Mechanical forces play an important role in initiation, development, and remodeling of cardiovascular disease. Computational biomechanics has been widely applied in cardiovascular research in recent years, and extensive work has demonstrated that it has not only improved our understanding of the disease progression and remodeling following treatment, but also helped with diagnosis and treatment as well as design of medical devices. Heart and artery are active areas where computational modeling and biomechanics have made remarkable advances in investigating mechanisms governing disease development and finding optimal strategies for the management and treatment of those diseases and improving public health. Multi-disciplinary collaborations from medical imaging, bioengineering, computational modeling and clinical sciences may lead to advances in clinical applications. The goals of the Research Topic on “Computational Biomechanics of the Heart and Vasculature with Potential Clinical and Surgical Applications” were to: (a) advance ventricle biomechanical modeling to better understand ventricle function, disease initiation and development, ventricle remodeling, optimization of surgical treatment; and (b) advance vulnerable plaque biomechanical modeling to better understand mechanisms governing plaque development and rupture, plaque remodeling, optimization of surgical treatment including stenting, graft and others. Research in other areas related to cardiovascular disease was also encouraged. The Topic collects novel studies of the cross-disciplinary research in biomechanics and cardiovascular diseases. It covers a wide range of research work from disease mechanism, computational modeling, material characterization, imaging to clinical studies. Articles include new methodologies that have been recently developed to better model the hemodynamics and mechanical properties of cardiovascular system. A significant number of articles in this Research Topic are computational biomechanical simulations of the heart and vasculature with a focus on clinical and surgical applications for better patient outcomes. The second focus is using computational biomechanics to better understand the important role of hemodynamics on disease progression. There are also collections of articles demonstrating how computational modeling has contributed to medical device design and optimization. This Topic provides a comprehensive overview of the advances in biomechanical technologies and their applications in cardiovascular diseases.Seddighi and Han established the theoretical foundation for non-circular blood vessel bent buckling and simulated the buckling behavior of arteries with elliptic and eccentric cross-sections using finite element analysis. Their results demonstrated that oval or eccentric cross-section increases the critical buckling pressure of arteries. To better characterize the mechanical behavior of coronary plaque, Torun et al. developed a framework of material characterization pipeline by combining ex vivo inflation test, inverse modeling, ultrasound/MR measurements and a machine learning-based Bayesian optimization. This could be a promising way in the future to investigate the non-linear mechanical properties of soft tissues. The importance of incorporating pre-stress in biomechanical simulation is well recognized and Fonken et al. executed time-resolved three-dimensional ultrasound-based fluid-structure interaction (FSI) simulations on an extensive set of patient data to quantify the influence of the pre-stress estimation on wall mechanics and hemodynamics. The results underline the importance of incorporating pre-stress in FSI simulations and demonstrated that their framework could provide an important tool for personalized modeling and longitudinal studies on abdominal aortic aneurysm growth and rupture risk. Moreover, considering pre-stress and anisotropy, Vignali et al. presented a new method based on a linearization strategy to perform patient- specific FSI simulations and demonstrated a significant reduction of computational costs for FSI analysis of ascending thoracic aortic aneurysm. Recent research has shown physics-based mathematical modeling and inverse method to be a useful predicting tool for hemodynamic monitoring. To verify this method in clinical setting, Bikia et al. validated a mathematical inverse-problem solving method for acquiring non-invasive estimates of mean aortic flow and stroke volume using age, weight, height and measurements of brachial blood pressure and carotid-femoral pulse wave velocity. Chen et al. established a multiscale modeling approach to reveal the vascular remodeling behavior under the interaction between the macroscale of wall shear stress (WSS) loading and the microscale of cell evolution by employing computational fluid dynamics (CFD) and agent-based model.To better understand artery buckling behavior, Li et al. used 0D/3D geometric multiscale modeling method to investigate the long-term hemodynamic effects of enhanced external counterpulsation on the vascular intima in coronary artery disease treatment and demonstrated that a longer pressurization duration might result in an excessive WSS, which could easily damage the vascular intima at coronary stenosis. Banerjee et al. proposed a new hemodynamic factor - pressure drop coefficient and suggested that it could be an alternate diagnostic index for decision-making in the cardiac catheterization laboratory, in addition to fractional flow reserve. Cao et al. investigated coronary hemodynamic characteristics and suggested that patient-specific hemodynamic characteristics may play an important role which should be considered as an important factor rather than depending on coronary stenosis alone.Computational biomechanics has been widely applied in clinical applications to improve diagnosis and treatment planning. This Research Topic collects a wide arrange of such applications in different cardiovascular diseases. Coronary is one of the most active areas of research. Kan et al. applied a virtual stent-graft deployment model in patient-specific type B aortic dissection to investigate the impact of stent-graft length on wall stress distribution post thoracic endovascular aortic repair. The study demonstrated the potential of using the virtual stent-graft deployment model as a pre-surgical planning tool to help select the most appropriate stent-graft length for individual patients. Hou et al. investigated the effect of valve height on the aortic valve opening and closing and suggested that it is important to select the appropriate range of valve height for a smoother blood flow through the aortic valve and valve closure in the case of continuous aortic root dilatation in patients with aortic valve disease. Xiong et al. investigated the effects of the patent ductus arteriosus (PDA) on the flow features of the modified Blalock–Taussig shunt (MBTS) to help with preoperative surgery design and postoperative prediction. These findings suggested that preservation of PDA is not recommended during MBTS procedures because it negatively influences hemodynamics and may lead to pulmonary over-perfusion, inadequate systemic perfusion, and a heavier cardiac burden, thus increasing the risk of heart failure. Li et al. assessed the implant depth of a Venus-A prosthesis during transcatheter aortic valve replacement (TAVR) when the areas of eccentric calcification were distributed in different sections of the aortic valve. It was found that the implant depth of the Venus-A prosthesis is closely related to the distribution of eccentric calcification in the aortic valve during TAVR and suggested that future surgical strategy should consider aortic root morphology to prevent prosthesis migration.Aorta is another important research focus. Ahmed et al. performed a CFD analysis to investigate the hemodynamic effect of the endovascular Fontan revision based on anatomy, blood flow and pressure on a patient-specific model of a 2-year-old female with hypoplastic left heart syndrome. These results indicated that the proposed endovascular revision would lead to unfavorable hemodynamic conditions post procedure thereby confirming CFD modeling is a beneficial tool in surgical planning for single ventricle congenital heart defect patients. Fumagalli et al. quantified the effects of different types of hypertrophic cardiomyopathy on intraventricular blood flow and pressure gradients and suggested the computational approach is useful to guide surgical treatment of the disease.In heart applications, Tian et al. investigated of the influence of enhanced external counterpulsation (EECP) treatment on blood flow distribution and WSS-derived hemodynamic factors in the carotid bifurcation. Results indicated that EECP intervention increased internal carotid artery blood flow and WSS in the carotid bifurcation in patients with neurological disorders. Lu et al. evaluated contrast material concentration and transport in middle cerebral artery (MCA) stenosis and proposed contrast material remaining time could be a quantitative indicator to evaluate the changes in blood perfusion after the intervention for MCA stenosis. Zhang et al. quantified intracranial aneurysms hemodynamic parameters in the flow diverting stents after single- and dual-stent treatments and suggested that average pore size of stent wires at the aneurysm orifice could be a potential index for predicting the efficacy of flow-diversion treatments. Wang et al. investigated the influences of the anatomorphological features of the portal venous system on hemodynamic characteristics before and after splenectomy, with emphasis on identifying the specific anatomorphological features that make postoperative hemodynamic conditions more clot-promoting.Some other applications were also included. Wu et al. applied CFD to optimize the hydraulic and hemolytic performance of an inhouse centrifugal maglev blood pump with a secondary flow path through variation of major design variables, with a focus on bringing down intensity of turbulence and secondary flows. The results shed light on the impact of major design variables on the performance of modern centrifugal blood pumps with a secondary flow path. In a second article, Wu et al. reported a novel design of a wearable and portable extracorporeal centrifugal blood pump based on an in-house centrifugal maglev blood pump. Compared with the baseline pump, the hydrodynamic and hemolytic performance of the portable pump was maintained without serious degradation. Li et al. explored changes in the hemodynamic behavior of the flush flow with respect to the flow injection speed and the system design to aid the design of a novel flow-blockage-free intravascular endoscope using three candidate endoscope designs. The results suggested that the endoscope design with a diameter narrowing of 30% at the endoscope neck might yield images of a better quality. Qi et