Hepatocellular carcinoma (HCC) is a leading cause of cancer-related morbidity and mortality worldwide. Most patients are diagnosed with advanced disease, limiting their options for treatment. While current treatments are adequate for lower staged disease, available systemic treatments are limited, with marginal benefit at best. Chimeric antigen receptor (CAR) T cell therapy, effective in treating liquid tumors such as B-cell lymphoma, presents a potentially promising treatment option for advanced HCC. However, new challenges specific to solid tumors, such as tumor immunoanatomy or the immune cell presence and position anatomically and the tumor microenvironment, need to be defined and overcome. Immunotherapy currently in use must be re-engineered and re-envisioned to treat HCC with the hopes of ushering in an answer to advanced stage solid tumor disease processes. Future therapy options must address the uniqueness of the tumors under the umbrella of HCC. This review strives to summarize HCC, its staging system, current therapy and immunotherapy medications currently being utilized or studied in the treatment of HCC with the hopes of highlighting what is being done and suggesting what needs to be done in the future to champion this therapy as an effective option. Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related mortality and the leading cause of cancer-related morbidity worldwide ,2,3. RelUnfortunately, early diagnosis is rare due to the vague symptoms associated with liver tumors and lack of effective identification and screening of non-cirrhotic at-risk populations. These shortcomings are highlighted by the geographic discrepancy in expert opinion; currently, the United States and Canada do not recommend any screening owing to a lack of conclusive evidence proving a mortality benefit in these populations ,10. ConvCarcinogenesis in HCC is the result of a variety of ongoing and disparate insults stemming from heterogeneous disease processes. Regardless of the initial insult, HCC arises in a setting of sustained inflammation, generation and inadequate clearance of toxic reactive oxygen species and a host of genomic alterations all causing hepatocellular stress and dysfunction. These ongoing processes disrupt normal hepatocellular function and lead to several overarching processes, including progressive genomic instability, cell death and fibrosis of normal hepatocytes leading to cirrhosis and abnormal hepatocellular regeneration causing dysplastic and neoplastic cellular proliferation each specific to the causative etiology.While tumorigenesis in the liver stems from an insult that disrupts normal hepatic and hepatocellular structure and function, the liver itself is unique to most other solid organs due to the presence of systematically fixed phagocytotic cells which control bloodstream screening and clearance. This is known as the reticuloendothelial system (RES) or, more recently, the mononuclear phagocyte system (MPS) . These tMost current standard treatments for advanced stage HCC are associated with significant systemic side effects and a modest at best survival benefit . AdvanceWhile HCC is defined as a singular entity, the liver pathologies resulting in HCC carcinogenesis are highly variable depending on the inciting injuries. Each insult utilizes a different process to promote genomic instability, ongoing inflammation, cellular damage and neoplastic proliferation. Here, we will provide an overview of each of the main disease processes in the liver that may progress to HCC.Most cases of HCC are due to viral causes, namely hepatitis B virus (HBV). HBV is a partially double-stranded DNA virus and can either exist as an acute infection with longstanding immunity or a chronic infection with the potential for reactivation . HBV cauAlternatively, HCV, which is an RNA virus, cannot integrate into the host hepatocyte genome and thus requires continued replication . This coAnother etiology, non-alcoholic steatohepatitis (NASH), is a growing epidemiologic burden with the rising prevalence of obesity in the US and worldwide. NASH causes inflammation in hepatocytes by depositing excess circulating fat globules within the cells. These fatty hepatocytes become distressed and compete for oxygen leading to inflammation, loss of function and an immune response that results in subsequent fibrosis . NASH alAlcohol is a well-known carcinogen for a variety of cancers and contributes to their development in a multimodal fashion. Acetaldehyde, the metabolite of ethanol, impairs the cell’s DNA repair mechanisms through direct cross-linking . AlcoholWhile each etiology shares the overall theme of insult leading to fibrosis, degeneration, regeneration and finally mutagenesis ending in HCC tumor cells, the process by which these stages are carried out is clearly quite heterogeneous. As such, it becomes extremely important to strive to tailor treatment options based on the specific causative etiology, a task which, as we will see, has yet to be achieved.Current HCC treatment pathways depend on the stage at which a patient is diagnosed. The most commonly used staging system in HCC is the Barcelona clinic liver cancer (BCLC) system. The BCLC system classifies patients into five prognostic groups in order of advancing disease stage—0, A, B, C and D. The staging system incorporates liver function (Child-Pugh class), tumor burden  and patient performance status (using the Eastern Cooperative Oncology Group [ECOG] status) . PatientSurgical management of HCC is divided into two categories, resection or surgical removal of part of the liver based on anatomic segments or transplant, the complete removal of the native liver and replacement from deceased- or live-donor tissue. The decision to resect depends on tumor size, location and degree of underlying hepatic decompensation. Patients eligible for resection often have preserved liver function and tumors which are localized anatomically without any vascular invasion, falling into BCLC categories 0 and A ,32,33. ATransplant for HCC is usually reserved for patients in BCLC categories 0 and A who do not meet the above resection criteria. In the US, the Milan criteria is often used to determine transplant eligibility for HCC patients. The Milan criteria states that patients who have—(1) a single tumor with diameter less than 5 cm OR (2) not more than three foci of tumor, each one not exceeding 3 cm AND (3) no angioinvasion AND (4) no extrahepatic involvement are eligible for liver transplantation ,35,36. IRadiofrequency ablation (RFA) is being increasingly utilized to treat small, early stage BCLC 0 and A tumors as well as to treat patients who are at risk to progress while waiting on the transplant list . RFA is As HCC BCLC stage progresses to B, the patient is often no longer eligible for surgical resection, transplant or ablation. Instead, reducing tumor burden becomes the goal of treatment. This scenario is also the case for patients who may be in lower BCLC stages but their diminished overall health renders them ineligible for safe surgical intervention. Treatments designed for this purpose include the direct administration of either intra-arterial chemotherapy ) or radiotherapy ) and are collectively referred to as locoregional therapy ,42. SpecTARE, on the other hand, utilizes selective intra-arterial injection of radioactive yttrium-90 (Y-90), iodine-131 (131I) or rhenium-188 (188Re) to cause radiation-induced cell necrosis . SimilarThese locoregional methods of treatment for advanced disease, while showing some benefit to patients, ultimately do not lead to a sustained response. Residual tumor and local recurrence often necessitate additional TARE or TACE procedures and progression of disease sometimes renders these locoregional therapies ineffective with no response seen . Also, tFor advanced staged HCC tumors (BCLC B & C), surgical and locoregional therapies are not recommended. Instead, the disease is so advanced only systemic therapies are applicable in this patient population. Sorafenib, one of the medications which is FDA approved for advanced HCC, is a systemic oral therapy which inactivates multiple kinase proteins, including vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR) and rapidly accelerated fibrosarcoma (RAF) kinases, halting pathways responsible for angiogenesis and cell growth ,47. It iIn order to understand how immunotherapy could work in treating HCC, it is first important to characterize the nuanced complexity behind liver immunogenicity in a healthy state. Liver parenchyma is characterized by a rich, dense network of hexagonal lobules consisting of a triad of biliary, arterial and venous structures surrounding a central vein. Within these lobules, 60% of liver parenchyma is composed of hepatocytes. Amongst the remaining tissue are sinusoids lined with Kupffer cells. The liver serves numerous critical functions such as protein synthesis, metabolism and detoxification. The ultimate function of the Kupffer cells is to phagocytose cellular debris from these processes and remove circulating endotoxins, sometimes