Added flavors are a marker for ultra-processing of food and a strong link exists between the intake of ultra-processed food and the development of obesity. The objective of the present article is to assess animal and human data elucidating the impact of added flavors on the regulation of food intake and body weight gain, as well as to define areas for future research.Mechanistic studies suggest that added flavors induce overeating and body weight gain by two independent mechanisms: Added flavors promote hedonic eating and override homeostatic control of food intake, as well as disrupt flavor-nutrient learning and impair the ability to predict nutrients in food items. Supporting these potential mechanisms, added flavors increase feed intake and body weight as compared to non-flavored control diets in a broad range of animal studies. They are actively promoted by feed additive manufacturers as useful tools to improve palatability, feed intake, and performance parameters. In humans, added flavors are extensively tested concerning toxicity; however, no data exist concerning their impact on food intake and body weight.Added flavors are potential contributors to the obesity epidemic and further studies focusing on their role in humans are urgently required. These studies include obesity interventions specifically targeting food items with added flavors and cohort studies on independent associations between added flavor intake and metabolic, as well as cardiovascular, morbidity, and mortality.The online version contains supplementary material available at 10.1186/s12916-022-02619-3. Food additives are substances added intentionally to food to preserve flavor, as well as to enhance taste, appearance, or other sensory qualities . ExampleAdded flavors are used in food for different reasons. Within the last decades, industrial processing of food has led to flavor losses which were compensated by added flavors , 5. TheyIn the present opinion article, arguments are presented that added flavors contribute to the obesity epidemic in recent decades. Thus, mechanistic studies suggest that added flavors induce overeating and body weight gain by two independent mechanisms: Added flavors promote hedonic eating and override homeostatic control of food intake, as well as disrupt flavor-nutrient learning and impair the ability to predict nutrients in food items. Supporting these potential mechanisms, added flavors increase feed intake, as well as body weight, in animals and are actively promoted by feed additive manufacturers. In humans, added flavors are extensively tested concerning toxicity; however, no data exist concerning their impact on food intake and body weight.These arguments are based on the literature search summarized in Additional file Added flavors promote hedonic eatingAdded flavors disrupt flavor-nutrient learningThere are two potential mechanisms by which added flavors might induce food intake and body weight gain:Added flavors might induce overeating and weight gain by promoting hedonic eating and overriding homeostatic control of food intake Fig. . Food inAdded flavors might induce overeating and weight gain by disrupting flavor-nutrient learning and impairing the ability to predict nutrients in food items Fig. . Flavor-Added flavors potentially cause inconsistency between orosensory characteristics of a meal and associated nutrients. Products might taste similar despite differences in their nutritional composition which causes a higher variability in food composition linked with a specific flavor. Convincing evidence suggests that variability leads to disruption of flavor-nutrient learning which subsequently impairs both formation of reliable flavor-nutrient associations and proper regulation of food intake causing overeating and subsequent body weight gain .Figure Added flavors have been used in the production of animal feed for more than 40 years . In pigsAdded flavors are actively promoted by feed additive manufacturers. One manufacturer states that its flavors “are used by some of the biggest feed companies in the world” . The samThe first kind of studies tested the preference of animals regarding flavored and non-flavored feed. The same animals were exposed at the same time period to one or more feeds with added flavors (intervention) and a non-flavored feed (control). Feed intake per time unit was the primary outcome in these experiments. It was significantly higher for at least one added flavor tested in the intervention as compared to the control in studies in goats  and poniIn a second type of studies using a within-subject design, the same animals were exposed consecutively to one or more feeds with added flavors (intervention) and non-flavored feed (control). Feed intake and body weight gain per time interval were the primary outcomes in these experiments. They were higher in the intervention as compared to the control for orange flavor in a study with calves but no difference was found in the feed intake of second-lactation cows . In anotIn a third kind of studies, applying a between-subject design, groups of different animals were exposed to either one or more feeds with added flavors (intervention) or non-flavored feed (control). Comparisons of feed intake, body weight gain, and final body weight between the intervention and control groups were the primary outcomes in these experiments. Feed intake was significantly higher in the intervention as compared to the control for at least one added flavor in various studies in pre-weaning piglets –50, postVarious studies have assessed the impact of added flavors on feed intake, as well as body weight, in animals and the main results are summarized below and in Additional file Combined, these studies indicate that added flavors can increase feed intake, body weight gain, and final body weight. However, none of these animal studies focused on the mechanisms for these effects. Therefore, additional studies should assess how promotion of hedonic eating, disruption of flavor-nutrient learning, and other potential mechanisms contribute to increased feed intake, body weight gain, and final body weight. Moreover, results were heterogeneous and changes in feed intake, body weight gain, and final body weight were not always in the same direction. A major drawback is that several between-subject design studies did not assess all three endpoints  as compared to unflavored feed (control) in animal experiments. Additional file 3. Obesity prevalence in the US and inflation-adjusted US flavor and fragrance industry sales since 1960.