al. presented the evaluation on a novel design of balloon post-dilation catheter using an experimentally verified finite element method, by comparing a newly designed spherical-tip catheter with a traditional conical-tip catheter. The study indicated that the finite element model could be a helpful tool for future optimization and evaluation of novel catheters, so as to save time and budget in product development and reduce/replace animal studies.Biomechanical application to medical device design is promising. Pagoulatou et al. investigated acute and long-term effects of proximal aortic compliance decrease on central hemodynamics by leveraging a computational model of the cardiovascular system and demonstrated that the main mechanism that drives hypertension acutely after banding is enhancing the forward wave, which becomes even more significant after the heart remodels itself to match the increased afterload. Wang et al. conducted a quantitative CFD simulation to elucidate the local hemodynamic effects of a short-term left ventricular assist device in a patient-specific aorta model. The study provided a strategy for further predicting and assessing the effects of these hemodynamic signals on the aorta. Yan et al. compared the differences in hemodynamics and mechanical properties of bicuspid aortic valve (BAV) with different phenotypes throughout the cardiac cycle by FSI modeling. It demonstrated that the hemodynamic parameters may be critical for prediction of other subsequent aortic diseases and differential treatment strategy for certain BAV phenotype.Computational models have also been applied to investigate cardiovascular disease progression. In aortic diseases, Deng et al. determined the mechanical effects in the formation of apical aneurysm by comparing the myofiber stress on the apical wall between healthy, subaortic obstruction, and midventricular obstruction models. The results suggested that the midventricular obstruction significantly increase the myofiber stress in the apex of left ventricle, which might directly initiate the apical aneurysm. Ghasemi et al. proposed a local stress modulated remodeling algorithm to explore the mechanical response of arterial tissue to the remodeling of collagen fibers and to understand the collagen fiber patterns required in carotid artery and plaque tissue to maintain plaque stability. Using the remodeling algorithm, the optimum fiber patterns in various patient specific plaques are identified. Wang et al. attempted to quantify patient-specific in vivo coronary plaque material properties based on IVUS-based multi-patient study. It was found that there is a large inter-patient and intra-patient variability in the in vivo plaque material properties. Wang at al. investigated the effect of flow alterations on endothelial cell distribution in the presence of gap between two struts within the parallel flow chamber. It was found that flow disturbance related to the presence of the gap and the strut orientation angle might affect the reendothelialization process on the side surfaces of the strut between the gap areas. Qing et al. analyzed the hemodynamic changes in endovascular aneurysm repair stent grafts under different configurations, angular directions, and angles. It was found that the use of the cross-limbed technique did not increase the risk of thrombosis under poor neck anatomy and the main factor affecting the hemodynamic index was the angle of the aneurysm neck, which suggested that the hemodynamic changes caused by the angle of the aneurysm neck could not be ignored when investigating the effect of the morphology of stent graft on hemodynamics.This exciting collection of papers represent the recent developments in biomechanical modeling for cardiovascular diseases with state-of-the-art contributions on the new advances in this growing field, with an emphasis on the interface between engineering and clinical medicine. With the growing clinical needs for more accurate diagnosis of intravascular lesions, computational tools have promising potential in the field of interventional planning. The articles published in this Topic help readers understand the great potential of biomechanical simulations in improving the diagnosis and treatment of heart and vasculature diseases and create more research opportunities for clinical translational research. We are delighted to be a part of this effort and witness the rapid expanding of biomechanics in clinical applications, providing more opportunities for future discussions and exchanges of ideas and experiences in translating biomechanics into a wider range of applications. This Topic, undoubtedly, reveals the necessity to further investigate the biomechanics of the heart and vasculature with potential clinical and surgical applications. The articles in this Topic will help to promote research in this area which eventually will lead to advancement of better treatment and management technologies and strategies.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.ZL received funding from NSFC  and ARC (DP200103492 and DP200101970).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


TOR, the master regulator of cell growth, is conserved in all eukaryotes, including the highly diverse microalgae. We review current knowledge about the conservation and regulation of TOR signaling in microalgae. 2 mitigation. Microalgae integrate light and nutrient signals to regulate cell growth. Recent studies identified the target of rapamycin (TOR) kinase as a central regulator of cell growth and a nutrient sensor in microalgae. TOR promotes protein synthesis and regulates processes that are induced under nutrient stress such as autophagy and the accumulation of triacylglycerol and starch. A detailed analysis of representative genomes from the entire microalgal lineage revealed that the highly conserved central components of the TOR pathway are likely to have been present in the last eukaryotic common ancestor, and the loss of specific TOR signaling elements at an early stage in the evolution of microalgae. Here we examine the evolutionary conservation of TOR signaling components in diverse microalgae and discuss recent progress of this signaling pathway in these organisms.Microalgae constitute a highly diverse group of photosynthetic microorganisms that are widely distributed on Earth. The rich diversity of microalgae arose from endosymbiotic events that took place early in the evolution of eukaryotes and gave rise to multiple lineages including green algae, the ancestors of land plants. In addition to their fundamental role as the primary source of marine and freshwater food chains, microalgae are essential producers of oxygen on the planet and a major biotechnological target for sustainable biofuel production and CO Together with photosynthetic prokaryotes (cyanobacteria), microalgae are thought to generate about half of the oxygen produced on the planet . In addiEmiliania huxleyi (Haptophyta). Green algae have also generated two algal lineages through secondary endosymbiosis with unrelated hosts, the relatively rare marine Chlorarachniophyta, and Euglenida  kinase is a clear example of a highly conserved eukaryotic protein that promotes cell growth when nutrients are available. TOR signaling has been well characterized in yeasts and mammals, but our current knowledge about this pathway in microalgae is still limited  to 11 detected in the Glaucophyta Cyanophora paradoxa. FAT, FRB, kinase, and FATC domains can also be identified in microalgal TOR proteins  at Thr389, which is located at the well-conserved domain FLGFTYVAP. In turn, phosphorylated S6K phosphorylates the C-terminus of the 40S ribosomal protein S6 (RPS6) at conserved Ser residues to activate translation initiation . This br Imamura , anotherEmiliania huxleyi   and starch. Remarkably, inhibition of TOR signaling by rapamycin, AZD8055, or Torin1 results in the accumulation of TAGs and starch in divergent groups of microalgae, indicating that algal TOR may play an important regulatory role in the synthesis of these storage molecules. In a rapamycin-sensitive strain of the red alga P. tricornutum . The up-regulation of the ppGpp level inhibits rRNA synthesis, leading to decreased protein synthesis in the chloroplast (C. merolae following TOR inactivation, suggesting a possible regulation of ribosome biogenesis in different cellular compartments by TOR (Photosynthesis provides ATP and NADPH required for COth algae . Proteomth algae . QuantitThe biotechnological use of microalgae as a source of biofuels and value-added products demands a better knowledge in these organisms about the signaling pathways and proteins that regulate cell growth and metabolism such as TOR. However, we are just starting to elucidate the function of TOR in microalgae, and most of our knowledge in this field comes from a few model species, which certainly narrows down the high potential associated with the evolutionary divergence of these organisms. The main regulators of TORC1 signaling in yeasts and mammals such as Rheb, RAGs, or Ragulator are not conserved in microalgae, raising the question of whether specific regulatory mechanisms and proteins evolved in these organisms. Building on this hypothesis, TORC2 seems to be absent in microalgae, although the presence of a TOR complex functionally similar to TORC2 constituted by highly divergent proteins compared with their yeast and mammalian counterparts cannot be ruled out. The answer to this question will probably come with the identification of TOR-interacting proteins in microalgae.C. merolae (2, nitrogen, and phosphorus regulate TOR activity in microalgae (TOR governs important cellular processes including translation, autophagy, and the synthesis of energy storage molecules. How TOR coordinately controls anabolic and catabolic processes and the specific underlying mechanisms are largely unknown in microalgae. Significant progress has been made in connecting TOR with starch and lipid synthesis in


With increasing interest in exploring narratives of aging through literary/oral sources, a less prominent area of inquiry has been possibilities afforded by narratives inherent in music across the life-span and into later-life. Two possible areas of interest are the arc of development and change over the life-course, and inherent content of the music. The latter element is less prominent in narrative analysis, providing a potentially rich field of discovery. While popular song parallels these possibilities, further opportunities arise from instrumental, orchestral and operatic music, allied to strengths in musicological analysis. Later compositions whose elements provide insights into life course review  as well as romantic love in later life across generations . The analysis will seek to delineate to possible avenues of interpretation of musicological analysis in understanding the phenomenology and opportunities arising from aging.