activating cytokines and recruiting immune cells in response . BetweenAnother critical feature of the liver is its immune regulation and relative immune tolerance ,55. ImmuThe HCC tumor utilizes the relative state of immunotolerance afforded to the liver to evade detection, creating what is known as a tumor microenvironment. This microenvironment is comprised of similar immunologic elements as the liver in its healthy state but with differing local recruitment, cellular crosstalk and immunoanatomy. A schematic of normal versus tumor microenvironment is represented in In addition, there are cancer-specific immune responses. For example, there is the recruitment of tumor-infiltrating lymphocytes (TILs), which normally reside in the tumor cells’ immediate environment or sometimes within the stroma of the tumor . TILs arThese factors create a variety of unique tumor microenvironments which help explain the heterogeneity of the HCC tumors. In fact, the microenvironments are likely different even amongst HCC tumors resulting from the same etiology. For example, one study showed distinct and differing populations of immune response cells have been found in HBV versus HCV HCC tumor microenvironments ,76.Besides the HCC immunomodulation, cancer cells, in general, play a large role in shaping the microenvironment based on their metabolic requirements. Cancer cells require accelerated cellular growth, progressive mutagenesis and altered cellular metabolomics, creating a distinct microenvironment around them to support these requirements characterized by relative hypoxia, acidosis and aerobic glycolysis (known as the Warburg effect) . This crThe tumor microenvironment is vital for mutagenesis, tumorigenesis and neoplastic proliferation in HCC. The distinct nature of the microenvironments could provide specific targets, in the form of TILs, modified APCs, activated macrophages or other unidentified antigens for immunotherapeutic regimens . This woSimilar to the development of routine vaccines, vaccines can also be created to prime a patient to target tumor cells. These vaccines can be targeted to specific nucleic acids or polypeptides so that when they present in a patient, the cells will be targeted by the hosts’ immune response ,85. TherDirect targeting of immunosuppressive receptor-ligand interactions offers a potential method of slowing tumor progression. PD-1 is a T cell receptor which inhibits T cell activation promoting tolerance which, in the tumor microenvironment, allows unchecked tumor proliferation and has been found on TILs in the HCC tumor microenvironment . PreviouCTLA-4 or CD152, is an immune system checkpoint receptor which downregulates immune system response by limiting co-stimulation, activation and subsequent proliferation of T cells through competition against the normal co-stimulatory signal and were first studied in mesothelioma . TremeliFirst identified as a means to study T cell activation, chimeric antigen receptors (CARs) fused to T cells were used by a group of scientists, including Carl June from the University of Pennsylvania, to successfully treat a 5 year old girl named Emily Whitehead with relapsing acute lymphoblastic leukemia (ALL) in 2010 . CAR T cAFP is secreted and sometimes utilized as a tumor marker for HCC. Previously believed to be too non-specific and non-functional to target, all proteins are presented in class I MHCs and, thus, the opportunity to program CAR T cells to recognize this complex was established ,104. TheMucin-1 (MUC-1) is also known as polymorphic epithelial mucin and is a group of glycoproteins with high molecular mass that is overexpressed in human cancer cells including HCC cells . A PhaseEpithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell signaling, migration, proliferation and differentiation. It has been identified as a marker for stem/progenitor cells of the liver and is disproportionately upregulated in HCC . For thiGlypican-3 (GPC3) is a member of heparan sulfate proteoglycans and is located on the cell surface. GPC3 is known to be upregulated in HCC as well as other solid tumors . This prIn the treatment of advanced HCC cancer, CAR T seems to overcome many barriers that current therapies cannot. However, CAR T cell therapy creates a new set of obstacles that must be addressed. Concerns with this mode of therapy include a reported “on-target, off-tumor” destruction of normal cells as well as an exaggerated cytokine response when CAR T cells are infused, both of which can be extremely harmful to the patient ,120. In Additionally, while targets may be accessed via infusions in hematologic malignancies or cancers of circulating blood cells such ALL, solid tumors are static and surrounded by a tumor microenvironment. As we have discussed, HCC tumor cells are particularly adept at altering and evading immune responses, ultimately hindering CAR T immune cell trafficking and diminishing efficacy significantly ,120. AnoThere has been parallel work done in the field of immunotherapy on the use of autogenously-generated anti-tumor lymphocytes in the treatment of advanced HCC. Preliminary studies using various HCC targets have been performed in patients, with moderately improved responses. Some studies reported increases in survival from 30 months to 44 months on average as well as increases in disease-free survival over 1-year (RR = 1.23), 2-year (RR = 1.37) and 3-years (RR = 1.35) in patients who received adjuvant autogenously-generated activated cytokine-induced killer T cells and natural killer cells after either surgical resection, radiofrequency ablation or percutaneous ethanol injection . In addiSingle-modality therapy options each have their own set of limitations leading to decreased efficacy. Only 10–20% of treated HCC patients show a durable response to current treatment. However, a multimodal approach has been explored in HCC treatment, with the most promising combinations involving immunotherapy.One strategy trialed was the combination of checkpoint inhibitors and systemic therapies, in the hopes of augmenting the effect of systemic therapy. PD-1 inhibitors, as well as other checkpoint inhibitors, have been trialed in combination with sorafenib and have shown increased anti-tumor effects in in vivo models. Unfortunately, in humans, disease-free and overall survival were not statistically increased and the combination requires more broad randomized control trials ,127.To overcome the aforementioned immune cell trafficking barriers of systemically infused CAR T therapies, techniques developed for TARE are being used to deliver CAR T cell infusions . Highly In addition, radiofrequency ablation and irradiation can be combined with either systemic or directly infused CAR T cell to improve the efficacy of CAR T cells by generating a localized immune response which leads to increased antigen presentation ,130. ThiThough most immunotherapy options are presented to patients that are not surgical candidates, for the growing population of post-transplant patients there is a risk of developing recurrent HCC which would require systemic treatments. Immunosuppression, a requirement after transplant, has historically excluded the transplant population from clinical trials using immunotherapy due to concerns for decreased efficacy and increased harm in this patient population using this modality. PD-1 inhibitors were contraindicated in patients with previous solid organ transplantation as there is a documented risk of organ rejection, quoted to be 37.5% in liver transplants, in a systematic review . HoweverHepatocellular carcinoma is a significant cause of morbidity and mortality worldwide, with no reliable method to detect the disease early in its progression. This is combined with the fact that systemic treatments available offer modest, at best, improvements in overall survival and disease-free survival in advanced or recurrent HCC, highlighting the need for novel therapeutic strategies to treat advanced stage HCC patients. To date, the most promising modality to offer this solution is immunotherapy.While multi-modal treatment may offer augmented HCC response rates when tested properly, this option likely will not address the heart of the issue—HCC is a heterogeneous disease of multiple and often-times interwoven, etiologies. Its complex immunomodulation and tumor microenvironment require even more specific and individual treatments, something most obtainable by immunotherapy. The future of HCC treatment must strive for a patient-specific, tumor-specific treatment and do so in a reproducible and cost-effective manner.If we can correctly identify and exploit druggable targets utilizing immunotherapy on an individual level, we can begin to slow disease progression, downstage patients to be eligible for surgical resection, combine it with surgical resection for improved results or eliminate the need for surgical resection altogether. In this regard, CAR T cell therapy, with its unlimited programmable potential, could be the key to unlock the personalized HCC treatment needed to treat this heterogeneous disease.