Mycobacterium tuberculosis (Mtb) infections is particularly arduous. One challenge to effectively treating tuberculosis is that drug efficacy in vivo often fails to match drug efficacy in vitro. This is due to multiple reasons, including inadequate drug concentrations reaching Mtb at the site of infection and physiological changes of Mtb in response to host derived stresses that render the bacteria more tolerant to antibiotics. To more effectively and efficiently treat tuberculosis, it is necessary to better understand the physiologic state of Mtb that promotes drug tolerance in the host. Towards this end, multiple studies have converged on bacterial central carbon metabolism as a critical contributor to Mtb drug tolerance. In this review, we present the evidence that changes in central carbon metabolism can promote drug tolerance, depending on the environment surrounding Mtb. We posit that these metabolic pathways could be potential drug targets to stymie the development of drug tolerance and enhance the efficacy of current antimicrobial therapy.Treatment of  Mycobacterium tuberculosis (Mtb) is one of the leading causes of death world-wide by an infectious agent , argininte (DHAP), aspartate (DHAP), methionte (DHAP), lysine te (DHAP), and thrte (DHAP) are sevein vitro . Mtb can pathway . The glyin vitro cultures where Mtb can utilize multiple different carbon sources, Mtb isolated directly from infected mouse lungs was found to preferentially metabolize fatty acids over other carbon sources such as glucose or glycerol  and cholesterol are abundant lipid carbon sources available to nfection . Mtb libinyl-CoA . TherefoMtb drug tolerance  and sulfolipid-1 with increased chain lengths   Figure 2te (PEP)  H-generating steps of the TCA cycle, likely to prevent production of NAD(P)H in conditions that these cofactors cannot be re-oxidized. This altered flux is caused by re-routing of acetyl-CoA to fatty acid and subsequent TAG biosynthesis , increasarvation . These marvation . The Δtgolerance Figure 2olerance . Redirecolerance . A Δicl1 glucose . TherefoMtb is unable to grow on glycerol as the sole carbon source in low pH (Mtb growth in low pH, prevents the pH-induced drug tolerance (glpK mutant exhibited decreased sensitivity to isoniazid and rifampicin compared to wild-type Mtb, further supporting that decreasing glycerol metabolism increases drug tolerance (glpK is dispensable in the mouse model of Mtb infection, suggesting that glycerol is not a primary carbon source in mice (Mtb would have access to glycerol in the host (glpK mutant survived better than wild-type Mtb during treatment with pyrazinamide or any drug combination involving pyrazinamide, but not during isoniazid or rifampicin monotherapy, in a mouse model of infection (Mtb drug tolerance specifically to pyrazinamide. Since the glpk mutant does not exhibit increased tolerance to pyrazinamide in vitro, the pyrazinamide-specific tolerance in the glpK mutant is dependent upon the microenvironment within the host (glpK mutations in Mtb clinical isolates (glpK mutations are associated with but do not confer drug resistance themselves. However, if these mutations promote drug tolerance, they may enable Mtb to survive during antibiotic therapy, extending the time wherein Mtb may acquire a drug resistance mutation.n low pH . This isn low pH  (Figureolerance . Glyceroolerance . glpK is in mice . Howeverthe host . Furthernfection . Therefothe host . Multiplisolates , and in isolates . The glpMtb undergoes changes in carbon metabolism in response to host derived stresses that render Mtb more tolerant to antibiotics. However, the observed drug tolerance is rarely pan-antibiotic. For example, in hypoxic conditions, while Mtb becomes extremely tolerant to some antibiotics, including isoniazid, rifampicin, and streptomycin, it remains susceptible to antibiotics that target ATP synthase, and in some cases becomes more sensitive to killing by ATP synthase inhibitors and other antibiotics that target the electron transport chain (Mtb mutants with altered sensitivity to either isoniazid, rifampicin, pyrazinamide, or ethambutol during mouse infection, the majority of mutants identified only exhibited significantly altered susceptibility to a single antibiotic (In this review, we have highlighted studies demonstrating that rt chain . Furthertibiotic . TherefoMtb that result in increased drug tolerance are often correlated with growth arrest, including toxicity mediated by propionyl-CoA or MCC intermediate accumulation (glpK mutant had no fitness defect in mice yet had altered antibiotic susceptibility to pyrazinamide (Metabolic changes in mulation , hypoxiamulation , and decmulation . Howevermulation . Specifizinamide . In addizinamide .Mtb metabolism impacts tolerance to specific antibiotics, particularly in the host environment, could lead to novel therapeutic approaches. We have highlighted several studies that demonstrate it is possible to manipulate metabolic pathways to reverse tolerance to a number of frontline antibiotics. For example, the Mtb Δtgs1 mutant is more susceptible to killing by isoniazid, rifampicin, or streptomycin during stress and in mice (Mtb by isoniazid (Mtb can potentiate killing by frontline antibiotics. Our review focused on central carbon metabolism, however these pathways are intricately connected to other metabolic networks, such as amino acid biosynthesis. De novo biosynthesis of amino acids from the TCA cycle and other pathways is essential for Mtb virulence and recent studies have shown that inhibiting amino acid biosynthesis is a promising approach for therapeutic development (Understanding the mechanisms by which  in mice . This susoniazid . Thus, celopment . AlthougAll authors participated in the conceptualization and writing of this mini-review.CS is supported by NIH grant AI134847 as well as a Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease Award. GH is supported by National Science Foundation graduate research fellowship DGE-1745038 and NIGMS cell and molecular biology training grant GM007067. EW is supported by NIAID award T32AI007172. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.We are grateful to H. Eoh for kindly sharing his unpublished manuscript for this issue while we prepared our final versions.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.