Surgical process modelling is an innovative approach that aims to simplify the challenges involved in improving surgeries through quantitative analysis of a well-established model of surgical activities. In this paper, surgical process model strategies are applied for the analysis of different Minimally Invasive Liver Treatments (MILTs), including ablation and surgical resection of the liver lesions. Moreover, a generic surgical process model for these differences in MILTs is introduced. The generic surgical process model was established at three different granularity levels. The generic process model, encompassing thirteen phases, was verified against videos of MILT procedures and interviews with surgeons. The established model covers all the surgical and interventional activities and the connections between them and provides a foundation for extensive quantitative analysis and simulations of MILT procedures for improving computer-assisted surgery systems, surgeon training and evaluation, surgeon guidance and planning systems and evaluation of new technologies. Improving these highly complex surgical procedures is a shared concern of experts with different backgrounds. However, without a solid knowledge of these treatment processes, they can hardly be improved2.For many years, surgery has been considered an art, treating surgery as an artist-driven process. This agrees with the fact that many of the processes during surgery are processed mentally inside the artist’s/surgeon’s brain. To better expose this process, expert consensus meetings, national and international guidelines provide generalized recommendations on a high abstraction level based on the pillars of evidence-based medicine. In recent years, with the introduction of new technologies, tools and hybrid operating rooms (ORs), surgeries became increasingly convoluted3, which can support analysis and predicting surgical actions. Analysis of surgical process models can reveal the bottlenecks and potential improvements to the surgeries, aiding further advances9. Such process models are a great means for finding the structural coherence of complex surgical procedures and for obtaining a profound qualitative and quantitative understanding of the relations within the surgical procedure, its variation parameters and its output parameters13. Hence, these are great tools for training surgical teams and educating young surgeons.In surgical process modelling, surgeries are treated not as an artist-driven process but as a sequence of tasks and steps that are followed by the clinical team15. Although the less invasive nature of MILT compared to open surgeries benefits the patient17, various challenges that can increase the risk of surgical errors remain, including inadequate visualization of the patient’s internal structure, lack of tactile feedback and complex navigation towards target treatment lesions19. Moreover, the continuous change of the liver shape and location due to, e.g., pneumoperitoneum, patient respiration and manipulation of the liver during an intervention, add to these challenges1. Over the last three decades, a broad range of MILT techniques has been introduced. These techniques can be categorized into three methods: laparoscopic liver resection (LLR)24, laparoscopic liver ablation (LLA)29 and percutaneous ablation (PA)34 and robot-assisted resection35. This paper focuses on the first three categories. As a result, different surgeons and interventionists use different methods and techniques, which can all be executed with large process variations. Furthermore, procedures are further dependent on factors such as:medical team skills, experience and preferencespatient-specific properties, such as patient’s body topography, patient health condition and clinical historytype, size, and location of the treatment areas.Minimally Invasive Liver Treatment (MILT) is an example of a procedure were different clinicians use different methods and techniques to treat liver lesions through surgical/interventional liver manipulations when non-surgical methods (non-invasive and chemotherapy treatments) are not adequate. After the introduction in the previous century, minimally invasive approach for liver surgery has only in recent years changed the way how benign and malignant lesions are treated36. Therefore, the aim of this study is to establish a generic surgical process model  of MILT that covers the entire procedure for a variety of MILT methods and their corresponding techniques. The proposed generic process model provides the relation between entities and allows quantitative and qualitative studies of surgical procedure. The process model was developed in a modular way to increase its usability and efficiency and to facilitate aspects of data acquisition, analysis and procedure improvement39.These all add to the inherent complexity of MILT procedures. A detailed generic process model of MILT is crucial for assessing these complexities, educating new surgeons and improving MILT procedures. Yet, to the best of our knowledge, such model has not yet been established. The sole study available on modeling the MILT process is a qualitative study describing radiofrequency percutaneous ablationmethods. Within each method several variations, referred to as types, can be distinguished:Laparoscopic Liver Resection (LLR): Resecting the necessary region of the liver parenchyma using the minimally invasive approach. Depending on the size and location of the resection region, three types of operations can be applied: formal resection41, anatomical resection44 and atypical resection, also known as parenchyma sparing47.Laparoscopic Liver Ablation (LLA): Laparoscopic ablation of the tumor by placing one or several needles inside or around the target lesion, aiming to destroy target cells by means of burning, electrifying, freezing, or chemicals. The clinician manipulates the internal structures through small incisions to make the treatment region accessible and to ensure that the treatment is performed on the right location. LLA has four different types: Radiofrequency Ablation (RFA)48, Microwave Ablation (MWA)51, Irreversible Electroporation (IRE)52, Cryoablation (CA)55 and Ethanol Injection (EI)58.Percutaneous Ablation (PA): Similar to LLA, but without laparoscopic manipulations and ablation needles are inserted directly through the skin into the treatment area. PA has the same treatment types as LLA.This study focuses on three commonly distinguished MILT 10 were applied as described below.To establish a generic process model of MILT, the modeling strategies proposed in our previous workProcedure Considering the entire procedure as a single process, starting from patient intake until the end of the intervention. Highest abstraction level, lowest granularity.Phase (P) Contains groups of modules and decisions that all share a goal or purpose. Intermediate abstraction level, intermediate granularity.Module (M) A chain of actions and decisions aiming to fulfil a specific goal within a phase. Low abstraction level, high granularity.The generic process model of MILT was established at three levels of abstraction and granularity, see Fig. Data of the MILT procedures were collected through live observations and offline video observations, literature study and interviews.Sixteen live observations at Oslo University Hospital (OUH), Oslo, Norway and Erasmus Medical Center, Rotterdam, Netherlands (Erasmus MC), performed by experienced teams. The live observations were composed of twelve laparoscopic and four ablation treatments.Eight interviews with clinical experts at Erasmus MC and OUH.Nine offline observations using endoscopic video recordings of laparoscopic liver surgeries and OR recordings of ablation procedures.The data were acquired from:65 has been investigated.The process model was primarily designed based on the live observations in the OR. Interviews with the surgical team members were conducted to verify that the observed procedures were representative for MILT methods in general. To obtain a thorough understanding of surgical methods and to let the teams get used to the observer, the observer also attended several laparoscopic resection procedures of other organs in the aforementioned hospitals. Furthermore, the procedure description of MILT procedures in Refs.After establishing the MILT process model, endoscopic video recordings of laparoscopic liver surgeries of fifteen extra procedures were used for verification. In addition, the author (MG) has attended six intervention sessions in Erasmus MC and Bern University Hospital (BUH).For final verification, the proposed process model the process model was presented to clinicians and the validity and correctness of the generic process model for different techniques of performing MILT were discussed with the participating clinicians in OUH and Erasmus MC. Example surgical videos were used to discuss how the process model mimics every activities in performing different technics of MILT in clinical practice. Video Marker Software was used to discuss the registered surgical data for the entire duration of sample surgeries over the endoscopic videos.Ethical approval was obtained from each of the clinical centers in which the data were collected and observations were done for design and verification of the process model  and Erasmus MC and BUH with following the hospitals ethical rules). Based on these hospitals rules, informed consents were obtained from patients for further investigation on their surgical procedure. All methods for data acquisition and verification were performed in accordance with the relevant guidelines and regulations of the hospitals.10). Based on the data from OR observations, interviews and literature studies, a top-down approach was first used to establish a global overview of the surgical workflow. Next, the endoscopic videos were used as low-level data to model the details of the process model and to improve the initially established general overview bottom-up. This modelling process was iterated until no process model changes resulted from new iterations anymore.A combination of top-down and bottom-up approaches was used to benefit from the advantages of both approaches . The individual procedures were merged in the generalization process, covering all events of the treatments and not only the most probable events.10.The generic MILT process model was concretized by using workflow and process model diagrams. The