Across a wide variety of research environments, the recording of microsaccades and other fixational eye movements has provided insight and solutions into practical problems. Here we review the literature on fixational eye movements—especially microsaccades—in applied and ecologically-valid scenarios. Recent technical advances allow noninvasive fixational eye movement recordings in real-world contexts, while observers perform a variety of tasks. Thus, fixational eye movement measures have been obtained in a host of real-world scenarios, such as in connection with driver fatigue, vestibular sensory deprivation in astronauts, and elite athletic training, among others. Here we present the state of the art in the practical applications of fixational eye movement research, examine its potential future uses, and discuss the benefits of including microsaccade measures in existing eye movement detection technologies. Current evidence supports the inclusion of fixational eye movement measures in real-world contexts, as part of the development of new or improved oculomotor assessment tools. The real-world applications of fixational eye movement measurements will only grow larger and wider as affordable high-speed and high-spatial resolution eye trackers become increasingly prevalent. To date, most studies of fixational eye movements—FEMs, the small movements we make while we attempt to fixate our gaze on a target—have been conducted in laboratory settings. Though invaluable for their controlled and reliable conclusions about well-defined contexts, such restricted scenarios may provide limited insight into the roles and applications of FEMS in real-world conditions.FEMs consist of three main kinds of movements: microsaccades, drift, and tremor. Of these, microsaccades have been best studied, largely due to their comparatively larger sizes and speeds, which make them easier to detect and characterize than drift and tremor —especially with noninvasive commercial video trackers. Thus, changes in microsaccadic dynamics have been linked to changes in perception , 2, 3, 4Extending and expanding this research into ecologically-valid contexts has been particularly important because debates about FEMs have often been ecological in nature: For example, after early studies demonstrated the existence of microsaccades, some researchers questioned whether these movements were a laboratory artifact without any real-world function e.g., , 19. We Accordingly, a growing body of research has started to highlight multiple real-world implications of microsaccades—and to a lesser extent drift—, from monitoring of fatigue to the prevention of occupational health and safety hazards, to the identification of differences in perceptual judgments by novice and elite athletes. Thus, FEM measures in applied domains provide researchers with valuable information about the dynamics of cognitive and perceptual performance within those domains, while helping to clarify the real-world functions of FEMs. Here, we offer a comprehensive review of the literature on applications of FEMs to ecologically-valid scenarios and real-world contexts. Due to the difficulty of measuring tremor, even inside the lab, and the resulting scarcity of studies including tremor measurements, this review focuses primarily on microsaccades, and secondarily on drift. Table 1 lists the microsaccade detection criteria and the eye tracking methodologies used in the studies we examine here.Whereas the great majority of early studies relied on simple, artificial stimuli (but see Winterson and Collewijn ) and higMicrosaccades follow the saccadic “main sequence” relationship between amplitude and velocity, not only during prolonged fixation, but also in the fixation periods that occur between saccades during free-viewing and visual search , 27, 53.Another area of interest in microsaccade research, which has started to extend to more realistic experimental contexts, is the relationship between microsaccadic features and covert attention, the shifting of attention without a corresponding shift in gaze. After some early debate, there is overall consensus in the field that attention and microsaccades are intimately related, with biases in microsaccade direction pointed to the spatial location of covert attention in the visual field , 8, 20. Barnhart et al observed biases in microsaccade direction in the ecologically-valid context of attentional misdirection during the performance of magic . Participants viewed a magic trick where a coin vanished from beneath a napkin and reappeared under a different napkin. Microsaccade direction served as an index of covert attention, helping researchers determine how participants divided their attention across the two napkin locations, while being fooled by the magician’s misdirection. Microsaccade direction has also been linked to social orienting in both humans and rhesus macaques. Guerin-Dugue, Roy  found th Several recent studies have moreover used microsaccade direction to assess differences between the attentional focus of novice versus elite athletes . Attempts have also been made to quantify drift parameters in ecologically valid conditions. Recent work has replicated with dynamic stimuli the previous finding that drift can be successfully modelled as a self-avoiding random walk , 57. To As the use of natural, ecologically-valid stimuli grows, the usefulness of FEM measures in a diversity of research scenarios is likely to become increasingly apparent. One consideration with many potential practical applications is that, whereas microsaccade magnitudes are relevant for close inspection of small objects or object features at short viewing distances, the same slight changes in angular eye rotation may shift an observer’s gaze between large objects at longer viewing distances Poletti and Rucci (59).The field of automotive design holds great promise for the application of FEM measures: eyesight is the primary source of information during driving , and eyeBenedetto, Pedrotti  measuredand moving. Participants wore a head-mounted EyeSeeCam eye tracker and followed a set driving route around the Chubu University Kasugai campus. The vestibulo-ocular reflex altered the gaze data during the drive, as the head moved by itself or as a result of changes in elevation of the road. Microsaccades were detected—though at much lower rates than in other studies—including during the compensatory slow-phase of the vestibulo-ocular reflex.Miki and Hirata  tracked Mental fatigue is a common cause of errors in critical situations such as while driving, flying a plane, or performing surgery. Multiple research studies have found saccadic and microsaccadic eye movement measures to be valid and objective indicators of fatigue in a variety of scenarios, both inside and outside the lab (61-66). Di Stasi, McCamy  first foAs with driving, fatigue is a cause of accidents and errors in aviation, and studies of FEMs in aviation have focused on detecting changes in fatigue. Di Stasi, McCamy  determinAcute hypoxia, defined as decreased oxygen in the body at the tissue level, is a serious hazard in aviation that can lead to visual dysfunction, rapid pulse, dyspnea, syncope, and mental disturbances including delirium and euphoria , 70, 71.A more recent study has reexamined the relationship between microsaccadic velocity and hypoxia. By using a flight simulator housed within a hyperbaric chamber, Thropp and Buza  exposed Air traffic control is another task in which fatigue can result in severe consequences . DecreasObservations of astronauts’ oculomotor behavior provide additional evidence of changes to FEMs in response to flight conditions. Yet, spaceflight differs from atmospheric flight in many ways, including a substantial difference in gravitational forces. On earth, the orientation of the head relative to gravity is constantly signaled by otolith organs, which respond to changes in linear acceleration . Eye movements are modulated in response to changes in these signals as part of the vestibulo-ocular reflex, which maintains gaze stability when the head is moved. The low-gravity conditions present in spaceflight might therefore alter FEMs and decrease gaze stability.Reschke, (2004)  presenteKornilova and Kozlovskaya  tested tKornilova (2004) again reported increases in drift in cosmonauts during spaceflight, as well as increases in nystagmus (both in spontaneous eye movements and while performing stepwise saccades to target points). In addition, the amplitudes of vertical stepwise saccades were reduced, relative to pre-flight baselines, so that 47% of the cosmonauts made additional (micro)saccades to reach the targets. Loss of smooth pursuit movements was also observed in some cosmonauts. Vestibular activation—via active head movements—resulted in improved smooth pursuit tracking, indicating that the oculomotor impairments observed were due to vestibular sensory deprivation from prolonged microgravity exposure (rather than from some other microgravity-related problem). In a follow-up study of cosmonauts during long spaceflights (126–195 days), Kornilova, Alekhina (44) again found similar oculomotor impairments, and noted a link between increased drift and nystagmus, and loss of smooth pursuit.More recently, Kornilova and colleagues  found inTaken together, the studies above indicate that prolonged exposure to low gravity conditions causes gaze instability, with measurable alterations in drift, nystagmus, SWJs, (micro)saccades, and smooth pursuit movements, both during free viewing and in tasks that require the controlled allocation of gaze.Over the past several decades, researchers have increasingly used eye tracking measurements to study cognitive and motor processes in athletes during sports performance . Few of Piras et al. (2015) investigated microsaccade dynamics in table tennis players. A professional table tennis coach was filmed while swinging the racket and striking balls. Expert and novice players watched the coach hit the balls on video and tried to predict, after each hit, whether the balls would end on the left or right side of the table. While conducting this task, participants had to constrain their gaze to a constant fixation target in the dynamic scene: a red dot on the middle of the coach’s chest.During the ‘post-bounce period’ , both experts and novices made more frequent and larger microsaccades than in other time periods. In the case of incorrect predictions, the post-bounce microsaccade rates of experts and novices were comparable. In the case of correct predictions, the post-bounce microsaccade rates of experts were lower than those of novices. Whereas post-bounce microsaccade durations did not differ between experts and novices, experts made longer-duration microsaccades than novices during the ‘response period’ , though only in the case of incorrect predictions. No differences in microsaccade rates were observed between experts and novices during the response period, however. Throughout the viewing task, observers were more likely to direct their microsaccades to the left than to the right of the fixation target—which was the side of the display that most often contained the ball or the racket. No relationship between microsaccade direction and response accuracy or expertise was found, for any task period. In a subsequent study by the same group, observers again viewed pre-recorded table tennis videos, but their fixations were unconstrained . That isboth their foveal vision and parafoveal vision to sample the scene. Whereas experts had higher overall microsaccade rates than novices, both experts and novices made more microsaccades in more frequently fixated regions, consistent with prior reports of increased microsaccade production in consistently fixated natural scene regions . Experts made fewer and longer fixations than novices, perhaps due to having learned to “anchor” or peg their gaze on a given location for extended time periods in order to use During forehand strokes, the hand/racket area attracted higher microsaccade rates from experts, whereas the head area attracted higher microsaccade rates from novices. During backhand strokes, the trunk area attracted higher microsaccade rates from experts, whereas the head area again attracted