in the preclinical and clinical realms.There are limitations to the present review. Most studies predominantly focused on associations between beta-blockers and mediator enzymes (such as MMP-9) instead of examining their direct effect on clinical  or physiological endpoints . As such, no inferences can be drawn on the direct association between beta-blockers and these endpointsPreclinically, almost all studies were performed in vitro. Therefore, future research on the effect of beta-blockers in addition to conventional treatment of glioma should ideally be performed in vivo. Preclinical studies could further investigate the effect of beta-blockers in vivo by measuring clinically relevant outcomes  and physiological endpoints  in mice. Preliminary results of an ongoing clinical study suggest that propranolol in combination with etodolac (VT-122) might have a positive effect on survival in recurrent glioblastoma patients. Hypothetically, VT-122 attenuates inflammation and thereby increases tolerability for anticancer therapy , 52. AddAlthough preclinical research provides limited evidence for the effectiveness of beta-blocker usage in glioma care, this review identifies potential mechanisms through which beta-blockers might affect glioma proliferation, migration, drug sensitivity, and programmed cell death. However, the effect on patient outcomes remains unclear due to the limited body of clinical evidence. In addition to identifying potential mediators, future preclinical research should further explore the effect of beta-blockers on physiological and clinical endpoints. Including medication and beta-blocker usage in large prospective neuro-oncological registries could be a valuable step for examining the direct or conjunct effect on cancer-related patient outcomes.ESM 1(PDF 221 kb).


Multichannel near-infrared spectroscopy (NIRS), including 52-channel NIRS (52ch-NIRS), has been used increasingly to capture hemodynamic changes in the brain because of its safety, low cost, portability, and high temporal resolution. However, optode caps might cause pain and motion artifacts if worn for extended periods of time because of the weight of the cables and the pressure of the optodes on the scalp. Recently, a small NIRS apparatus called compact NIRS (cNIRS) has been developed, and uses only a few flexible sensors. Because this device is expected to be more suitable than 52ch-NIRS in the clinical practice for patients with children or psychiatric conditions, we tested whether the two systems were clinically comparable. Specifically, we evaluated the correlation between patterns of hemodynamic changes generated by 52ch-NIRS and cNIRS in the frontopolar region. We scanned 14 healthy adults with 52ch-NIRS and cNIRS, and measured activation patterns of oxygenated-hemoglobin [oxy-Hb] and deoxygenated-hemoglobin [deoxy-Hb] in the frontal pole while they performed a verbal fluency task. We performed detailed temporal domain comparisons of time-course patterns between the two NIRS-based signals. We found that 52ch-NIRS and cNIRS showed significant correlations in [oxy-Hb] and [deoxy-Hb] time-course changes in numerous channels. Our findings indicate that cNIRS and 52ch-NIRS capture similar task-dependent hemodynamic changes due to metabolic demand, which supports the validity of cNIRS measurement techniques. Therefore, this small device has a strong potential for clinical application with infants and children, as well as for use in the rehabilitation or treatment of patients with psychiatric disorders using biofeedback. Near-infrared spectroscopy (NIRS) has been widely used as an appealing technology in the field of neuropsychiatry . NIRS haBased on these clinical advantages and disadvantages, multichannel NIRS is thought to be most useful for research with infants and children, and for rehabilitation or biofeedback treatment of psychiatric disorders . RecentlFourteen healthy adults participated in this study . Participants who had psychiatric conditions, neurological disorders, head injury, or alcohol or other substance dependence, were taking psychotropic medication were excluded from the study. Following an explanation of the study, we obtained written informed consent from all participants. This study was conducted with the approval of the ethics committee of Keio University Hospital.The task used in the present study was similar to that in Takizawa et al. . ParticiDuring the task period, participants were instructed to produce as many nouns as possible, without using repetitions or proper nouns, beginning with a designated syllable. One of these three sets of initial syllables  were presented in counterbalanced order to the participants. Initial syllables were changed every 20-s during the 60-s task. The linear subtraction method (task measurements