process model was made to have a modular structure to increase model usability and efficiencyQualitative verification was performed to confirm that the path options in the established process model fit any observed order of possible actions and decisions occurring during MILT in clinical practice. This was done by registering the sequence of the encountered process model elements (phases and modules) throughout the entire treatment procedure of fifteen offline observation of MILT procedures from OUH. In addition, the author (MG) has attended four intervention sessions at Bern University Hospital and two at Erasmus MC. Furthermore, interviews with clinical teams were done and the process model was discussed with highly experienced surgeons with at least 10 years of surgical experience in OUH and Erasmus MC.Quantitative verification was performed to confirm that the sum of the encountered workflow elements  duration was equivalent to the total procedure times of fifteen offline MILT procedures from OUH.Qualitative and quantitative verifications were done to confirm that the proposed generic process model of MILT reflects the procedure in clinical practice:As each treatment procedure can be composed of thousands of steps, in-house process model data registration software was developed to facilitate registration of data on the videos of the endoscopic camera Fig. .Figure 2Operation: the entire process in the OR, from when OR and patient are being prepared, until when the patient is moved out of the OR to the recovery room.Intervention: starts with the first ablation needle manipulation or first incision in the abdomen by the interventionist/surgeon and ends when the last incision is closed.Surgery: starts with the first incision in the abdomen by the surgeon and ends when the last incision has been closed.Treatment: the actual physical treatment (resection or ablation) of the target region.Within the MILT treatment procedure, including its preparations, three hierarchical sub-phases are distinguished clinically:The generic process model of MILT procedures at the lowest granularity level (highest abstraction) is displayed in Fig. Phase 01: Intake—The patient is admitted to the hospital and complete anamnesis is collected.Phase 02: re-operative Imaging—Medical images of the abdominal region are made for planning the MILT procedure prior to a possible operation. Phase 02 can take place right before operation up to a few months prior.Phase 03: Pre-operative Planning—Planning includes all decisions about things like treatment approach, incision locations and resection paths or possible needle placements, size of the target region, etc. prior to the possible operation. The planning is based on the patient anamnesis (from Phase 01), medical images (from Phase 02), available equipment and technical resources and experiences.Phase 04: Intra-operative Preparation—On the day of operation, prior to the intervention, the patient, OR equipment and surgical instruments are prepared for the operation.Phase 05: Intra-operative Imaging—Medical images can be acquired in the OR, before and during the intervention.Phase 06: Intra-operative Planning—The treatment plan can be generated or updated in the OR just before and during the intervention. Any pre-operative data and new images were taken in the OR (from Phase 05) aid in making decisions in this phase. The MILT method and type might also be changed during the operation. The MILT procedure is considered aborted if it is converted to a non-MILT procedure, such as open surgery.Phase 07: Operative field Access—If LLR or LLA is the preferred method, the surgeon first makes the operative field accessible.Phase 08a/b: Isolation of the treatment area consists of activities to separate the target region from surrounding structures and prepares the target region for the treatment. Based on the nature of these activities and how they affect the patient’s anatomy, isolation can be performed in two ways:Phase 08a: Treatment Area Isolation: Destructive—Isolation by destructive (permanent) dissection or closure of surrounding structures. Only applies to LLR and LLA.Phase 08b: Treatment Area Isolation: Non-destructive—Isolation with temporary effects, using actions such as temporarily closure of vessels or hydro dissection.Phase 09: Needle Manipulation—Maneuvering ablation needle(s) to the desired position.Phase 10: Treatment—The actual treatment of the target region by either resection or ablation.Phase 11: Intra-operative Complications—Handling any complications that might occur during the operation. Such actions may include, for example, blood transfusion and hemostasis (e.g. bleeding vessel ligation) or surgical drainage.Phase 12: Miscellaneous—Other clinical activities that do not directly serve the MILT procedure might take place, such as biopsy and catheter placement.Phase 13: Intra-operative Wrap-up—All activities aimed at wrapping up, such as removal of un-absorbable materials, closing the incisions, etc. symbols. Apart from the continuous support of nurses and anaesthesiologist in the entire intra-operative phases and the act of blood suctioning in parallel to other treatment activities during surgery, based on the current data, the parallel activities are associated with two phases: intra-operative preparation phase (Phase 4) and intra-operative imaging (Phase 2) activities. In intra-operative phases, we plotted the connections associated with the imaging phase, where there was a high chance of performing imaging routines. In other places where this is less likely to happen, we used a symbol  to show the possibility for imaging. A brief walkthrough of the module-level MILT process model including the contents of the modules in the process model is provided in Supplementary material-part The generic process model of MILT procedures at the module level, including the phases, modules and decisions linking the modules, is provided in Fig. The result of the quantitative and qualitative verifications of the process model confirmed that the process model provides a pathway for all encountered sequences of actions and decisions that were observed to occur during MILT procedures in clinical practice. Supplementary material-part In sessions with two highly experienced surgeons and two assistant surgeons in OUH and Erasmus MC, discussing the validity and correctness of the generic process model for different techniques of performing MILT, it was confirmed that the proposed process model mimics the activities in the clinical practice.Surgical process models bring several advantages and pave the way for further improvement of operations. The presented generic process model covers a broad range of MILT procedures and associated techniques. No deviations from the proposed process model were found in the treatment procedures that were analysed in the verification process. The proposed process model provides relationships between different entities of MILT procedures at the proposed levels of details. Thus, the process model provides the possibility for extensive quantitative as well as qualitative analysis of the procedures at the desired level of detail.In intra-operative phases, distinguishing between planning and other treatment activities is a complicated task. Planning during operation is an ongoing mental activity and can be considered as an element inside all intra-operative phases. Modelling planning activities in a separate phase in the generic process model provides the foundation for further analysis and improvement of planning. Recognizing the points where planning occurs in the surgical process model and deriving the sequential relationships between planning and other intra-operative activities, show how and to what extent planning is associated with different activities and reveals the possible bottlenecks of planning.https://www.youtube.com/c/DrSergeyBaydo/videos) and Dr Riccardo Memeo (https://www.youtube.com/channel/UCdhB0tuE3EC_iNipn1A3ltg/videos) YouTube Channels.) to make the process model as generally applicable as possible. Moreover, in verification process, the endoscopic videos of fifteen additional surgeries performed in OUH were analysed and six live observations of MILT procedures were performed in Erasmus MC and BUH. For these reasons, process model should conform to the procedures in other institutions as well. In this study, we did not take videos from the OR. These recording would make further quantitative validation of the model possible, but also requires special ethical approval, since sensitive information is recorded. In an earlier study, we investigated the consequence of recording in the OR66. In this study the pre-operative and not the post-operative phase was included in the generic process model, because the former has a direct influence on performing the treatment, which is the focus of this work. All concepts associated with different techniques of MILT are defined and categorized as different phases and modules. Thus, we expect that variations of performing actions in different institutions by using different techniques/instrument, will hardly cause any deviations from the proposed process model. However, lack of instruments, equipment or knowledge might change the course of actions or introduce innovative ways to tackle problems , which may not be considered in the presented process model. Recognizing and registration of surgical activities are crucial for performing analysis on surgical procedures, generating and verifying surgical process models and training machine learning methods to develop AI systems for the future hybrid ORs67. The in-house developed Video Marker Software in this work aided efficient registration and verification of data over the endoscopic video. The extracted data using the Video Marker Software from surgical videos that are acquired from OUH has been presented in Supplementary material-part 69. Such systems can be of great use to aid gathering surgical data for different purposes of process model analysis and verifications71.Imaging activities can occur at any moment in the intraoperative phases. Although imaging activities could be defined as a green phase in the proposed generic surgical process model, it was decided to model sequential and parallel dependencies between entities as it highly benefits further analysis of process model and performing possible simulations. Live observations and interviews with experts in two institutions (OUH and Erasmus MC) were performed to determine the low granularity level structure of the process model. The process model was initially established based on the data from endoscopic video analysis and live observations in aforementioned institutions. The data was complimented with literature studies and analysis of videos of procedures available on the web from different institutes 74 are being widely used in the process of the development of technologies. These methods aid smooth adaptation to changing requirements throughout the development process by using iterative planning and feedbacks from developers and the end users75. With the process model and computer simulations, analysis of the effect of possible changes and their eligibility aids making right decisions and adaptations during the agile sessions.The presented process model aids different aims of analysis for improvement of surgeries/interventions in follow-up studies. Analysis of process models and providing connections between every entities of the surgical procedures, identify the points where AI and software/platform systems can be beneficial, predicts how big the benefits are and determines how these systems can be designed and developed to be employed in clinical practice, see e.g. Ref.78. The optimal treatment option for each surgery with specific conditions can be derived and novice surgeons can be trained based on the probable sequence of events and the possible deviations for each operation. The experienced surgeons can review the steps and possible deviations before or during an operation as a roadmap. For this purpose, real-time recognition of surgical steps over the endoscopic videos is required, a topic which has attracted wide attentions in recent years80. The process model benefits analysis of surgeons’ learning curves82. Durations and occurrence frequencies of surgical steps and deviations from nominal surgery paths can be used as criteria for learning curve analysis, as well as surgeons’ skills evaluations. In recent years navigation platforms for guiding surgeons in performing MILT attracted broad attention88. Analysis of the proposed surgical process model can reveal the optimal treatment options to guide surgical teams using navigation systems by suggesting/predicting next surgical steps and the time required for performing each surgical action90. Currently, prior to operation the lead surgeon/interventionist goes into the details of the patient’s organ-specific anatomy and mentally pre-visualizes the whole procedure and all its key steps. The complexities of such pre-operative planning activity, can be reduced by the process model which brings the possibility to propose the treatment options for individual procedures. Analysis of surgical process model can prevent extra costs of trial and error in the development phase of technologies and introduction of new technologies into clinical practice. With the process model, it is possible to provide scientific evidence for the possible enhancement of surgeries by the proposed technology for specific methods/types/techniques of performing surgeries. The effects and eligibility of any adjustment in the new technologies can be analysed on the surgical procedure, prior to actual implementation of technologies, resulting in a more efficient business model.The process model can widely contribute in the training and skill evaluation of surgeonsA generic surgical process model for MILT was established by applying the modelling strategies developed in prior work. The presented model covers MILT methods for laparoscopic liver resection, laparoscopic liver ablation and percutaneous ablation, with their types, techniques and variations as observed in data obtained from various sources. As the presented model was established using a numerical model representation, it can be used for extensive quantitative and qualitative analysis and improvement of MILT procedures through various ways, such as the introduction of new technologies in the OR, training of clinical teams, analysis of learning curves and skills evaluations, optimization of OR management and medical team activities in the OR.Supplementary Information.


This work studies the dynamics of a three dimensional Hopfield neural network focusing on the impact of bias terms. In the presence of bias terms, the models displays an odd symmetry and experiences typical behaviors including period doubling, spontaneous symmetry breaking, merging crisis, bursting oscillation, coexisting attractors and coexisting period-doubling reversals as well. Multistability control is investigated by employing the linear augmentation feedback strategy. We numerically prove that the multistable neural system can be adjusted to experience only a single attractor behavior when the coupling coefficient is gradually monitored. Experimental results from a microcontroller based realization of the underlined neural system are consistent with the theoretical analysis. Different stability regions distributed in the parameter plane, multistability of asymmetric multi-stable patterns of the spiral chaotic patterns with different dynamic amplitudes, are demonstrated in the model [al,. In 2021 presents a Hopfield neural network where the authors performs an analytical study and reveal interesting features such as existence of bistability, and antimonotonicity. Complex dynamics of the global coexisting multiple firing activities are explored in a bi-neurons HNN with different gradients [The human brain represents a complex system composed of a multitude of neurons whose study is important to understand different mechanisms of biological function in neural systems . More phhe model . Yang etradients  proposesradients  with parThe rest of this work is organized as follows, the following section presents a theoretical study where we analyses the stability of equilibrium points. The numerical study will be discussed in Section III to show the details generated by the dynamics of this model. In Section IV, microcontroller-based implementation are performed to support our investigations. Section V concludes this paper.1.1We consider the Hopfield neural networks model made of three neurons proposed in literature . When thHere Equilibrium state are obtained if all the derivative are equal to zero Asymmetric case (The case 3.Control of multistabilityIn the presence of the excitation current in (arameter ,24. For .001 see , Fig. 12. This control is presented in Coexistence of multiple attractors offers great flexibility in the performance of dynamical system while not requiring a large change of parameter , and the3The Arduino MEGA board build around the AT MEGA 2560 Microcontroller was used during the experimental phase for its speed, deep price, and accessibility. The programming language in the Arduino software IDE is C/Arduino. This language is very closed and compatible with the C programming language. Here digital components replace analog circuits as the latter is influenced by the temperature of the components, atmospheric conditions, and pressure ,33,34,354The dynamical behaviors of a class of three dimensional Hopfield neural networks has been explored focusing on the effect of bias current. In case of perfect symmetry (Bertrand Frederick Boui A Boya: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Balamurali Ramakrishnan: Conceived and designed the experiments; Analyzed and interpreted the data.Joseph Yves Effa: Contributed to analyze and interpret the data; Wrote the paper.Jaques Kengne: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Karthikeyan Rajagopal: Conceived and designed the experiments; Analyzed and interpreted the data.This work is partially funded by Center for Nonlinear Systems, Chennai Institute of Technology, India vide funding number CIT/CNS/2022/RD/006.No data was used for the research described in the article.The authors declare no conflict of interest.


We comprehensively explore different optical configurations of a radio-frequency atomic magnetometer in the context of sensor miniaturisation. Similarities and differences in operation principles of the magnetometer arrangements are discussed. Through analysis of the radio-frequency and noise spectra, we demonstrate that all configurations provide the same level of atomic polarisation and signal-to-noise ratio, but the optimum performance is achieved for significantly different laser powers and frequencies. We conclude with possible strategies for system miniaturisation. Detection systems based on radio-frequency (RF) atomic magnetometers have been successfully implemented in a wide range of measurement scenarios covering explosives detection , geomagn2 range) ,9 over sThe principle of operation of rf atomic magnetometers relies on the detection of magnetically driven atomic coherences between neighbouring Zeeman sublevels of a given ground state level in the alkali–metal atoms. The atomic coherence is generated by an rf field resonant with the energy splitting of adjacent Zeeman sublevels. The sublevels are tuned into resonance with the rf field through the Zeeman effect, i.e., via the application of a bias magnetic field. Precessing atomic coherence is detected through its mapping onto the polarisation of a linearly polarised probe beam propagating through the medium. Generation of the atomic coherences requires the presence of a population imbalance among the Zeeman sublevels. Here, we concentrate on generating a population imbalance representing ‘orientation’, i.e., the majority of a population transferred to the sublevel with the maximum or minimum magnetic number (stretched state), and off-resonance probing .For many practical applications, Size, Weight, Power, and Cost (SWaP-C) considerations are as important as sensor performance. Our study intends to partially address the former without compromising the latter and provides the options to optimise sensor architecture according to the target application. For scenarios where the power consumption sets the main limit, the independent and degenerate pump-probe configuration may be applicable. Improvements to size and cost might be possible with the reduction in the number of laser diodes and relevant electronics, which is achievable in the degenerate and single beam cases. It needs to be stressed that our studies were performed at ambient temperatures in a paraffin-coated cell. In our opinion, miniaturisation of the sensor would have to include wafer-based