higher microsaccade rates from novices. In addition, microsaccades were largest and fastest on the head and trunk regions, for both experts and novices. Microsaccade directions shifted between forehand and backhand strokes when both experts and novices fixated the ball, and also when experts (but not novices) fixated the hand/racket region. One possible explanation for this difference is that experts fixating the hand/racket region might allocate their covert attention to the opposite side of visual field (where the trunk/head are located), but that novices lack the skill to do so. Gonzalez, Causer  suggesteOne often-asked question in FEM research has been whether microsaccades help improve performance in tasks requiring high-acuity judgments, such as the threading of a needle. any real-world tasks, measured the eye movements of participants while they aimed and fired an air rifle, or threaded a sewing needle. The experiments were designed to mimic the real-life scenarios as closely as possible, while providing objective measurements. Sewing task trials required participants to thread a copper wire into a needle. Trials started when the needle was illuminated, and ended as soon as the wire touched the needle . Shooting task trials similarly began when the rifle was illuminated, and ended with the participant’s pulling of the trigger. As participants conducted these tasks, their microsaccade rates not only did not increase above baseline , but dropped to ~0.5/second at the end of both types of trials (needle-threading and rifle-aiming). This pattern suggested that microsaccades were not important to the performance of high-acuity tasks. An early study by Winterson and Collewijn , aimed aIn a follow-up “needle-threading” study, Bridgeman and Palca  asked paIn contrast to these pioneering studies, later experiments showed that microsaccades can improve performance in active visual tasks, including high-acuity perceptual judgments. Ko, Poletti  reexaminFigure 2. Consistent with prior research . However, this coordination was found in a control experiment, designed for such specific purpose. Participants were instructed to shift their gazes between marks on a slated plane . The researchers reasoned that the needle-threading task did not result in a comparable coordination of microsaccadic movements due to having relatively impoverished 3D depth cues . However, it is possible that vergence movements, version movements, or their coordination, were not critical for the needle-threading task.The combined findings from the above studies indicate that microsaccades enhance performance in certain high-acuity tasks by moving gaze very precisely to key locations, at the right times. Further research is needed to understand the contributions of FEMs to aiming and shooting. Whereas microsaccades could be transiently suppressed just the trigger is pulled, possibly aiding the shooter’s aim, this possibility has not been tested with actual aiming and shooting tasks, where sports-specific demands can differ from those in other high-acuity tasks. For example, elite shooters not only have extensive practice in precisely aligning their aim to small targets, but they also may need to track targets moving in unpredictable directions from unpredictable starting locations (as in clay shooting).One study explored visuo-motor differences between high-level clay target shooters and inexperienced controls in a visually guided saccade task: participants were instructed to quickly saccade only towards red targets (at 6° eccentricity), and to maintain fixation if green distractors appeared instead (at 1.5° or 3° of eccentricity) . Expert This same study also assessed the fixational stability of expert and novice shooters, in terms of their gaze deviation from the fixation point. Whereas the fixation patterns of the two groups were comparable in the presence of shooting targets, novice shooters had more unstable fixations than experts in the presence of distractors. Future research might assess the relative contributions of microsaccades and drift (as opposed to larger saccades) to the gaze (in)stability of experts and novices. Ideally, this work should be conducted in real-world scenarios and involve elite shooters, given that expertise in shooting tasks may not result in oculomotor differences in contrived experimental settings. FEMs, by definition, impede precise fixation. The resulting retinal motion can create difficulties in certain applied and clinical domains. For example, some neuro-ophthalmic examinations (such as pupillographic campimetry) require the patients’ accurate fixation during testing, which can be challenging—particularly in the presence of pathological FEMs. Thus, accounting for FEMs is necessary for the meaningful evaluation of visual field defects, as well as for tracking their progression . ImportaFigure 3.FEMs can also hamper the creation of medical images of a patient’s retina, such as with scanning light ophthalmoscopy (with or without adaptive optics), by generating image warping artifacts and the ensuing image processing difficulties , 85—see Post-processing removal of retinal motion relies on an important prerequisite: having accurate FEM measurements to begin with. Attempts to solve these problems have resulted in an innovative means of recording FEMs with arcsecond resolution, via adaptive optics. only able to effectively measure small eye movements (rather than large saccades), because their fields of view are very limited in size . Adaptive optics scanning laser ophthalmoscopes can provide measures of retinal position at the level of individual cones, and record the movement of these cells during microsaccades, drift and tremor , 87. In FEMs also produce “fixation jitter” in gaze-assisted computer cursors, making eye movement cursors less precise than standard mouse cursors . Except In situations where tasks are impossible or difficult for humans to complete, robots and other devices are increasingly controlled via teleoperation. Gaze-based commands can complement systems relying on visually evoked potentials to operate wheelchairs, robots, or drones , 92. HowInadvertent microsaccades can also introduce artifacts in EEG recordings: these include small corneo-retinal artifacts , 95, as While the study of FEMs promises fruitful applications to everyday life situations, some intrinsic limitations exist due to the nature of FEM themselves. For example, conditions involving excessive blinking would limit the usefulness of all eye movement measures, including FEMs. The link between microsaccades and the state of the observer  has been consistently observed, but it does not appear to produce perfect correspondence, as microsaccade production may jointly depend on low-level visual stimulation features , 49, 98,Though various microsaccadic features have been linked to task performance and fatigue in a variety of tasks—including during fixation, guided viewing and visual search , 28, 65,FEM research has often been limited to artificial contexts, largely due to equipment and methodological limitations: Most head-or desk-mounted high-resolution and high-frequency eye trackers cannot follow gaze while a participant is walking, running, or engaged in many daily activities. However, improvements in commercially equipment, and the increasing affordability of high-speed eye trackers, have expanded the range of feasible scenarios for eye-tracking data collection. Some innovative choices in experimental paradigms and camera setups have further allowed for increasingly realistic experimental contexts. Accordingly, microsaccadic features (among a range of FEM dynamics) are being considered in many research scenarios, in and out of the lab, where they were not traditionally studied. Below, we discuss further future possibilities.A common challenge in image processing is the difficulty in identifying task-relevant image regions. Microsaccade dynamics have been linked to image informativeness, which combines bottom-up and top-down factors . Thus, mLarge saccades have been applied to improve customers’ ‘quality of experience’ with product/services, in contexts ranging from predicting intent to playing a movie  to loweMicrosaccade measurements may be likewise useful in educational settings, by assessing the user experience of students. FEM recordings could help identify what elements of an e-learning platform best capture student interest, as well as help detect when students become fatigued due to excessive cognitive load and/or time-on-task. Additionally, improved models of microsaccadic biomechanics may be used to help prevent visual fatigue in a variety of displays, including virtual environments .FEMs are altered in a wide variety of ophthalmic and neurologic conditions . HoweverThe development of low-cost, noninvasive eye movement-based screening tools for medical conditions could have significant real-world impact. For instance, the inclusion of FEM measurements in screening methods could expand and improve the early identification of disorders, leading to timely diagnoses and treatments. Modelling FEMs in virtual or augmented environments may moreover provide insights into how fixation dynamics change in diseased or injured states .Mental health counselling may also benefit from FEM measurements. FEM dynamics have been linked to physiological states such as arousal , 65, 66.Tracking large and small eye movements may also help assess fatigue in healthcare workers and provide insight into fatigue-related errors , 110. FaIn the COVID-19 pandemic, the healthcare system has shifted towards virtual care  and someFatigue assessments in healthcare workers may be a more pressing issue for in-person consultations, however, in that worker fatigue could increase the risk of infection from emerging diseases . ExtensiPeople generally produce FEMs unintentionally and are unaware of their occurrence . It follEye movements, including microsaccades, are critical to designing visual prosthetics that faithfully reproduce natural vision , 121. ThAn important path for future FEM applications will be to gain further understanding of gaze parameters in professional environments and task scenarios where FEMs are already known to be relevant. For example, though microsaccades are known to be affected by fatigue—as discussed earlier , 31, 34—FEMs may also be used to harness the professional knowledge of workers engaged in specialized visual tasks, particularly those performed in restricted visual areas  where larger eye movements are less relevant. In addition, FEMs might help ascertain differences in expertise among operators. For example, because microsaccade dynamics change with cognitive workload , microsaWe reviewd the literature on real-world and ecologically-valid applications of FEMs, with a focus on microsaccades. Analysis of FEMs has revealed relationships between these movements and fatigue, hypoxia, and vestibular sensory deprivation, as well as other changes in health status. FEM measurements also provide insights into the allocation of covert attention, and what information is important during a given task. Measuring FEMs in ecologically-valid contexts has moreover improved our understanding of the role that these motions play in visuomotor performance and perceptual judgments. Attempts to counteract unwanted FEMs have additionally led to certain technological innovations, such as the creation of novel eye-tracking systems that allow the removal of FEM-based retinal motion artifacts from medical images. Future research may explore not only how FEMs affect performance in applied contexts, but also how FEM measurements can be harnessed to improve execution and/or reduce work hazards in a variety of real-world scenarios—especially as eye tracking technologies continue to advance. http://biblio.unibe.ch/portale/elibrary/BOP/jemr/ethics.html and that there is no conflict of interest regarding the publication of this paper. The author(s) declare(s) that the contents of the article are in agreement with the ethics described in This work was supported by the Empire Innovator Program, State of New York and the National Science Foundation . We thank Dr. Katherine Alexander for feedback during the preparation of this manuscript.