The use of magnetic resonance imaging (MRI) for liver radiation treatment planning, adaptation and response assessment is an active area of clinical research. Particularly, research on the use of functional MRI to optimise radiation dose distribution and perform mid-treatment adaptation has grown in recent times. The aim of this review is to provide the current state of evidence on the most relevant functional MRI methods being investigated for liver radiation therapy applications aimed at the development of individualised, adaptive treatments for patients with liver cancer.Stereotactic body radiation therapy (SBRT) is an emerging treatment for liver cancers whereby large doses of radiation can be delivered precisely to target lesions in 3–5 fractions. The target dose is limited by the dose that can be safely delivered to the non-tumour liver, which depends on the baseline liver functional reserve. Current liver SBRT guidelines assume uniform liver function in the non-tumour liver. However, the assumption of uniform liver function is false in liver disease due to the presence of cirrhosis, damage due to previous chemo- or ablative therapies or irradiation, and fatty liver disease. Anatomical information from magnetic resonance imaging (MRI) is increasingly being used for SBRT planning. While its current use is limited to the identification of target location and size, functional MRI techniques also offer the ability to quantify and spatially map liver tissue microstructure and function. This review summarises and discusses the advantages offered by functional MRI methods for SBRT treatment planning and the potential for adaptive SBRT workflows. Stereotactic body radiation therapy (SBRT) is increasingly utilised for treatment of primary liver cancer as well as liver metastases, most often in patients who are not eligible for other local therapies such as surgical resection, ablation or embolization techniques ,2,3. SBRTraditionally, normal tissue complication probability (NTCP) models that predict liver toxicity are based on dose-volume parameters . HoweverIn this review, we provide an overview of the various methods used for liver function assessment, focusing on imaging techniques for the estimation of regional liver function. We will then introduce magnetic resonance imaging (MRI) techniques for functional liver imaging and their current application in liver SBRT. Finally, we will discuss the challenges, limitations and potential future directions for functional MRI in liver SBRT.Liver function is currently assessed via scoring systems such as the Child-Pugh (CP), Albumin-Bilirubin (ALBI) and Model for End-stage Liver Disease (MELD) scores, all of which are based on biochemical markers indirectly linked to liver function ,9. LiverThe Indocyanine Green (ICG) test is a biochemical test, where the retention of the intravenously administered fluorescent ICG dye at 15 min (ICG-R15) provides a more direct measure of overall liver function. The ICG is cleared from the bloodstream exclusively by the liver, and if more than 15% of the ICG remains in the circulation 15 min after injection, then liver function is considered to be impaired. ICG tests are routinely performed to assess liver function in patients after liver transplantation or to assess functional liver reserve prior to surgical liver resection ,14. ICG Computed tomography (CT) techniques such as dynamic contrast-enhanced computed tomography (DCE-CT) with pharmacokinetic modeling have shown that portal venous perfusion, described by the portal venous blood flow, correlates with overall liver ICG-R15 measurements, which also reflect liver perfusion. Reduction in portal venous perfusion has been investigated as a measure of liver function dose response, given that RILD is characterised by the development of venous occlusions. Furthermore, regional portal venous plasma flow is correlated with local radiation dose and is a predictor of post-treatment radiation-induced liver dysfunction ,19,20. Ahttps://www.australianclinicaltrials.gov.au/anzctr/trial/ACTRN12619001499178, accessed on 27 September 2022). However, the limited availability of specialist and expensive nuclear medicine facilities is a downside of SPECT and PET based methods.Various single-photon emission computed tomography (SPECT) function . The radng liver . SPECT ing liver ,25. 99mTs scores . 99mTc-Hfunction . Positrofunction ,28. Tracfunction . A cliniOn the other hand, MRI is widely available and most patients with liver cancers already undergo several MRI scans as part of diagnostic, treatment and follow-up procedures. MRI offers high spatial resolution and full liver coverage, with the ability to acquire several functional images giving complementary information on tissue perfusion, cellular function and microstructure in a single imaging session. In the next section, we will discuss some of the functional MRI techniques that are used to assess liver function.A typical liver MRI protocol consists of T2-weighted (T2w) imaging, in- and opposed-phase (IP/OP) T1-weighted (T1w) imaging, T2*-weighted (T2*w) imaging, diffusion-weighted imaging (DWI) and dynamic contrast-enhanced MRI (DCE-MRI) . The anaDWI is a functional MRI technique which derives image contrast from the differences in the amount of diffusion of water molecules in different tissues. Diffusion is a physical process of random, microscopic movements of water molecules due to their thermal energy (Brownian motion). As tissues with different micro-structures, cell densities, vascular networks, and composition present different levels of barriers to water molecular diffusion, DWI can provide an indirect assessment of tissue characteristics such as cellularity, micro-circulatory perfusion and fibrosis. DWI is being increasingly applied for improved confidence in lesion detection and characterisation as well as in predicting and monitoring tumour response to treatment . HCC les2 or more. A quantitative diffusion parameter, the apparent diffusion coefficient (ADC), derived via mono-exponential modeling of the DWI signal at b = 0 s/mm2 and two or more b-values, is widely used to evaluate liver tumours. Malignant liver tumours have a reduced ADC compared to benign tumours and healthy liver tissue, although the values can overlap with the ADC in other solid benign lesions such as hemangioma and hepatocellular adenoma [2, 0 and 500 s/mm2, and 0 and 750 s/mm2) in healthy volunteers before and after a meal [2 reflected the expected post-prandial increase in liver portal venous perfusion. This is because at low b-values, typically less than 200 s/mm2, the DWI signal is additionally sensitive to micro-circulatory perfusion.DWI is performed using strong gradients that sensitise the MRI signal to the diffusion. The sensitivity of the signal to diffusion is controlled by varying the strength and timing of the gradients  during imaging. Several DWI images are acquired at different b-values, commonly ranging from b = 0 to 800 s/mm adenoma . An incr adenoma . Koinuma adenoma . Other s adenoma ,40, demor a meal . They fo2 can be used to separate