buffer gas cells, where the required cell heater might become a significant, if not leading, component of the power consumption budget. Activities involving wafer cell development and optimisation are underway in our lab.A typical orientation generation scheme involves an atomic ensemble’s interaction with a circularly polarised laser beam propagating along the bias magnetic field. Optical pumping also describes the transfer of optical angular momentum to the target atoms via spin-exchange collisions (SEC) ,12. AnotIn this paper, we compare the performance of three rf atomic magnetometer configurations from the perspective of possible system miniaturisation. The focus is on the efficiency of atomic polarisation generation , sensor sensitivity , and the laser system parameters required to achieve this sensitivity. In all cases, we study configurations based on indirect pumping ,17. The All measurements described here are performed in a shielded setup. Caesium atomic vapour is housed in a cross-shaped . The polarisation for the probe beam light is initially cleaned using a polariser (Glan-Thompson X) and quadrature (Y) component of the signal. The amplitude (An rf magnetic field resonant with the Larmor frequency (set by the bias static field) creates coherences between the Zeeman sublevels. This leads to precession of the collective atomic spin generated through optical pumping. Paramagnetic Faraday rotation maps the value of the collective atomic spin onto the polarisation state of the linearly polarised probe beam ,9,10. ThThe ambient magnetic field is suppressed by the use of Twinleaf cylindrical shields. A pair of solenoids inside the shield generates a well-controlled bias magnetic field with a relative homogeneity at the level of X (red curve) and Y (dark blue curve) components. The resonance frequency, linewidth, and amplitude of the spectral profile have been extracted by fitting a Lorentzian profile to the rf spectrum. We are aware that the Lorentzian profile is produced by a purely orientated state [Radio-frequency spectroscopy is used to characterise the generation efficiency of the population imbalance (orientation) and the magnetometer signals amplitude. The rf spectrum is created by monitoring the magneto-optical signal as the rf field frequency is scanned across the magnetic resonance. ed state ,21 and iThe configuration with independent pump and probe beams is used as a baseline for performance evaluation of the other magnetometer arrangements studied, since it is well understood and has demonstrated high performance ,17. In tOptimisation of the rf atomic magnetometer signal begins with a selection of test rf field amplitude values that will be used in all further experiments. The rf field amplitude is set such that it does not perturb the linewidth of the spectroscopic measurements. The value is selected by recording the dependence of the rf spectroscopy signal on the rf field amplitude. This dependence is also used in rf field calibration .The dependence of the rf atomic magnetometer signal on the probe beam power, as shown by the blue squares in r powers ,14. The The phenomenon is similar to the well-known alignment-to-orientation conversion ,23,24 anTo evaluate the signal-to-noise ratio, noise spectra, such as presented in The probe beam power amplitude dependencies of the signal and noise were used to evaluate the signal-to-noise ratio. The ratios for the three discussed configurations are presented in The degenerate configuration refers to a case where the pump and probe beams are produced by a single laser source, i.e., have the same frequency, but different polarisations, as shown in Previously, we have shown that changing the probe beam detuning does not affect the maximum achievable signal-to-noise ratio . With inTo address the issue of optimum pump beam power more specifically, we measured the rf spectral profile parameters as a function of the pump beam power for fixed probe beam power and detuning in the vicinity of the optimum detuning from Detuning scans, such as  in ref. ). We sawThe single beam configuration refers to a case where orientation within the F = 4 caesium ground state level is produced and probed by a single linearly polarised laser beam, as shown in lignment . This reRf spectra were systematically recorded for various laser detunings from the Similarly to other cases, the noise spectra were recorded with a spectrum analyser for the laser beam power values used in the previous measurement. In agreement with the fluctuation–dissipation theorem, the changes in the character of the rf spectroscopy profiles were also observed in the atomic noise profiles. In particular, the noise spectra recorded below The three magnetometer configurations presented here represent three different indirect pumping mechanisms to generate atomic orientation. The first two involve circularly polarised pump beams, while the third is solely based on a single linearly polarised beam. While the efficiency of pumping in terms of the rf resonance linewidth is comparable, the mechanisms differ and are highlighted by the conditions required to achieve optimum performance. The independent pump and probe configuration and degenerate arrangement differ in terms of pumping not only in the power of the pump beam that is required to optimise the process but also in the range of powers for which the optimum is observed. This is a direct consequence of the difference in pump beam detuning from the While discrimination between different magnetometer configurations through direct comparison of the signal amplitudes is meaningless because of the different probe beam detunings and powers, the signal-to-noise ratio provides a tool for the evaluation of different arrangements’ performance. The standard configuration with independent pump and probe beams offers the optimum signal-to-noise ratio at the lowest power, which is easily accessible with a standard vertical cavity surface emitting laser (VCSEL) diode. This comes at the price of the relative complexity of the instrumentation: two separate diodes, drivers and stabilisation. Frequency locking of the pump beam is achievable with standard absorption spectroscopy, but securing stable off-resonant detuning (GHz range) of the probe beam is more complex. A method used in atomic clocks  involvesTo summarise, we have systematically explored different laser configurations of an rf atomic magnetometer, based on similar pumping mechanisms, with variation in the role the probe beam plays. We showed the generation of a similar level of orientation in all three cases through various types of pumping mechanisms. Pumping involves a circularly polarised beam for the independent pump and probe configuration, both circularly and linearly polarised beams in the degenerate case, and solely a linearly polarised beam when there is a single beam. We demonstrate the same signal-to-noise ratio in all three arrangements. As mentioned, the presence of non-linear interactions driven by the linearly polarised beam creates options for improved orientation within the F = 4 manifold, operating an all-optical internally driven non-linear spin maser  and spin


Alginate microcapsules are one of the attractive non-invasive platforms for handling individual cells and clusters, maintaining their isolation for further applications such as imaging cell sorter and single capsule qPCR. However, the conventional cell encapsulation techniques provide huge numbers of unnecessary empty homogeneous alginate microcapsules, which spend an excessive majority of the machine time on observations and analysis. Here, we developed a simple alginate cell encapsulation method to form content size-dependent alginate microcapsules to eliminate empty microcapsules using microcapillary centrifugation and filtration. Using this method, the formed calcium alginate microcapsules containing the HeLa cells were larger than 20m, and the other empty microcapsules were less than 3m under 4000 rpm centrifugation condition. We collected cell-containing alginate microcapsules by eliminating empty microcapsules from the microcapsule mixture with simple one-step filtration of a 20 m cell strainer. The electrical surface charge density and optical permeability of those cell-encapsulated alginate microcapsules were also evaluated. We found that the surface charge density of cell-encapsulated alginate microbeads is more than double that of cells, indicating that less voltage is required for electrical cell handling with thin alginate gel encapsulation of samples. The permeability of the alginate microcapsule was not improved by changing the reflective index of the medium buffer, such as adding alginate ester. However, the minimized thickness of the alginate gel envelope surrounding cells in the microcapsules did not degrade the detailed shapes of encapsulated cells. Those results confirmed the advantage of alginate encapsulation of cells with the centrifugation method as one of the desirable tools for imaging cell sorting applications. Cell separation and isolation are fundamental for single-cell-based analysis and further applications, such as using those single cells as parts of regenerative medicine. Because of its many advantages against conventional in vitro dish experiments, microdroplets are one of the suitable methods for isolating single cells for their analysis in minimal volumes. For example, microdroplets have an isolated compartment for each target cell identification and easy handling, prevention from spatial dispersion for quantitative analysis and further operation and a small reaction volume for faster concentration increase with minimum amounts of reagents . The com2+ for cross-linking sodium alginate [2+-alginate gel microdroplets and Ba2+-alginate gel microdroplets were digested according to the difference in chelating efficiency of linkage-divalent cations of those chelating buffers [Following those water droplets, encapsulation technologies have also been developed to improve single-cell analysis. For example, microfluidics channels provide homogeneous polymers and gel capsules regardless of the difference of contents in those capsules ,21,22,23alginate ,26. Sevealginate ,28 and aalginate ,30. One  buffers . We also buffers .In this study, we aim to optimize an alginate encapsulation technology for imaging cell sorter applications because the cell encapsulation into alginate microcapsules can potentially maintain cells