Notch signaling is an evolutionary conserved cell-cell communication pathway. Besides regulating cell-fate decisions at an individual cell level, Notch signaling coordinates the emergent spatiotemporal patterning in a tissue through ligand-receptor interactions among transmembrane molecules of neighboring cells, as seen in embryonic development, angiogenesis, or wound healing. Due to its ubiquitous nature, Notch signaling is also implicated in several aspects of cancer progression, including tumor angiogenesis, stemness of cancer cells and cellular invasion. Here, we review experimental and computational models that help understand the operating principles of cell patterning driven by Notch signaling. First, we discuss the basic mechanisms of spatial patterning via canonical lateral inhibition and lateral induction mechanisms, including examples from angiogenesis, inner ear development and cancer metastasis. Next, we analyze additional layers of complexity in the Notch pathway, including the effect of varying cell sizes and shapes, ligand-receptor binding within the same cell, variable binding affinity of different ligand/receptor subtypes, and filopodia. Finally, we discuss some recent evidence of mechanosensitivity in the Notch pathway in driving collective epithelial cell migration and cardiovascular morphogenesis. Notch signaling is central in cell fate decisions and therefore it is one of the most well-conserved transduction pathways in metazoans . In its Each of the abovementioned steps in this cascade raises unanswered questions that would improve our understanding of several developmental processes and may also provide key insights to alleviate many pathological conditions, including cancer . Here, wIn this review, we offer a bird’s eye view on how to interpret cell-level and tissue-level dynamics with simple concepts such as lateral inhibition and lateral induction, discuss the limitations of these models, and highlight a novel set of questions that require integrating experimental investigation with concepts from quantitative mechanistic modeling. In doing so, we bring together the analysis of several biological systems as well as theoretical modeling approaches that highlight the emergence of common themes in the Notch pathway. For the sake of simplicity, technical details of the underlying biology and mathematical models have been occasionally omitted, and relevant literature has been suggested. Furthermore, given the extensive set of topics covered in this review, we have focused on certain experimental and/or theoretical models that are representative of a particular system, and pointed the interested readers to relevant reviews for in-depth discussions of specific areas of research.First, we review some aspects of the Notch signaling cascade that are necessary to understand Notch-driven pattern formation. It is followed by a discussion of various modeling approaches that can be used to understand the operating principles of Notch. After these two introductory sections, we discuss the principles of Notch-driven patterning. We analyze how Notch signaling gives rise to divergent cell fate – lateral inhibition – and convergent cell fate – lateral induction – among neighboring cells. Experimental evidence and theoretical modeling have contributed to understanding the competition and synergy between these patterning mechanisms in various physiological and pathological systems, including angiogenesis, inner ear development and cancer metastasis. Moreover, we review the oscillatory dynamics of Notch signaling that can arise due to coupling with other signaling pathways, for instance, during somitogenesis. Further, we examine the role of various molecular and morphological features that introduce additional layers of complexity to the canonical Notch signaling outcomes. The scenarios discussed here include the role of cell shape and packing geometry, cis-interactions between molecules within the same cell, mechanisms that alter the binding affinity between ligand and receptor paralogs, and beyond-nearest neighbor signaling through filopodia. In the final section, we review evidence pointing to a role for mechanosensitivity in assisting Notch-driven cell-fate decision. Relevant examples discussed here include collective epithelial cell migration and cardiovascular morphogenesis.In this section, we discuss the main components and steps of the Notch signaling cascade. We will avoid excessive details on the molecular structure of the Notch receptor and ligands that are not required for the topics discussed in this review.The main steps of the Notch signaling cascade are very well conserved across several organisms and include production and targeting of the Notch receptor to the cell membrane, ligand-receptor binding, conformational rearrangement of the receptor, release of the intracellular domain (NICD) and downstream transcriptional regulation . First, Notably, NICD regulates the transcription of the Notch receptor and its ligands, either in a direct or indirect manner. Specifically, NICD promotes the transcription of Hey/Hes1  – an inhAlthough the main steps of the signaling are quite general, there are specific aspects that differ from one organism to another, or even from cell to cell – these will be the focus of the section on “Non-canonical Modulation of Notch Signaling.” First, the signaling depends on cell-cell contact area because physical contact is required for juxtacrine signaling . We willFurther details on the signaling cascade will not be considered here; additional information can be found in several excellent reviews . Here, wIn this section, we briefly overview different classes of theoretical models that have been applied to Notch signaling.X), which can either represent a concentration or copy number. In many cases, since molecular copy numbers are large, X is treated as a continuous variable that obeys an ODE of the form:Many mathematical models aim at reconstructing the dynamics of mutually interacting biochemical species and/or genes in the Notch pathway with ordinary differential equations (ODEs). In these models, each chemical species/gene is described by a variable , there is only constitutive production (Kprod = K0). Conversely, at high NICD (NICD≫S0), the transcription rate is scaled by a fold-change (Kprod = K0λ). Therefore, λ < 1 implies a decrease of transcription rate (inhibition), while λ > 1 (activation) implies an increase of transcription rate  of a ligand or receptor that binds to X, and k is the ligand-receptor binding rate constant.Γ binding . TherefoN interacting biochemical species or genes, such as the intracellular signaling network sketched in X1,X2, …, XN) and a set of N ODEs of the form of Eq. 1. In such system of equations, the production term for Xi  is determined by the incoming signal from all other chemical species/genes that interact with X.It is worth mentioning that biochemical and gene regulatory networks are sometimes modeled with Boolean, rather than continuous, variables. A Boolean variable can only assume two states These models of intracellular dynamics can be generalized to a multicellular scenario by arranging cells in a discrete lattice . In thesLattice model, however, assume a rigid arrangement of cells on a grid, and cannot take into account biophysical processes including cell migration and cell growth. Biochemical signaling and cell-level behavior can be integrated in agent-based models . In agenN points connected together according to a pre-defined rule, such as elastic springs . Secreted VEGF molecules bind to VEGF receptors (VEGFR) in the endothelial cells at the boundary of an existing blood vessel . ActivatMathematical models suggest that tip-stalk differentiation can be understood as an example of lateral inhibition where external VEGF inputs activate Notch-Dll4 signaling . MoreoveA binary model of Notch-Delta driven tip-stalk differentiation, however, cannot fully explain some experimental observations . For insVarious models have been developed to explain deviations from classical Notch-Delta driven angiogenesis. in vitro  that activates Jag1 in vitro . Strikinin vitro . Mathemain vitro . While hin vitro . Therefoin vitro .The dynamics of Tip-Stalk differentiation receives several signaling inputs besides VEGF and TNF. In a pathological context, cancer cells can stimulate the sprouting of new blood vessels in the tumor microenvironment to supplement tumor growth . TypicalAs we gain a better understanding of the complex spatiotemporal dynamics of normal and tumor angiogenesis, the advantages and disadvantages of combining drugs targeting angiogenesis with other standard-of-care therapies demand further investigation. Limited exposure to vasculature potentially protects the tumor from therapeutic agents that directly target cancer cells. Thus, perhaps counterintuitively, a transient renormalization of the tumor vasculature, timely synchronized with antitumor drugs, could serve as a potential strategy to alleviate tumor progression .Due to the strong coupling between signaling and cell mechanics observed during angiogenesis, several mathematical models have explored the connection between molecular mechanisms and cell- and organ-level behaviors. Further information on these models, which are not discussed here, are reviewed by Lateral induction and lateral inhibition operate progressively at different stages of the inner ear development to turn an initially homogeneous population of non-sensory cells into a refined mosaic of cells with specific phenotypes. The inner ear is composed of hair cells that convert external stimuli into electrical signals, and supporting cells that provide tissue scaffolding, maintain a stable electrochemical environment, and occasionally differentiate to replenish the hair cell population after an injury . During Metastases represents the most critical step during tumor progression. Typically, cancer