the perfusion and diffusion components of the DWI signal, giving a pseudo-diffusion coefficient (D*) which reflects the rate of tissue perfusion and a perfusion fraction (f) which reflects the amount of perfusion, in addition to the diffusion coefficient D [The intra-vascular incoherent motion (IVIM) model fitted to DWI image series acquired at several (at least 5) b-values with 3 or more less than 200 s/mmicient D . Althougicient D . Howevericient D .2) and is described by the kurtosis of the distribution, which is incorporated into the Kurtosis model of diffusion [2) are fitted to the Kurtosis model, to calculate diffusion kurtosis Kapp along with a corrected diffusion coefficient DK. It is recognised that ADC values calculated at b-values below 1000 s/mm2 primarily assess diffusion in the extracellular space. In contrast, Kapp and DK additionally describe the interactions of water molecules with cell membranes and intracellular structures [app correlates moderately with CP score and ICG-R15 measurements [app reflects the heterogeneity of tissue microstructure, diffusion kurtosis imaging (DKI) shows promise for functional liver imaging. A summary of the various DWI models and their parameters is given in Both the mono-exponential and IVIM models rely on the assumption that diffusion is gaussian, which is false in biological tissues. The deviation from the gaussian model is more apparent at higher b-values  is a functional MRI technique that involves the acquisition of several images before and after the intravenous injection of an MRI contrast agent. MRI contrast agents are typically paramagnetic Gadolinium ion  the area under the curve (AUC), usually calculated as the the amount of enhancement over a period of time, typically 60 or 90 s (AUC60 or AUC90), (b) the maximum or peak enhancement ratio, which is the maximum relative increase in the T1w signal from baseline, (c) the wash-in slope, which determines the rate of enhancement, (d) the mean transit time (MTT), which describes the average time taken for the blood to perfuse the tissue and relates to the blood volume and flow, and (e) the wash-out slope, which relates to the rate of excretion ,52. Semitrans, is adequate for assessment of tumour perfusion in liver metastases [trans, and the outflow rate constant. The arterial flow fraction, which describes the proportion of supply from the hepatic artery, is derived as the ratio of the arterial inflow rate to the total inflow rate . Often, two additional parameters, the arterial and venous delays, describing the delay in arrival of the contrast agent from the larger hepatic artery and portal vein to the parenchyma are also included in the model [Quantitative DCE-MRI is based on tracer kinetic modeling of the image time series. Standard tracer kinetic models assume a single inlet for the contrast agent from the circulatory system to the tissue vascular space, with passive exchange of the contrast agent between the vascular and extracellular spaces. A two-compartment Extended Tofts model, providing model parameters such as the volume transfer constant Ktastases . Howeverhe model . This mohe model ,60.Pharmacokinetic models that describe the kinetics of hepatobiliary contrast agents are necessarily more complex than perfusion-only models. These models are typically dual-inlet, two compartment models b, and inOther specialised or advanced functional techniques include magnetic resonance elastography (MRE) which is used to measure liver stiffness for assessment of liver fibrosis and cirrhosis, and arterial spin labeling (ASL) which is an alternative perfusion imaging technique. MRE uses an external vibration generator to measure the elastic properties of the liver tissue, which have been correlated with liver functional reserve measured using ICG tests . In ASL,MRI offers excellent soft-tissue resolution for the detection and characterisation of liver tumours. While CT remains the workhorse of radiation therapy, MRI has several advantages such as zero ionising radiation exposure, superior tissue contrast, availability of hepatocyte-specific contrast agents and the ability to obtain anatomic and functional images within a single scan. MRI is, therefore, increasingly being used to provide complementary information to CT images for SBRT planning, although it is not mandated in most liver SBRT guidelines ,73,74. IRegional liver function measurements are not currently used to guide liver SBRT planning. While research in this area is still in its infancy, preliminary studies have showed promise. Proof of concept studies using retrospective data from small groups of patients (<20) with HCC and liver metastases have demonstrated benefits of using spatial liver function maps to identify and spare high functioning liver from irradiation in terms of significant dose reduction to functional liver, while maintaining target coverage and OAR sparing comparable to plans designed based on the anatomy only ,91,92,93In studies conducted thus far, liver function was measured semi-quantitatively—as a percentage of maximum tracer uptake in SPECT images, normalised iodine density  in DECT images, and liver-to-spleen intensity ratios in gadoxetate DCE-MRI images. Only one study used pharmacokinetic modeling of gadoxetate DCE-MRI to derive quantitative perfusion and uptake rates . In studctioning . There ictioning , Simeth al liver . Wei et al liver . The relal liver .Despite correlations observed between chronic liver disease and DWI-derived parameters, there have been no studies using functional DWI parameters to guide liver SBRT planning, and the role of this functional MRI technique in liver SBRT planning remains unexplored.In the clinic, response assessment after liver SBRT follows general assessment criteria for liver cancers, such as the Response Evaluation Criteria In Solid Tumours (RECIST), modified RECIST (mRECIST), the European Association of Study of the Liver (EASL) or the Liver Imaging Reporting and Data System (LI-RADS) criteria. These criteria typically assess changes in tumour size and in the case of mRECIST, EASL and LI-RADS criteria, also include observations on arterial enhancement of the tumours in contrast-enhanced CT or MRI images. Imaging is usually performed every 3 months after treatment for response evaluation. After SBRT, HCC tumours shrink slowly, however, change in size is not always correlated with treatment success . Price eThere are no assessment criteria that account for the radiation-related response of the liver parenchyma. However, several imaging studies report morphological and physiological changes in the immediate surrounding liver parenchyma following SBRT . These cTumour ADC (in both HCC and liver metastases) has been observed to increase significantly compared to baseline as early as 1 week into treatment. In HCC patients treated with SBRT, a tumour ADC increment within 6 months following treatment has been observed to be indicative of favorable response, with estimated minimum effect size ranging from 20% to 25% ,110. HowQualitative assessment of DCE-MRI images is already part of