minus pre- and post-task baselines) minimized the vocalization effects during the VFT. The total number of correct words generated during the VFT was taken as a measure of task performance. The order of measurements by 52ch-NIRS and cNIRS was randomized for all participants.We used a 52ch-NIRS machine  with two wavelengths (695 and 830 nm) and a time resolution of 0.1 s to measure the relative changes in absorbed near-infrared light. These changes were transformed into changes of [oxy-Hb], [deoxy-Hb], and total-hemoglobin  concentrations as indicators for brain activity using a modified Beer–Lambert law  uses three different wavelengths  with a time resolution of 0.13 s. The device is 62 mm long, 82 mm wide, 28 mm thick, and weights 111 g . It has All data were analyzed using the “integral mode.” The pre-task baseline was determined as the mean over a 10-s period just prior to the task initiation, and the post-task baseline was determined as the mean over the last 5-s of the post-task period. Linear fitting was applied to the data between these two baselines. After applying linear fitting, to directly compare the data that was recorded at different temporal resolutions, the 52ch-NIRS data (sampling rate = 0.1 s) was linearly up-sampled to a sampling rate of 0.01 s and then down-sampled to 0.13 s, which was the sampling rate of the cNIRS data. Finally, these data were used in following correlation analyses.A measuring point of activation (channel) was defined as the region between one emitter and one detector. Thus, the probe set for 52ch-NIRS covered brain regions that included the bilateral prefrontal {approximately dorsolateral , ventrolateral , and frontopolar [BA 10]}, superior temporal, and middle temporal cortical regions. The cNIRS optodes were set on the forehead located above the frontal pole (BA10). The correspondence between the optode positions and the measurement areas on the cerebral cortex was confirmed based on a previous multi-subject study of anatomical craniocerebral correction via the international 10-20 system  to compahttps://www.scipy.org) package for python.For each of the four channel pairs for each participant, we calculated the Spearman correlation coefficients in the NIRS-based signal time-courses (for [oxy-Hb] and [deoxy-Hb]) between 52ch-NIRS and cNIRS. Overall, we obtained 56 correlation coefficients for both the [oxy-Hb] and [deoxy-Hb] measurements (4 channel pairs × 14 participants). Considering the statistical issue of multiple comparisons, we set the threshold for statistical significance at 0.00089 (0.05/56 pairs). Preprocessing (linear correction and resampling) and statistical analysis were conducted using in-house code based on the SciPy .We calculated the signal to noise ratio (SNR) using the same methods of the previous NIRS study . In partP < 0.00089; corrected for multiple comparisons). In particular, 91% of channels (51/56) revealed significant time-course correlations in [oxy-Hb] and 77% of channels (43/56) showed significant time-course correlations in [deoxy-Hb]. Moreover, the means of the correlation coefficients were 0.44([oxy-Hb]) and 0.39([deoxy-Hb]) across all 56 pairs. We then confirmed that there were no significant differences in mean correlation coefficients between [oxy-Hb] and [deoxy-Hb] (P = 0.287). Furthermore, there were no significant differences of SNR between two devices in four channel pairs for both [oxy-Hb] and [deoxy-Hb] . The high correlation values might have resulted from using similar probe measurement systems and the same experimental paradigm. In addition, no significant SNR differences between two devices may also support our findings of high correlation values. In particular, distance from the scalp (and brain) does not differ significantly between the two systems among the optode pairs. Channel location for the devices was not exactly the same because of their structural differences, but because of the relatively low spatial resolution of NIRS (between 2 and 3 cm), we believe that the effect of location difference on [oxy-Hb] and [deoxy-Hb] activation patterns was minimal. A previous NIRS-fMRI correlation study demonstrated that longer distance from the scalp and brain negatively affects NIRS SNR and reduces NIRS ([oxy-Hb])-fMRI(BOLD) time-course correlations . As a potential reason of these observations, artifacts due to head motion might have contributed to these findings. Even though SNR is comparable between two devices, we assume that artifacts are more prevalent in 52ch-NIRS because its probe set covers broad regions from bilateral prefrontal to temporal cortices, which may induce lower correlation values. cNIRS has only 4 channels located in the frontal pole, is lighter and smaller than 52ch-NIRS, and fastening it tightly to the frontal scalp is