cells invade the circulatory system, reach anatomically distant sites and give rise to a secondary tumor . These cGenerally, epithelial cancer cells partially or completely lose their cell-cell adhesion and acquire motility by undergoing the epithelial-mesenchymal transition (EMT) . EMT canAn effort to elucidate the coupled dynamics of Notch signaling with the EMT gene regulatory network  suggestsBesides, Jag1 can also stabilize a hybrid epithelial/mesenchymal (E/M) cell phenotype . Such hyin vitro that knockdown of Jag1 inhibits the formation of tumor emboli in hybrid E/M inflammatory breast cancer (IBC) – a rare but highly aggressive form of breast cancer that moves largely collectively through clusters . Typically, cells undergoing a partial or complete EMT also show traits of CSCs . Mathemaclusters  – cells clusters .Notch signaling can also regulate spatiotemporal pattern formation at the level of a tumor tissue. Analysis of breast cancer tissues highlighted subsets of mesenchymal CSCs at the tumor invasive edge, while subsets of hybrid E/M CSCs were largely localized in tumor interior . A recenFinally, the transition to a mesenchymal phenotype is not exclusive to epithelial cells. Besides undergoing tip-stalk differentiation in sprouting angiogenesis, endothelial cells can alternatively undergo Endothelial-to-Mesenchymal Transition (EndMT) . While tSo far, we discussed mechanisms of spatial patterning. Due to its crosstalk with other signaling pathways, however, Notch can exhibit non-trivial temporal patterns. As an example, here we discuss somite segmentation, a well-known example of Notch oscillatory dynamics. During somite segmentation, the embryo’s body axis is segmented into somites – blocks of epithelial cells that later give rise to vertebrae and tissues in the adult body . SegmentOscillations in gene expression are generated in a cell autonomous manner via an autoregulatory negative feedback by Hes/Her proteins. Upon protein productions, Hes/Her molecules dimerize and suppress their own transcription . The delSeveral experimental observations suggest a role for Notch-Delta signaling in synchronizing oscillations in neighboring cells, due to the biochemical coupling between the Notch and Hes/Her pathways. As previously discussed, NICD transcriptionally activates the family of Hes/Her molecules, which in turn, represses Delta . TherefoNotch-Delta binding potentially provides information about the phase of the Hes/Her clock in neighbors. Mathematical modeling of the Notch-Hes/Her circuit developed by Lewis and colleagues  suggestsIt remains unclear whether Notch’s unique role is to ensure robust temporal correlation among neighbors. While it is generally accepted that Hes/Her self-inhibition is sufficient to generate temporal patterns, a number of studies in mouse models suggest that Notch might be required for oscillations. For further details, a comprehensive review on the role of Notch signaling in the somite segmentation clock is offered by In the previous section, we discussed mechanisms of lateral inhibition and lateral induction guided by biochemical feedbacks between Notch and its ligands. In this section, we review mechanisms that modulate Notch signaling besides canonical positive and negative transcriptional feedbacks. These include dependence on cell-cell contact area and cell packing geometry, binding between receptors and ligands within the same cell, specificity in the affinity between receptor and ligand paralogs, and mechanisms enabling signaling beyond nearest neighbor. From a phenomenological standpoint, these mechanisms can be viewed as additional features beyond the simple nearest neighbor signaling mechanism.In the previous section, we developed a geometrical intuition on lateral inhibition that is based on alternate arrangement of Sender and Receiver cells. Mathematical modeling of Notch-Delta signaling helps understand these patterning dynamics on idealized ordered lattices. For instance, Notch-Delta signaling leads to a very specific pattern where Senders are surrounded by six Receivers on a perfect hexagonal lattice see . DisordeThe development of the basilar papilla, the avian equivalent of the mammals’ organ of Corti, exemplifies how fluctuations in cell arrangement modulate lateral inhibition. The fully developed basilar papilla consists of a hexagonal mosaic where Sender cells  are surrounded by six Receiver cells  . GoodyeaThe size of shared contact area between neighbors is expected to fine-tune Notch signaling. Shaya and collaborators investigated the relation between cell size and cell fate by integrating experimental and computational methods . By incoAlthough Notch has evolved as a cell-cell signaling mechanism, receptors and ligands can bind within the same cell. Ligand-receptor binding within the same cell, or cis-interaction, does not lead to downstream signaling, but rather to ligand-receptor complex degradation (cis-inhibition) . DespiteAlthough cis-interactions are mostly known to degrade Notch signaling without any contribution to signaling, experiments recently reported cell autonomous activation of Notch, such as in the cases of Drosophila bristle precursor cells and cell cycle regulation in T cells . These eGiven the role of cis-inhibition in enforcing robust lateral inhibition, it can be postulated that a switch from cis-inhibition to cis-activation would compromise precise cell patterns of Sender and Receiver cells. The number of Notch receptor and ligand subtypes varies considerably in different species see . TypicalFirst, binding affinities depend on the molecular structure. For instance, Notch1 has a greater affinity to Dll4 than to Dll1 and Jag1 . MoreoveMechanisms that modify the binding affinity between the various subtypes of receptor and ligand can potentially result in a shift in cell fate by introducing an asymmetry between Delta and Jagged ligands. One such well-characterized mechanism is the glycosylation by Fringe proteins that results in a conformational change in the extra cellular domain of the Notch receptor . GlycosyAlthough the Notch pathway is primarily designed as a pairwise signaling mechanism among nearest neighbors, beyond nearest neighbors’ interactions are occasionally enabled by filopodia.Filopodia can extend up to several cell diameters and thus introduce contacts beyond nearest neighbor . For ins3/s) from cell to cell in hamster ovary cells (b), however, the signaling strongly depends on the influx of Delta ligands in the contact area but only weakly on the size of the contact area  . IncorpoThe coupling between Notch signaling and mechanical forces is not unidirectional: Notch signaling can, in turn, regulate the function of vascular barriers that separate blood from tissues. For instance, Notch drives the assembly of adherens junctions in a non-canonical mechanism  . ConsistNotch signaling is one of the most ubiquitous transduction pathways in vertebrates. Despite the variety of biological systems and processes, both physiological and pathological, that Notch signaling regulates, its structure and function are incredibly well-conserved.Notch signaling has drawn incredible attention from the physics and mathematics community because, besides regulating cell-fate at a single cell level, it offers fertile ground to dissect the principles of spatiotemporal pattern formation in a tissue. To the eye of a physicist/mathematician, Notch signaling gives rise to the modes of lateral inhibition and lateral induction similarly to a system of spins that align together or in opposition in a magnet. However, unlike magnetism, these different outcomes of cell states emerge from underlying molecular interactions that are often non-linear and can be separated in time-scale as well. The geometrical intuition about Notch patterning via lateral inhibition and lateral induction provides a key to interpret experimental observations in physiological processes such as embryonic development and angiogenesis . For exaMoreover, most of the theoretical efforts toward understanding the operating principles of Notch have focused on deterministic models. Cell-to-cell variability, however, can arise due to both stochasticity in the intracellular biochemical signaling (intrinsic noise) and fluctuations of other cellular components and/or in the extracellular environment (extrinsic noise) . FollowiAdditional factors such as cell size and shape, affinity of ligand subtypes, molecular interactions within the same cell, and filopodia modulate the signaling. These mechanisms can be generally seen as details that add further complexity to the simple nearest neighbor’s communication mechanism. For example, it is still not completely understood how trans- and cis-interactions integrate to establish cell fate. Cis-interactions between receptor and ligands of the same cell can typically lead to mutual degradation . Recent vice versa.Moreover, early experimental findings suggest a role for mechanosensitivity in modulating Notch. The effects of extracellular forces on Notch activation are more quantified at the single molecule level ; it remaNotch signaling has also received attention as a therapeutic target to curb cancer progression . While tAnother exciting direction concerns the widespread development of single cell sequencing techniques. Recently, Notch signaling has been studied at the single cell resolution in Zebrafish development, hematopoiesis and cancer stem cells . For insOverall, insights from experimental and theoretical models continue to unravel the operating principles of Notch signaling, a master regulator of spatiotemporal cell patterning in development and tumor progression.All authors discussed the content of the manuscript, agreed to be accountable for the content of the work, and discussed and edited the manuscript. FB wrote the manuscript. JO and MJ led the research.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.