routinely used evaluation criteria, with early arterial, portal venous and hepatobiliary phase enhancement used as indicators of tumour response and parenchymal reaction to radiation as described above. Correlations of tumour and hepatic response with semi-quantitative and quantitative DCE-MRI parameters have also been reported in some studies. For example, an increased wash-in slope and peak enhancement in HCC tumours as early as two weeks following radiotherapy has been linked to improved local response . SimilarRILD is the main complication from liver SBRT and results from radiation induced damage to the liver parenchyma. The reported complication rate for liver SBRT is <5%, when strict dose-volume constraints are applied and patients without cirrhosis or well-compensated cirrhosis are selected for treatment. RILD is seen most often in patients with pre-existing chronic liver disease and is typically defined by an increase in the CP or ALBI score after treatment ,108. CurApproaches for treatment planning guided by the spatial distribution of baseline liver function have been already described in a previous section. In most studies, while the radiation dose distribution was modified to spare high functioning liver, dose–response and post-treatment liver function were not considered during plan optimisation. However, Wu et al. found that plans that are designed to spare highly functioning regions of the liver do not necessarily retain the most post-treatment global liver function . The authttps://clinicaltrials.gov/ct2/show/NCT02460835, accessed on 27 September 2022, NCT02460835: A pilot study of individualised adaptive radiation therapy for hepatocellular carcinoma) aims to develop a function-based planning and adaptive SBRT approach using portal venous perfusion maps to estimate regional liver function and is due to complete in 2023. Another clinical trial commencing in 2022 at the University of Sydney ) is examining the feasibility of using liver function measurements from pharmacokinetic modeling of gadoxetate DCE-MRI for liver SBRT planning and mid-treatment adaptation.Implementation of individualised, mid-treatment adaptation of liver SBRT has been realised using ICG tests for measurement of liver function . In a PhFunctional MRI offers the ability to spatially map liver function in patients with liver cancers, enabling optimisation of SBRT dose distributions to preferentially spare high functioning liver regions, without compromising target coverage and OAR dose constraints. Incorporation of dose–response information on functional MRI parameters can also enable adaptive treatment approaches, leading to personalised dose escalation to improve the therapeutic ratio or de-escalation to reduce the risk of treatment-induced toxicity.Current research has highlighted the promise of functional MRI for personalisation of liver SBRT, however, there is much work to be done. As there is no ’ground-truth’ measurement of liver function, considerable efforts will be required for the validation of functional parameters as biomarkers of liver function. Various studies have used DCE-MRI parameters such as arterial and portal venous perfusion and hepatocyte uptake rates interchangeably to define liver function and validated them against global liver function measures such as ICG-R15 and CP scores. At present, classification of high and low functioning liver regions is based on relative differences in the spatial distribution of these parameters. This is because there is no absolute threshold to separate parameter values indicating high and low liver function. Ideally, the minimum threshold for high liver function should be identified based on the immediate and long-term dose–response of the functional MRI parameter. Previous studies have reported on longitudinal changes in DWI and DCE-MRI parameters in liver cancer tumours following treatments such as chemotherapy, chemo-embolisation and radio-embolisation ,124,125.For a functional MRI parameter to demonstrate a clear dose–response, it is also essential that it is reproducible. It is well known that DWI and DCE-MRI derived quantitative parameters suffer from a lack of reproducibility. Considerable efforts are underway to reduce variations in image acquisition and post-processing methods to improve the accuracy and precision of these parameters. However, many of the functional MRI parameters described in this review are novel and their technical performance has not yet been well characterised. Therefore, clinical trials aiming to utilise functional MRI in liver SBRT workflows must consider the uncertainty associated with the measurement of each functional parameter. Quality assurance should be performed in multi-centre studies to measure and monitor parameter uncertainties throughout the period of the clinical trial and establish performance benchmarks.Finally, researchers interested in personalised liver SBRT using functional MRI should aim to answer the question—“What is the benefit of this approach to the patient?”. Functional MRI involves advanced image acquisition techniques, combined with complex mathematical modeling of the MRI signal to derive quantitative functional parameters. SBRT requires precise delineation of tumour volumes and accurate treatment delivery. Typically, margins to account for uncertainty in both are less than 5 mm. Current standard of care treatments for liver SBRT require multiple low dose CT scans to characterise tumour motion due to respiration, and if using breath hold techniques, it is necessary to confirm reproducibility of target position. Treatment volume margins are then applied to account for residual uncertainties. Functional MRI acquisition can take longer than a typical breath-hold duration. Advanced motion correction methods will need to be developed to align functional maps acquired in free breathing to the CT scans used for dose calculation and characterise tumour motion. These techniques are not currently implemented on MRI scanners and, therefore, will require offline image processing utilising considerable computational resources. Optimisation of liver SBRT planning based on spatial liver function distribution and mid-treatment plan adaptation is also non-trivial. Future studies must be performed not only to validate the methodology and establish the feasibility of the technique, but also provide robust evidence of real improvements in patient outcomes.Liver SBRT is an attractive treatment option for patients with unresectable liver cancers, with promising outcomes demonstrated for patients with good overall liver function. In the case of patients with impaired baseline liver function, curative treatment options are limited. Functional MRI, however, offers the opportunity for safe delivery of personalised and adaptive liver SBRT using spatial liver function maps to optimise radiation dose distribution. Prospective, longitudinal studies using standardised MRI protocols and the development of dose–response models of liver function will enable patients with impaired overall function to also be offered curative treatment.