in isolated conditions even under the fully packed dense concentration and can prevent contamination during sorting procedures. In the conventional microfluidic alginate microcapsule formation methods, homogeneous same-size alginate capsules were formed regardless of the cells inside. Therefore, if we apply this encapsulation technology for screening, the total number of microparticles increases significantly more than the total number of cells. Hence, we developed a simple centrifugation method to acquire the alginate microcapsules containing cells with a cell strainer before the cell sorting procedure. With this method, the size of alginate microcapsules relies on the contents; the alginate microcapsules containing cells are larger than 10 Alginic acid is, in nature, a hydrophilic polysaccharide and forms soluble hydrogels with monovalent metal ions such as sodium and alginates. Alginates change to water-insoluble gelatinous calcium alginate by adding the aqueous calcium salt as described in Applying this soluble to insoluble gelation change of alginate droplet surfaces, we can acquire alginate microcapsules having an insoluble alginate layer surrounding the contents inside as solutions. For example, as shown in A mixture of 1% sodium alginate  and 5% glucose  was prepared with mild stirring. The mixture was used as the sodium alginate buffer for alginate microcapsule formation.2 in DMEM medium  supplemented with 10% heat-inactivated fetal bovine serum  and 100 U /mIn this experiment, we used HeLa cells  as standard cells ,34,35. AWe used 10 First, we prepared a tapered glass micropipette having a 100 Next, the micropipette for sodium alginate droplet formation was prepared. Using a syringe , we filled glycerin  from the tip to We also prepared the two-layer buffer reservoir, in which 50 After setting the tip of the micropipette filled with sodium alginate on the two-layer buffer reservoir, we formed calcium alginate microcapsules as follows: First, as shown in Next, as shown in The images of samples were observed with the inverted microscope  and recorded with a CCD camera . We measured the particle size from the acquired images by using the image analysis software ImageJ  and then calculated the frequency distribution.Two carbon rod electrodes  of 100 particle samples for each experiment. The result of fitting linear equations to a set of data points was achieved using ordinary least squares regression (OLS) in Microsoft Excel .In the popular microfluidic pathway-based alginate encapsulation technology, the homogeneous same-sized alginate microcapsules are formed for a stable reagent reaction environment regardless of the cells inside. However, this approach forms many empty alginate microdroplets during cell encapsulation in the microfluidic pathway. Moreover, because of the similarity of alginate microcapsule sizes, separating empty microcapsules and cell-encapsulated microcapsules from their shape difference is challenging. Therefore, to reduce the number of alginate microcapsules, some other method for eliminating empty microcapsules was required to reduce the number of microparticles screening.To maximize those potential advantages of alginate encapsulation of cells for further analysis, such as imaging cell sorter applications, we developed a simple content size-dependent alginate microcapsule formation method to eliminate empty alginate microcapsules exploiting the surface tension and centrifugal force to determine the formed droplet sizes. The centrifugation method has been one of the popular methods to form alginate microcapsules before the microfluidic method becomes popular ,38,39,40First, we evaluated the output of glycerin from the end of the capillary tube with changing centrifugal force by controlling the rotation number of the centrifuge. To confirm the contribution of glycerin caps to reduce the formation of unexpectedly large alginate microcapsules, we compared the size distribution of empty calcium alginate microcapsules with/without glycerin at the tip of the capillary tube. Then, we examined the size distribution of HeLa cell-encapsulated alginate microparticles using the same centrifugation method. Figure F shows the size distribution of HeLa cells we used for encapsulation. The above results indicate that (1) the size of empty alginate microcapsules becomes smaller according to revolution velocity increase and (2) the size of HeLa cell-encapsulated alginate microcapsules maintains the identical size distributions reflecting the size distribution of HeLa cells regardless of the increase of revolution velocity. Hence, when we set the faster revolution velocity, the size distributions of empty alginate microcapsules and HeLa cell-encapsulated alginate microcapsules can be split into certain two-peak distributions with enough size gaps to separate them. For example, this experiment suggests that these two types of alginate microcapsules were divided easily into empty microcapsules less than 9 crobeads B into alcrobeads C, the Hecrobeads D and thecrobeads E, a thincrobeads D and theocapsule E, a thinAs the empty microcapsules distributed less than 9 We evaluated the potential damage of HeLa cells in alginate microcapsules to estimate the allowed time for this sample microcapsule filtration and subsequent cell sorting procedure. We used two types of inside solutions of alginate microcapsules, DMEM medium and 5% glucose mixed with 1% sodium alginate solution. As described in We used EDTA to release encapsulated HeLa cells because EDTA can chelate divalent cations and exchange calcium alginate gel for alginate sol. We soaked encapsulated HeLa cells in 10 mM EDTA  buffer (pH = 8) for 15 min. Another potential advantage of alginate encapsulation of cells is providing a stable surface change of alginate envelope to samples. We can use these stable surface charges of microcapsules for handling samples regardless of the difference in contents. Hence, we evaluated the values of surface charges of alginate microcapsules and their encapsulated particle dependences.q and a radius r, the amplitude of oscillation L in the AC electric field T is the cycle period, which is slow enough to balance the motion and viscous resistance during the entire oscillation. Therefore, the surface charge density We estimated the effective electric charge density of the microparticles from the amplitude of the sample oscillations in the AC electric field as follows. When we place a spherical particle with a charge L in the experiment.Hence, we used Equation  to estimWe placed particles in 5% glucose solution and applied L at L to estimate surface charge density Equation . TherefoEquation .As described above, enhancement of the surface charge of cells using an alginate gel envelope was successful in HeLa cells. This is one of the potential advantages of alginate microcapsules for imaging cell sorter applications because we can reduce the voltage of handling samples in microfluidic pathways for separation. However, we need to mention the possibility of opposite results of surface charge density and its reduction in polystyrene spheres for practical applications. Although the ny works ,44, the One possible reason for charge density reduction might be the interference of the thin alginate gel layer to reduce the thickness of the electric bilayer formed on the surface of polystyrene spheres. In contrast, the increase of the surface charge of HeLa cells by encapsulation also might be caused by the assistance of the surface charge density of alginate gel because both the surface charge and the thickness of the electric bilayer of HeLa cells were significantly smaller than the alginate gel. High values of empty alginate microbeads also might be caused by the significant size difference of empty alginate capsules from HeLa cells and polystyrene spheres. Equation  shows thFor the imaging cell sorter application of alginate microcapsules, there is improvement in the permeability of the thin alginate gel layer surrounding samples in the microcapsule envelopes with regard to more precise observation in an imaging cell sorter. Hence, we examined the improvement of permeability of alginate microcapsules and encapsulated HeLa cells by optimizing the refractive index of the buffer.According to the above results, we concluded that the optimization of the refractive index of the buffer was not effective in making transparent alginate envelopes even in the 1% alginate ester buffer. However, the alginate envelopes surrounding HeLa cells did not degrade those cells’ images in phase-contrast and bright-field observations. This might be caused by the advantage of the centrifugation method, in which a minimum thin alginate gel envelope enclosed HeLa cells.We investigated the ability of alginate gel microcapsules to image cell sorter applications. In this study, we examined the possibility of forming a homogeneous thin alginate gel envelope on the cells exploiting the balance of two opposite forces, centrifugation force and viscose force. For this experiment, we developed a simple droplet formation method as the centrifugation method exploiting the micrometer-sized tip of the capillary tubes in the centrifuge. The results showed that the homogeneous envelope was coated surrounding the cells and empty alginate microcapsules were less than a few micrometers. The centrifugation method accomplished the size-dependent selective collection of alginate microcapsules containing cells at 4000 rpm centrifugation and filtration. Significant enhancement of surface charge density of HeLa cells by the thin alginate gel encapsulation on the surface of cells was also observed. Change in the refractive index of the buffer did not improve the permeability of alginate envelopes; however, the advantage of the centrifugation method for wrapping minimum thickness of alginate gel surrounding cells can cause the clear phase-contrast and bright-field images of cells with a negligible small influence of alginate gels. All the above results suggest the alginate encapsulation of cells can give more advantages to an imaging cell sorter, such as (1) maximized concentration of samples maintaining their isolated conditions in microcapsules without any empty capsules, (2) enhancement of surface charge of cells for easy handling of electrostatic force and (3) improvement of permeability for observation of contents in the alginate microcapsules with minimized alginate gel envelope thickness.