A large number of adenoviruses isolated from humans and non-human primates, which have low seroprevalence in humans, have been vectorized and tested as vaccines in animal models and humans. However, a distinct hierarchy of immunological potency has been identified between diverse Ad vectors, which unfortunately limits the potential use of many vectors which have otherwise desirable manufacturing characteristics. The precise mechanistic factors which underlie the profound disparities in immunogenicity are not clearly defined and are the subject of ongoing, detailed investigation. It has been suggested that a combination of factors contribute to the potent immunogenicity of particular Ad vectors, including the magnitude and duration of vaccine antigen expression following immunization. Furthermore, the excessive induction of Type I interferons by some Ad vectors has been suggested to impair transgene expression levels, dampening subsequent immune responses. Therefore, the induction of balanced, but not excessive stimulation of innate signaling is optimal. Entry factor binding or receptor usage of distinct Ad vectors can also affect their in vivo tropism following administration by different routes. The abundance and accessibility of innate immune cells and/or antigen-presenting cells at the site of injection contributes to early innate immune responses to Ad vaccination, affecting the outcome of the adaptive immune response. Although a significant amount of information exists regarding the tropism determinants of the common human adenovirus type-5 vector, very little is known about the receptor usage and tropism of rare species or non-human Ad vectors. Increased understanding of how different facets of the host response to Ad vectors contribute to their immunological potency will be essential for the development of optimized and customized Ad vaccine platforms for specific diseases.Adenoviral vectors are a safe and potently immunogenic vaccine delivery platform. Non-replicating Ad vectors possess several attributes which make them attractive vaccines for infectious disease, including their capacity for high titer growth, ease of manipulation, safety, and immunogenicity in clinical studies, as well as their compatibility with clinical manufacturing and thermo-stabilization procedures. In general, Ad vectors are immunogenic vaccines, which elicit robust transgene antigen-specific cellular (namely CD8 Adenoviridae and the genus Mastadenoviridae, and are further subdivided into species A-G . Accounting for the inclusion of many Ad recombinants (http://hadvwg.gmu.edu/) and >200 non-human Ad serotypes have been identified to date. Adenoviruses are non-enveloped viruses which contain a double-stranded DNA genome. The virion exterior is composed of three major structural proteins, the fiber, the penton base and the hexon .+ T cells responses in mice following immunization with an Ad vector encoding the Ii or β2-microglobulin fusion antigen. In a separate approach, we recently have shown that encoding vaccine Ag cassettes in which a model antigen, enhanced green fluorescent protein (EGFP), is fused to a protein domain known to tether proteins to the surface of extracellular vesicles (EVs), can dramatically improve the humoral immunogenicity of both Ad5 and chimpanzee Ad vector ChAdOx1 in mice following i.m. and i.n vaccination. EVs play important roles in regulating immune responses and are conveyors of cellular communication signals  immunization with Ad5 expressing Ebola virus glycoprotein (GP) could provide 100% protection from challenge in NHPs   who are at increased risk of severe RSV disease, with similar results  fusion (F), nucleocapsid (N), and matrix M2-1) antigens has also been tested in humans following stration . Neutral results . Immune  results , has bee results –174. Whe results , 176, bu results . ChAdOx- antigensClinicalTrials.gov, this platform has advanced into clinical studies as a vaccine against Ebola virus (Macaca mulatta) , heterologous prime:boost vaccination regimens should be evaluated using diverse Ad vectors, or Ads in combination with MVA or protein based immunogens at different intervals. It is difficult to predict which Ad vectors should take precedence as the encoded antigen will need to be tailored to elicit the correct phenotype of immunity against a defined correlate of protection for each specific disease target. However, underpinning the evaluation of all Ad-based vaccines in pre-clinical animal studies, should be the inclusion of species C Ad5 vector controls to represent a benchmark of immunological potency. In addition, Ad vaccine candidates should be compared at several doses to evaluate the maintenance of vaccine potency upon dose de-escalation. The field should also make efforts to improve our understanding of the basic biology of many of these novel Ad vectors, as insights into the receptor usage, interactions of Ad vectors with different cell types following immunization and subsequent stimulation of differential innate signaling pathways will all impact on their downstream immunogenicity and ability to confer protective efficacy. Unfortunately, one major challenge in performing these types of head-to-head comparisons is the lack of widespread availability of many of these rare species or non-human Ad vectors to academic investigators, as many of these are being developed by large pharmaceutical companies.It is clear that a hierarchy exists in the immunological potency observed between rare species human and non-human Ad vectors in various animal species. As outlined above, Ad vector immunogenicity is most likely dependent on a complex combination of factors, rather than any particular factor in isolation. An ideal Ad vaccine platform will combine the following attributes; (i) low seroprevalence in humans, (ii) robust immunogenicity with potential for dose-sparing, and (iii) suitable manufacturing characteristics . Better understanding of the mechanisms which define effective vaccines, will enable us to design and customize improvements to existing Ad vaccine vectors, enhancing their potential for future clinical use.LC: writing and editing—original and final draft.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages , cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on  Small hmunities , while oHere, we review how the application of the CARD-FISH technique helped to advance our understanding of microbial trophic interactions and energy fluxes through microbial food webs, with the focus on aquatic nanoplanktonic flagellates (NF). First, we shortly discuss the currently used CARD-FISH protocols for protists and their combinations with other fluorescence-labeling techniques that allow determination of feeding modes and preferred prey at a single cell level in natural environments without any sample manipulation. Subsequently, we demonstrate how these approaches revealed hidden ecological and ecophysiological traits of so far unknown and morphologically almost indistinguishable protistan lineages in marine and freshwater habitats. Based on this novel knowledge that considerably modifies the current views on the carbon fluxes in pelagic environments, we propose an updated model of microbial food webs that more realistically reflects the complex trophic interactions and specific roles of NF. Our overarching aim is to attract more attention of microbial ecologists to the intriguing aspects of protistan ecology that these single-cell resolution approaches enabled to discover, thus considerably advancing our understanding of microbial food web structure and functioning.The existence of protists was discovered in the 17th century by Antonie van Leeuwenhoek, who was the inventor of a light microscope, the main tool used in ecological studies of these captivating organisms until the late 20th century. This technique provided basic information on the morphology and gave preliminary hints on feeding modes of the most commonly observed and cultured microplanktonic protists (cell size > 20 μm) that possess diverse cell structures and shapes allowing for tentative discrimination of species. Thus, ciliates or dinoflagellates can be microscopically classified, and a large number of planktonic taxa have been described . ConsequSpumella-like,” “(Pseudo)Bodo ssp.,” or “Paraphysomonas spp.” functional guilds of key bacterivores in marine and freshwaters, they failed to uncover their phylogenetic affiliation and diversity  ribosomal RNA (18S rRNA) became widely used as a marker gene in community analyses of protists . Due to shwaters . HoweverTo circumvent this lack of ecophysiological information, HTS sequencing studies have been combined with advanced statistical analyses, e.g., multivariate or co-occurrence networks . Such coNannochloropsis salina (Eustigmatophyceae) to 315,000 in the peritrich ciliate Vorticella sp.   . FISH ann (FISH) . The keyn (FISH) . It has n (FISH) . Howevern (FISH) . The main (FISH) . Double n (FISH) . Furthern (FISH) .The main strength of the FISH technique is that it is based on SSU rRNA gene phylogeny, and thus, it allows for the direct detection of phylotypes, the presence of which was identified in the samples by sequencing. Probes for novel lineages discovered with the HTS methods can be designed using full-length sequences of the 18S rRNA genes that are identical or have high similarities to short reads obtained via HTS , obtainiin situ hybridization employs oligonucleotide probes that target short regions  of rRNA genes and bind to a specific sequence of rRNA molecules in intact ribosomes. The first step of the CARD-FISH approach is selecting an already published probe or designing a novel probe for the lineage of interest based on a phylogenetic tree. Although most probes have been so far designed for bacteria and archaea, protists can be targeted as well, requiring only slight modifications in the general probe-design process described in great detail by In general, fluorescence Sample fixation is the key step for the preservation of protistan cells for the CARD-FISH procedure , step 1.Samples for CARD-FISH should be filtered with low vacuum underpressure onto white polycarbonate filters that retain microbial cells on their surface , step 2.In contrast to prokaryotic cells , enzymatStringent hybridization and washing conditions are of crucial importance for obtaining the specific binding of probes. They need to be experimentally optimized for each probe. Stringency of the hybridization at a certain temperature is determined by the concentration of formamide in the hybridization buffer and an appropriate concentration of NaCl in the washing buffer . The staCatalyzed reporter deposition or tyramide signal amplification TSA; , step 8 Hybridized and dried filter pieces need to be counterstained with a DNA dye, such as commonly used DAPI stain , which cUnsuccessful CARD-FISH procedure can be identified by weak or lack of signals, high background fluorescence, and unspecific probe binding that can be recognized by diverse shape and size variability of hybridized cells, especially in case of very specific probes. For detailed troubleshooting guide, please refer to the As we show below, CARD-FISH is a powerful tool to unveil ecological traits of poorly studied and uncultured protists in their natural environments at a single cell level. However, as every method, it has its limitations. First, sample fixation might results in a certain cell loss, and fixatives do not have uniform efficiency for all protists . Moreovein situ distribution and seasonal dynamics of individual protistan groups is important for obtaining a clear picture of the microbial processes in any ecosystem  were also found in the sea-ice, but in rather low abundances , labyrinthulomycetes (2–25%), fungi (2–25%), diplonemids (1–3%), Syndiniales group II (2–8%), MAST-4 lineage (1–6%), and an unidentified HF with a peculiar nuclear morphology , 2013. FMicromonas, turned out to dominate the abundance of picophytoplankton in coastal oceans and seas, with contributions exceeding 80%  belonging to Rhizaria . Large Ris cells . Cercozois cells  can accois cells   than members of the MAST-4 lineage (<1.5 bacteria HF–1 h–1), with an overall average cell-specific rate slightly above 2 bacteria HF–1 h–1  HF. Trophic relationships between bacterivorous cercozoans of the Novel Clade 2  targetedtophytes , their Ctophytes , and kintophytes , but alstophytes  and Cerctophytes   in combination with novel probe design for both eukaryotic grazers and prokaryotic prey as highly relevant and useful approaches . This meKP and KŠ conceptualized the study. All authors contributed to drafting the manuscript, preparing the figures, making critical revisions, and approved the final version of the manuscript.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.