The small intestinal epithelium has classically been envisioned as a conduit for nutrient absorption, but appreciation is growing for a larger and more dynamic role for enterocytes in lipid metabolism. Considerable gaps remain in our knowledge of this physiology, but it appears that the enterocyte’s structural polarization dictates its behavior in fat partitioning, treating fat differently based on its absorption across the apical versus the basolateral membrane. In this review, we synthesize existing data and thought on this dual-track model of enterocyte fat metabolism through the lens of human integrative physiology. The apical track includes the canonical pathway of dietary lipid absorption across the apical brush-border membrane, leading to packaging and secretion of those lipids as chylomicrons. However, this track also reserves a portion of dietary lipid within cytoplasmic lipid droplets for later uses, including the “second-meal effect,” which remains poorly understood. At the same time, the enterocyte takes up circulating fats across the basolateral membrane by mechanisms that may include receptor-mediated import of triglyceride-rich lipoproteins or their remnants, local hydrolysis and internalization of free fatty acids, or enterocyte de novo lipogenesis using basolaterally absorbed substrates. The ultimate destinations of basolateral-track fat may include fatty acid oxidation, structural lipid synthesis, storage in cytoplasmic lipid droplets, or ultimate resecretion, although the regulation and purposes of this basolateral track remain mysterious. We propose that the enterocyte integrates lipid flux along both of these tracks in order to calibrate its overall program of lipid metabolism. In recent decades it has become apparent that intestinal fat handling extends beyond the purely absorptive. For one, the intestine senses and responds to bioactive signaling lipids, derived from the diet or the commensal microbes, but even as it pertains to bulk lipid homeostasis, we can now consider multiple facets of the enterocyte’s relationship with fat and how this intercalates into metabolism writ large. We therefore intend not to present an exhaustive recapitulation of the literature on this topic. Rather, we will synthesize the themes that emerge and analyze their potential connections to human health and disease. We focus on human data while using work from model systems to bridge knowledge gaps, and in both cases, we emphasize studies that are physiologically integrative. For expert review of the mechanistic details of intestinal lipid uptake, deposition, and remobilization, we refer the reader to multiple recent publications , 5, 6, 7reverse intestinal lipid trafficking, that is, transcellular efflux of cholesterol from the circulation into the intestinal lumen, began to emerge , implying a more dynamic role in the regulation of lipid metabolism.Suspicion that the healthy intestine siloes at least a portion of absorbed lipid for later release originated in the repeated observation of a “second-meal effect” (SME), in which one lipid-rich meal apparently triggers an early surge in circulating apoB-48-containing intestinal triglyceride-rich lipoproteins (TRLs) during a second meal that occurs too quickly to be accounted for by the latter , 26, 27.Alternative lipid sources may also contribute to the observed SME. For instance, enterocytes recover FAs derived from biliary phospholipid excretion for esterification to TGs  and biliexistence of the SME is clear in a phenomenological sense, its biological basis and purpose remain hazy. This has been difficult to sort out experimentally as the SME has been difficult to model in mice  bile acid stimulation of hepatic de novo lipogenesis (DNL)  , 65.While dietary lipids are the source of apical track substrates, the source of lipids for the basolateral track is less clear. Before considering the purpose of a basolateral track, it would be useful first to establish that lipids would enter the basolateral track through an actively regulated, singular process in enterocytes. Such possibilities include (re)esterification of basolaterally absorbed free fatty acids (FFAs), basolateral TRL-TG reuptake, or intestinal DNL from circulating carbohydrate precursors. Each of the possibilities is supported by existing data, albeit with varying degrees of directness.14C-palmitate infused intravenously was recovered within minutes in jejunal biopsy homogenates  fat supply and systemic demand, the latter inversely proportional to the basolaterally returning chylomicron-remnant TGs not already siphoned by other tissues. This could underpin, for example, the intestine’s observed role in feedback regulation of food intake . AlthougEnterocyte signaling of nutrient status by any means likely requires cooperation with enteroendocrine cells, whose role in satiety and facilitation of insulin-mediated nutrient disposal is well established. For example, enterocyte nutrient-status feedback may be mediated in part via cholecystokinin production by enteroendocrine cells , 106. BiThe enterocyte has access to a range of FA species from the diet that can be further strategically enriched by basolateral uptake of circulating lipoproteins and DNL , 86, 87.Balancing the FA reserve may also be important to curate appropriately structured phospholipids that sustain lipoprotein secretion , 34, 111(Re)secretion of basolaterally derived fat could also reflect a separate basolateral lipid-secretory pathway, perhaps based on VLDL . AlthougThe apparent spatial and functional separation of bCLDs from those containing newly absorbed dietary fat (aCLD) , 15, 16 If the enterocyte represents a qualified “safe haven” for short-term lipid banking, we imagine it, like adipose tissue or liver , will coSpeculation as to the relationship between insulin resistance and dysregulated intestinal lipid metabolism calls up the question of mechanism. Although the intestine is not generally considered a classic insulin target tissue, some evidentiary support exists for a direct effect of insulin on intestinal lipid handling , 140. FoSeveral potential mechanisms have been proposed to account for the postulated intestinal resistance to insulin. Unsuppressed FFAs themselves may produce insulin-desensitizing lipotoxic effects , as may These direct and indirect effects of insulin resistance on intestinal lipid metabolism are not mutually exclusive and may even reinforce one another. For example, insulin resistance appears to drive up the proportion of chylomicron-TG derived from recirculated  FFAs versus enteral  FFAs . Increasgreater apoB-48  production rate and pool size in obese humans with versus without insulin resistance  versus in jejunal explants (both lower) in patients with diabetes relative to those without it . These fsistance . This disistance , 146. A sistance , 148.Finally, we once again consider bile acids (BA) given their tight correlation with insulin resistance , 152 andWe have attempted to update and streamline a dual-track model of enterocyte lipid handling, with a particular emphasis on human physiology in health and disease. Although others have also proposed elements of a dual-track model , 15, 16,A central question raised by this hypothesis is the extent to which these two tracks interact. That is, do they carry out their activities purely in parallel or do they functionally intersect? We have speculated on interactions between the two tracks, including as an integrated energy-sensing circuit or as a hedge against systemic TG overload. However, these remain hypotheses in want of further testing. Key questions that remain unanswered include the precise mechanism of basolateral FAuptake , the reason for the apparent dissociation between basolateral track’s β-oxidative potential relative to its demand and why the enterocyte stores fat from each track in distinct CLD.The authors have declared that no conflict of interest exists.