Although developed anticancer drugs have shown desirable effects in preclinical trials, the clinical efficacy of chemotherapy against brain cancer remains disappointing. One of the important obstacles is the highly heterogeneous environment in tumors. This study aims to evaluate the performance of an emerging treatment using antiangiogenic and cytotoxic drugs. Our mathematical modelling confirms the advantage of this combination therapy in homogenizing the intratumoral environment for better drug delivery outcomes. In addition, the effects of local microvasculature and cell density on this therapy are also discussed. The results would contribute to the development of more effective treatments for brain cancer.Although convection-enhanced delivery can successfully bypass the blood-brain barrier, its clinical performance remains disappointing. This is primarily attributed to the heterogeneous intratumoral environment, particularly the tumor microvasculature. This study investigates the combined convection-enhanced delivery of antiangiogenic drugs and liposomal cytotoxic drugs in a heterogeneous brain tumor environment using a transport-based mathematical model. The patient-specific 3D brain tumor geometry and the tumor’s heterogeneous tissue properties, including microvascular density, porosity and cell density, are extracted from dynamic contrast-enhanced magnetic resonance imaging data. Results show that antiangiogenic drugs can effectively reduce the tumor microvascular density. This change in tissue structure would inhibit the fluid loss from the blood to prevent drug concentration from dilution, and also reduce the drug loss by blood drainage. The comparisons between different dosing regimens demonstrate that the co-infusion of liposomal cytotoxic drugs and antiangiogenic drugs has the advantages of homogenizing drug distribution, increasing drug accumulation, and enlarging the volume where tumor cells can be effectively killed. The delivery outcomes are susceptible to the location of the infusion site. This combination treatment can be improved by infusing drugs at higher microvascular density sites. In contrast, infusion at a site with high cell density would lower the treatment effectiveness of the whole brain tumor. Results obtained from this study can deepen the understanding of this combination therapy and provide a reference for treatment design and optimization that can further improve survival and patient quality of life. Brain tumors have posed a unique health concern worldwide. Glioblastoma is the most aggressive primary brain cancer that remains incurable. The median survival is limited to 15 months even if the maximum treatment is applied ,3. The eDrug delivery involves multiple physiological and physicochemical processes, determined by the transport properties of drugs, biological properties of tumors, and their interactions . Solid tMathematical modelling plays an increasingly important role in drug delivery research. Compared to in vivo experiments, this method is cost-effective for examining each drug delivery process individually, or in an integrated manner to explore the underlying drug delivery mechanisms and determine the impacts of influencing factors for optimization ,15,16. TThe current study applies an image-based transport model aimed to investigate the combined delivery of antiangiogenic and liposomal cytotoxic drugs to heterogeneous brain tumors upon CED. The 3D realistic geometry of a brain tumor and its heterogeneous characteristics are extracted from the patient’s DCE-MR images and imported into the mathematical model as input. The model covers the key processes in intracerebral drug delivery, including liposome release dynamics, transport in tissue interstitium by diffusion and convection, blood drainage, drug binding with proteins, cell uptake, and drug elimination due to physical degradation and bioreactions. The antiangiogenic effects on blood vessels and tissue interstitium are governed by a kinetic model. The treatment is evaluated in terms of the effective distribution volume, where the local drug concentration is sufficient to kill 90% of tumor cells.Microvasculature networks can vary considerably in brain tumors depending on location and tumor growth stage. Given the dimensions of tumor and normal tissue (in cm) are usually about 3 orders higher than the distance between capillaries (in μm), the tumor and its holding tissue can therefore be treated as porous media in which the functions of capillaries are considered as distributed source terms . ConsequA brain, including the embedded tumor, consists of the microvasculature network and tissue; the latter can be further divided into the extracellular space (ECS), cell membrane (CM) and intracellular space (ICS). The transport of infused drugs between these compartments is schematically shown in The concentration of free drugs  ,38; (b) CFD,ECS) ,40. TherThe concentration of antiangiogenic drugs in the tissue ECS depends on the convective and diffusive transport, and elimination, asThe antiangiogenic effect can be described byMR scan of two brain tumor patients was performed on a 3.0-Tesla Ingenia MRI scanner  at Fortis Memorial Research Institute, Gurugram, India. MR imaging protocol included a standard protocol for brain tumor patients along with DCE MRI or T1-Perfusion MRI. DCE MR images of the brain were acquired using the 3D fast field echo (T1-FFE) sequence before, during and after contrast injection. In the current study, a systemic infusion of the contrast agent Dotarem  with a dose of All procedures performed in this study involving human participants were in accordance with the ethical standards of the Institutional Review Board (IRB No. 2020-001-19-28) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The demographic characteristics for the two exemplary patients are given in A representative DCE-MR image slice and reconstructed 3D configuration are represented in r volume ,42 of inThe final computational mesh consists of 82,944 uniform structured elements, obtained after the mesh-independence test . The dimThe quantitative analysis of DCE-MRI data is performed by computing the concentration of contrast agent from the signal intensity of the DCE-MR images on a voxel-by-voxel basis. This is based on the spoiled gradient recalled echo (SPGR)/FFE equation that is represented as (10)I(t)d in Ref  is adoptThe Leaky Tracer Kinetic Model (LTKM)  is applie in Ref . ReadersGiven that the treatment period is much shorter than the tumor growth rate, the drug transport properties and tissue biological properties are treated as constants. Bevacizumab (BEV) and temozolomide (TMZ), as the typical antiangiogenic drug and cytotoxic drug, respectively, are selected in this study. The baseline values of model parameters are summarized in The initial volume fraction of plasma  algorithm. The residual tolerances are set as The numerical simulations are performed using a 64-bit Intel(R) Core i7-10700 processor (Clock speed: 2.90 GHz), eight cores with 32 GB RAM. The total computational time involved in solving the fluid flow and drug transport equations is approximately 1.5 h.The external surface of normal tissue is assumed to be fixed with zero gauge pressure and drug flux, as it is the source-sink-driven flow in tissue ,42. The The delivery outcomes of this combination therapy under different regimens are evaluated by the following qualification indexes from the perspectives of drug accumulation, drug distribution and treatment effectiveness.The drug concentration directly reflects the amount of drugs accumulating in the tissue. It is determined by the aforementioned drug transport processes and varies throughout the brain tumor and its holding tissue. Spatial-averaged concentration The insertion of a catheter into the brain tissue has the potential to result in trauma and oedema. The fluid generated from damaged cells and the enhanced leakage from damaged blood vessels  can alte(b)The antiangiogenic effect is reflected by the reduction of blood vessel diameter. Although blood vessels shirk significantly in response to the antiangiogenic drugs , both th(c)Drug diffusivity can also be location-dependent in a brain tumor, subject to the local in vivo environment. This variation of drug property can also affect drug transport and accumulation. However, the diffusivity of each drug is assumed to be uniform in the brain tumor in this study due to the lack of relevant information that can be extracted from the applied medical images. This assumption can be relaxed by using diffusion-weighted MR images , where t(d)Liposome cell uptake is ignored in this study owing to its complex nature. This process is controlled by several factors including particle size , ligands(e)Only one catheter is placed in this study to examine the effectiveness of combination therapy in treating heterogeneous brain tumors. However, it is important to note that the cytotoxic drugs can penetrate a short distance from the infusion site; it is approximately 8 mm, as shown in (f)The infusate administrated into the brain tumor is able to push the soft tissue back to form backflow. Drugs in the backflow would transport along the track of the infusion catheter to the normal tissue, reducing the treatment effectiveness. The formation of backflow depends on the infusion rate, tumor location and more importantly, the local tissue properties. Given the difficulty to obtain the heterogeneous tissue mechanical properties from the applied medical images, this phenomenon is not simulated in this study. This limitation can be overcome by incorporating the tissue mechanics model, solid-fluid interaction and corresponding tissue properties ,98.(g)In the current study, the anti-angiogenetic and cytotoxic drugs were infused simultaneously inside the brain tumor due to the lack of clinical information regarding their infusion timings. Future studies in this direction should attempt to optimize the infusion schedule between antiangiogenetic and cytotoxic treatment to maximize the therapeutic output.The combined CED of liposomal cytotoxic drugs and antiangiogenic drugs to a heterogeneous brain tumor has been studied by image-based mathematical modelling. Results show that the co-infusion of antiangiogenic drugs can effectively improve the delivery outcomes of cytotoxic drugs. The reduced microvascular density inhibits the fluid exchange between the blood and tissue as well as the drug loss due to blood drainage. Moreover, the location of the infusion site plays an important role in determining the performance of this combination therapy. Better treatment can be achieved by infusing drugs at denser microvasculature sites. Infusing the drug in a place with high cell density risks reducing its effectiveness against the entire tumor. Results obtained from this study can deepen the understanding of chemotherapy using CED. Please note that this study is based on the DCE-MRI data of two patients, thus further studies using a larger patient cohort will be needed to validate the results and to provide a reference for improving the treatment design.