Determining the cause of stroke is considered one of the main objectives in evaluating a stroke patient in clinical practice. However, ischemic stroke is a heterogeneous disorder and numerous underlying disorders are implicated in its pathogenesis. Although progress has been made in identifying individual stroke etiology, in many cases underlying mechanisms still remain elusive. Since secondary prevention strategies are tailored toward individual stroke mechanisms, patients whose stroke etiology is unknown may not receive optimal preventive treatment. Cardioembolic stroke is commonly defined as cerebral vessel occlusion by distant embolization arising from thrombus formation in the heart. It accounts for the main proportion of ischemic strokes, and its share to stroke etiology is likely to rise even further in future decades. However, it can be challenging to distinguish cardioembolism from other possible etiologies. As personalized medicine advances, stroke researchers' focus is increasingly drawn to etiology-associated biomarkers. They can provide deeper insight regarding specific stroke mechanisms and can help to unravel previously undetected pathologies. Furthermore, etiology-associated biomarkers could play an important role in guiding future stroke prevention strategies. To achieve this, broad validation of promising candidate biomarkers as well as their implementation in well-designed randomized clinical trials is necessary. This review focuses on the most-promising candidates for diagnosis of cardioembolic stroke. It discusses existing evidence for possible clinical applications of these biomarkers, addresses current challenges, and outlines future perspectives. Due to an aging world population and a global shift from communicable to non-communicable disease, lifetime risk for stroke and the absolute numbers of stroke survivors are increasing all over the world , 2. IschStroke caused by cardioembolism (CE) accounts for ~20 to 30% of ischemic strokes with higher incidence in older patients . CardiacAF represents the main identifiable risk factor for CE strokes, and treatment with oral anticoagulation can prevent up to 70% of recurring events in those patients. Less commonly identified causes of CE stroke include severe systolic heart failure, patent foramen ovale (PFO), prosthetic heart valves, recent myocardial infarction, intracardial masses, and endocarditis. For those, different secondary prevention strategies apply, but levels of evidence for optimal preventive strategies in these cases vary greatly [reviewed by Kamel et al. ]. AdditiTo date, there is no gold standard for the determination of CE stroke etiology. The identification of a high-risk cardiac source of embolism in the absence of significant arterial disease therefore remains the cornerstone of diagnosis. Attempting to standardize categorization, several classification systems are in use. The most widely accepted tool remains the Trial of Org 10172 in the Acute Stroke Treatment (TOAST) classification system developed in the 1990s  stroke oper se but appreciates future stroke risk while inter-examiner reliability remains high  was developed in 2014. ESUS defines a subset of patients with cryptogenic stroke with a non-lacunar stroke based on infarct topography and sufficient diagnostic workup to exclude the presence of a high-risk cardioembolic source or occlusive atherosclerosis . The conBiomarkers of CE-stroke etiology include parameters from cerebral imaging, electrocardiography, echocardiography, advanced cardiac imaging, and blood-based markers. To date, possible clinical applications are mainly risk stratification and acceleration of etiological classification, consequently accelerating optimal treatment. In the presence of competing evidence, biomarkers could increase the level of certainty for a causal relationship of a specific etiology. In cryptogenic strokes (with no evidence for a specific etiology), biomarkers could justify a more extensive workup . Most importantly, some biomarkers have been implemented in the design of randomized secondary prevention trials aiming to characterize high-risk subpopulations for CE stroke . Consequently, biomarkers have the potential to support decision making in future secondary stroke prevention. Blood-based biomarkers share advantages over other types of biomarkers: They are rapidly measurable, they are cheaper compared to imaging or hands-on examinations, and—if possible confounders and demographic adjusted cutoffs are established—their interpretation is examiner-independent and does not require specific training. To date, blood-based biomarkers are not routinely measured in stroke care and due to a substantial overlap in the pathophysiology of possible stroke mechanisms and shared risk factors, yet no single marker can perfectly distinguish between CE stroke and other etiologies. However, they can still add valuable information for both clinicians and researchers.Blood biomarkers of myocardial stress may add information on subclinical cardiac pathologies, e.g., the presence of underlying atrial myopathy, systolic heart failure, or subclinical AF.Natriuretic peptides, namely, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) as well as their cleaved by-products MRproANP and NTproBNP, belong to the most widely studied blood biomarkers to distinguish CE from non-CE stroke. For BNP and NT-proBNP, individual patient-pooled data meta-analysis   as well post hoc analysis of the WARSS trial. This was a randomized trial comparing warfarin and aspirin for the prevention of recurrent stroke in patients with presumed non-CE stroke  and pro-inflammatory cytokines  have been mainly evaluated in regard to the diagnosis of large-artery atherosclerosis, growing evidence suggests a prominent role of systemic inflammation in the pathophysiology of AF  and the D-dimer, a degradation product of cross-linked fibrin, is formed during activation of the coagulation system. In CE stroke, it is hypothesized that D-dimer levels reflect the formation of fibrin-rich thrombus in the left atrium. Contrarily, thrombi originating from large arteries are believed to be mostly platelet-rich and therefore only promote a mild D-dimer increase . The assAlthough this review focuses on the most promising biomarkers that have proven consistent associations in multiple evaluations, it is to mention that preliminary data exists for a variety of other circulating proteins  markers may similarly reveal subclinical cardiac pathologies. For etiological classification, possible markers focus mainly on abnormalities of the cardiac atria, as the left atrium is considered the main source of thrombus formation both, in the presence and absence of AF. P-wave terminal force in lead V1 (PTFV1) is an established marker of left atrial abnormality and was found to be an independent predictor of incident ischemic stroke as well as incident AF in meta-analysis , 89. WhiIn conclusion, preliminary evidence strongly supports a possible role of ECG parameters for etiological stroke classification. However, further studies are required to confirm this association and unravel possible clinical implications.Transthoracic and transesophageal echocardiography, cardiovascular computer tomography (cvCT), and cardiac magnetic resonance imaging (CMR) may reveal previously undetected sources of cardioembolism during stroke workup, e.g., LA thrombus, ventricular thrombus, intracardial masses, aortic arch atheroma, and PFO , 101. DeThe gold standard for the evaluation of LA enlargement remains CMR as it allows for 3D reconstruction as well LA hemodynamics , 108. DeSince the failure of the NAVIGATE-ESUS and RESPECT-ESUS trials to demonstrate a benefit of oral anticoagulation in patients with ESUS, optimal secondary prevention still remains elusive in a significant amount of patients. A more rigorous etiological workup may lead to the identification of more patients with competing etiological factors [like it has been seen in the NAVIGATE ESUS trial ]. Thus, Biomarkers add relevant information for this endeavor, specifically CE biomarkers alone or incorporated into validated risk scores, could guide clinicians selecting patients for more excessive workup . Examples for existing risk scores are the STAF, iPAB, and ASAS scores –116. The2 on echocardiography). In Europe, the Midregional Proatrial Natriuretic Peptide to guide Secondary Stroke Prevention  randomized trial evaluates oral anticoagulation vs. antiplatelet therapy in patients without previous AF (selected by a MRproANP levels ≥ 200 pg/l) and the Apixaban for Treatment of Embolic Stroke of Undetermined Source  trial is evaluating apixaban compared to aspirin in ESUS patients with at least one non-major risk factor for cardioembolism including possible echo- and electrocardiography markers such as LA size >45 mm in parasternal axis, reduced LAA flow velocity ≤0.2 m/s, spontaneous LAA contrast, atrial high rate episodes, and PFO. If completed successfully, data from these trials not only could provide compelling evidence for the concept of atrial myopathy but also may prove the benefit of biomarker-guided therapy.Three trials that incorporate biomarkers to define target populations are currently enrolling: In the US, The Atrial Cardiopathy and Antithrombotic Drugs in Prevention After Cryptogenic Stroke  randomized trial evaluates apixaban compared to aspirin in ESUS patients with presumed atrial myopathy (defined by NTproANP levels > 250 pg/ml, PTFV1 > 5000V x ms or LA enlargement defined as LA diameter index ≥ 3 cm/mThus, biomarkers of CE-stroke etiology could ultimately evolve as a cornerstone in decision making regarding secondary stroke prevention.AM wrote the first draft of the manuscript. SV and KM wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.