Science,1 identifying a microbial sensing mechanism that regulates feeding behavior and host metabolism. In their study, a sex- and age-dependent gut-brain cross-talk is revealed. Their study confirmed that hypothalamic neurons can directly sense the structural components of the gut bacterial microbiota and regulate feeding and nesting behavior, affecting food intake and body temperature.Recently, Ilana Gabanyi and colleagues published an article in 2 The gut microbiota has the ability to regulate the host’s appetite, food preferences and feeding behaviors through satiety pathways. In addition, it was reposted that the gut microbiota and the brain participated in all the regulation of human activities and behaviors through the process of co-evolution.3It is known that human behavior is controlled by the brain. In recent years, a growing number of evidence suggests that gut microbiota can affect brain function and host behavior through neural pathways and immune system, forming the microbe-gut-brain axis. There are many potential communications between the gut microbiota and the brain, including immunomodulatory responses, enteroendocrine secretion, liver metabolism, neuronal innervation and signaling from microbial metabolites.5The hypothalamus integrates hormonal, sensory and gastrointestinal nutritional signals to regulate feeding behavior. The role of the gut microbiota in host metabolism depends on how it changes hypothalamic activity. Several animal studies suggested that the gut microbiota can change hypothalamic gene expression, levels of neuropeptide and neurotransmitter, and neuronal activity. Although little is known about the exact mechanisms by which the gut microbiota alters hypothalamic physiology, direct effects of microbial metabolism and related microbiota-derived molecules are likely involved. The gut microbiota regulates hypothalamic function may be related to SCFAs (short chain fat acid) and ClpB , that change host feeding behavior through hypothalamic neurons.However, it has not been confirmed whether brain neurons can directly sense bacterial components, and whether bacteria can directly regulate the physiological processes of the host by regulating neurons. Since the detailed mechanisms that drive gut microbes to affect brain function and control host feeding behavior are unclear, existing studies based on the effects of microbes on feeding behavior are difficult to be translated into clinical use.ARC neuron activity through Nod2 receptors and affects appetite by modulating hypothalamic circuits. Neuronal excitability was recorded with patch clamp, and it was found that MDP decreases the VgatARC neuronal activity in a cell-autonomous manner. Local ablation of Nod2 expression by injecting Cre-expressing virus into the hypothalamus of Nod2flox mice affected body weight and body temperature, indicating that Nod2 expression in hypothalamic neurons is necessary for feeding behavior and thermoregulation in aged female mice. The use of broad-spectrum antibiotics (AXB) resulted in increased body weight in Nod2 flox mice, confirming that microbe derived Nod2 ligands are involved in this regulation. This study demonstrates that the microbiota plays an important role in Nod2 ligand production and appetite regulation through Nod2-expressing hypothalamic neurons. The brain can assess food intake by sensing changes in gut bacteria. Excessive intake of certain foods may stimulate the disproportionate growth of certain bacteria or pathogens, compromising gut microbial balance  into the mice confirmed that MDP can reach the brain from the gut, possibly faster in females than in males. When neurons come into contact with bacterial wall peptides in the gut, the electrical activity of neurons is suppressed. MDP treatment caused the mice to eat less. Loss of Nod2 (gene knockout) in inhibitory γ-aminobutyric acid transporter-positive (GABAergic) neurons in aged female mice, these neurons are no longer inhibited by cell wall peptides, and the brain loses its ability to control food intake and body temperature, which increases appetite, render female mice to eat more and gain weight. However, this phenomenon was not found in male mice at the same age. In addition, it can reduce the nesting behavior of female mice and the range of daily body temperature. Moreover, the mice are more likely to develop diabetes and have shorter life span. This behavior is associated with heat retention, response to circadian rhythms, fasting, and adrenaline-stimulated thermoregulation. Therefore, Nod2 receptors expressed by inhibitory neurons play an important role in regulating human metabolism, especially in women. MDP induced different patterns of neuronal activation in young and old female and male mice, with more pronounced effects in older mice. Older female mice showed higher responsiveness to MDP in feeding behavior and thermoregulation, and MDP affected neuronal activation in a sex- and age-dependent manner. Eating and MDP gavage can inhibit GABA neuron activity. MDP increases Vgatnce Fig. .Fig. 1GuThis study revealed a new gut brain communication pathway, in which gut microbes regulate the feeding behavior of the host to stabilize its gut niche. Understanding the mechanism of gut microbiota affecting host appetite and metabolism will help better understand appetite-related disorders. Regulating the structure and composition of gut microbiota and modulating the feeding activities of appetite center, could be a new research direction for weight control and other metabolic disorders, especially for adult women. This study might lead to new treatments for brain diseases and metabolic diseases such as diabetes and obesity.


Mild traumatic brain injury (mTBI), also known as concussion, is a commoninjury which affects patients of all demographics. There is a global effort to accuratelydiagnose and identify patients at highest risk of prolonged symptom burdento facilitate appropriate rehabilitation efforts. Underreporting is common withlarge numbers not engaging with services, in addition to differences in treatmentoutcomes according to ethnicity, age, and gender. As patients recover, symptomologyevolves which challenges rehabilitative efforts with no clear definitionof ‘recovered’. This review describes key areas in mTBI such as diagnosticchallenges, epidemiology, prognosis, and pathophysiology which serves as anintroduction to “Eye Movements in Mild Traumatic Brain Injury: OcularBiomarkers.” Mild traumatic brain injury (mTBI), commonly referred to as concussion, is acomplex neurobehavioural phenomenon resulting from mechanical trauma. DespiteA single unifying diagnostic nosology for classifying mTBI representsone of the greatest challenges within the field. The term concussion andmTBI are often used interchangeably, however, some authorities suggestthat concussion should be considered a subset (milder form) of mTBI,although there is no consensus of such a classification . CurrentAlthough there is extensive discussion regarding the operationaldefinition of mTBI, the definition by the American Congress ofRehabilitation Medicine, revised by the World Health Organization (WHO),seems to be increasingly accepted by clinicians and researchers in thefield . The defth International Conference onConcussion qualifies SRC as a direct blow to the head, face, neck, orelsewhere on the body with an ‘impulsive’ force transmitted to the head(Sport-related concussion (SRC) is considered by some investigators tohave its own nosological framework. The Consensus