easier. In multichannel NIRS, such as 52ch-NIRS, optode caps can become uncomfortable to wear over time because of the weight of the cables and the pressure of the optodes on the subject's scalp, which may induce more motion artifacts. Thus, reducing caps and cable weights by using cNIRS can help reduce these artifacts. Moreover, optimum processing for reducing artifacts of NIRS signals requires advanced knowledge of computer science, signaling processing, and engineering, which may make more accessible for use in the clinical environment.Compared with other neuroimaging methods such as fMRI, NIRS has the disadvantage of lower spatial resolution and a lower SNR ratio . HoweverThis study had several limitations. First, fundamental system differences may affect our findings of lower correlation values in a few channel pairs. In particular, while both systems use the same theory of calculating [oxy-Hb] and [deoxy-Hb], they adopt distinct wavelengths, laser power, and sampling rates. Second, even though there were no significant differences in SNR among two devices, we observed relatively large variation in individual SNR data, which may be implicated in the low correlation values in some channels. Thus, considering statistical power due to relatively small sample size, the results of this exploratory analysis should be interpreted with caution and further research is warranted in a larger sample.In summary, we revealed that 52ch-NIRS and cNIRS demonstrated significant and high correlations in the patterns of hemodynamic change detected in the bilateral frontal pole, suggesting that cNIRS and 52ch-NIRS are similarly able to capture task-dependent hemodynamic changes. These findings indicate that cNIRS has the potential to be a novel treatment method for psychiatric conditions.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of Keio University Hospital. The patients/participants provided their written informed consent to participate in this study.JH, MM, and BY designed the study and wrote the manuscript. Data were collected by JH, AT, YY, and FM. JH and BY analyzed and interpreted the data. All authors have approved the final manuscript.This work was supported by a project grant of BrainEnergy Inc.MM has a potential conflict of interest; for the present study, BrainEnergy Inc. provided a project grant and material support [temporary rental of cNIRS (BrainEnergy System)]. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ABSTRACT IMPACT: Chronic kidney disease (CKD) affects ˜15% of the US population and the majority of patients are diagnosed too late to benefit from early intervention. We are developing a new diagnostic imaging tool (RadioCF-PET) for the kidney to enable early detection of diseases and to monitor overall kidney health. OBJECTIVES/GOALS: Nephron mass, or the number of functioning nephrons, is a measure of the functional capacity of the kidney. RadioCF-PET may enable early detection of nephron loss in patients with or at risk of CKD before changes are clinically detectable, facilitating early interventions to improve outcomes in these patients. METHODS/STUDY POPULATION: RadioCF-PET, labeled with Cu-64, has the advantage of using sub pharmacological doses for imaging, carrying low risk and can be used with the FDA’s exploratory IND (eIND) mechanism for early in human testing. We are developing the technology to be used in pre-eIND toxicology and pharmacology studies. We are also developing other aspects of translational science to propel this technology toward translation, including: market analysis, critical path to market, customer discovery, and commercialization strategy. RESULTS/ANTICIPATED RESULTS: Milestone 1: Apply technology in mouse model study and in human kidneys rejected for transplant. Anticipated Result 1: PET signal from RadioCF-PET correlates with glomerular density in healthy and diseased model male mice (R2 = 0.98). RadioCF-PET signal correlates with glomerular number in a donated human kidney (R2= 0.78). Milestone 2: Application to federal funding (STTR) and gap funding mechanisms to enable pre-eIND studies. Anticipated Result 1: Application for funding will aid to clarify and validate our market analysis and commercialization strategy. Milestone 3: Continued research and development with the technology in new studies. Other Anticipated Results: Future work with RadioCF-PET will enhance technology performance in preparation for pre-eIND studies. DISCUSSION/SIGNIFICANCE OF FINDINGS: We foresee a large clinical and commercial potential for RadioCF-PET to provide precise, early monitoring in patients at risk for or with CKD. The two biggest hurdles for clinical translation are validating safety and proving efficacy. This work targets both issues to facilitate RadioCF-PET toward clinical translation.