Statement onConcussion in Sport at the 4the head. TypicalHence, one of the challenges of mTBI research is that the presentdefinition encompasses a broad spectrum of injury. From the abovedefinitions it is clear that the limits of the definitions may overlapwith ‘moderate’ TBI at the upper end and trivial head trauma at thelower end. This has a significant impact in interpreting the research inparticular with respect to prognosis and management.Over 50 million people suffer traumatic brain injury each year  ; 62 and A meta-analysis of 15 prevalence studies  found that12% of the sample experienced TBI with loss of consciousness, with menbeing at more than double the risk of women . AnotheA significant burden of mTBI is related to sports-related concussion. In the A community-based study exploring multiple sources of referralsrevealed a significantly higher incidence of 170/100,000 andsport-related causes comprised 21% of these . In acomTBI predominantly impacts young males which is due to higher rates of contact-sports and riskier maneuverssuch as tackling . While mSome studies have investigated the relationship between racialdisparities and mTBI as well as sport-related concussion and haveidentified that there appears to be differences in concussion incidence,awareness, outcome, and morbidity. African American children under theage of 4 years old who sustained TBI have been shown to have mortalityrates twice those of Caucasians . Other rInequities in healthcare based on ethnicity have been identified inthe entire spectrum of mTBI. African American patients were less likelythan Caucasian patients to have emergency department visits for headinjuries and were less likely to be diagnosed with a concussion duringan emergency department visit . AfricanFuture epidemiological studies are required to investigate incidencelongitudinally with consideration of factors such as age, gender,ethnicity, and particular risk-factors in sport-related concussion suchas level of competition (elite vs non-elite) and player position whichis known to affect risk .mTBI is a heterogenous injury resulting in diverse clinicalpresentations which cluster into broad domains . ThesedConcussion-associated conditions are common symptoms which may bepresent irrespective of subtype. These include sleep disturbance andcervical strain. These patients may experience insomnia orhypersomnolence . CervicaHowever, these domains (and assocated conditions) have recently beenchallenged following a systematic review and meta-cluster analysis byLangdon and colleagues of 5592 athletes across 22 carefully selectedstudies . Their aRecovery from mTBI is variable depending on the population studiedand the definition of mTBI. The two week recovery of patients withsport-related concussion has been reported to range from 50% to 90%. At two In a cohort of 91 students, aged 13-19, a third of those who reportedproblems returning back to class following an mTBI had a history ofprevious head trauma. Similarly, a third of students struggled withreturn to school (57% of students took over 10 days recover and 29% tookover 21 days to recover). Of these, vision problems were correlated to a2.5x risk of difficulty at school  to ocularmotor dysfunction in patients with ongoing symptom burden. Maruta andcolleagues suggested that disrupted white matter integrity in the rightanterior corona radiata, uncinate fasciculus, and genu of the corpuscallosum correlated to decreased gaze accuracy in smooth pursuit eyetracking paradigms . TaghdirArguments for premorbid mental illness or maladaptive psychologicalphenomena following mTBI cite significant overlap and similarities tosomatization observed in psychiatric disorders such as depression,anxiety, and post-traumatic stress disorder (PTSD). The DSM-Vclassification for major depressive disorder includes difficultyconcentrating, headaches, indecisiveness, loss of energy, anxiety, andinsomina, all of which are mTBI symptoms . A studyOverall, PCS remains a contentious diagnosis among healthprofessionals despite an increase in literature rationalizing persistentneurological dysfunction. Individuals must continue to be treated on acase-by-case basis, accounting for multiple health-related factors.Following an mTBI, a series of complex biochemical, metabolic, andmicrostructural changes occur. Mechanical trauma  causesshear and stress forces within the brain parenchyma which disruptscellular membranes. This leads to extracellular shifts of potassium,intracellular shifts of sodium and calcium, glutamate release, anddiffuse neuronal depolarization ; 47; 75.After the insult, disruption to the blood-brain barrier results fromcerebral microvasculature trauma and loss of junctional adhesionproteins , causingMicrostructural changes to cytoskeletal architecture of dendrites,astrocytes, and axons (in particular microtubules and neurofilaments)leads to impaired neurotransmission. In particular, unmyelinated axonsare at greater risk of damage which explains why younger brains may beat greater risk of long term sequelae . InaddiFor an in-depth review of this topic, refer to Romeu-Mejia et al. andGiza et al. ; 80.mTBI is a common injury which represents an unmet health need on aglobal scale. There are challenges in not only identifying andsubsequently diagnosing these patients, but also grouping into sub-typesfor appropriate referral pathways. Further clarification of definitionsand criteria in mTBI is required for unified efforts  in this area. In future, ocular motorcharacteristics may serve as a diagnostic aid given emerging evidencewhich positively correlates to white matter tract disruption in thebrain. This is particularly relevant to a large group of mTBI patientssuffering from visual symptoms. Measuring the scale of theepidemiological burden of mTBI in communities is difficult with largevariation from hospital-based and primary care surveys. This is compounded by underreporting in sports leagues andadolescent populations. Racial disparities in health literacy,health-seeking behaviours, and treatment outcomes reflect wider societalissues. Further studies exploring these issues in greater detail mayelucidate contributing factors to facilitate policy and culture change.In addition, the lack of literature on mTBI in the elderly is alarming,considering the prevalence of falls in this population. Where symptomsmay be difficult to evaluate in the cognitively impaired, biomarkerssuch as quantitative eye tracking may prove useful. When studying mTBItrajectories across populations, prognosis is varied as diagnosing apatient as ‘recovered’ results in discharge from services and may notreflect complete resolution of symptomology or physiological recovery.This is evident from primary care surveys at one year and outcomes inschools as mentioned above. The spectrum of ongoing symptom burden andlabel of postconcussion syndrome may not be helpful with notableoverlaps in symptoms of mental illness. Unravelling the pathophysiologyand identifying a biomarker in neural recovery is key to improvedservice provision. Overall, mTBI is a common injury with significantgaps in both our primary understanding of this condition and treatmentmodalities.http://biblio.unibe.ch/portale/elibrary/BOP/jemr/ethics.htmland that there is no conflict of interest regarding the publication ofthis paper.The author(s) declare(s) that the contents of the article are inagreement with the ethics described inThis research was supported by the Health Research Council of NewZealand through the Clinical Research Training Fellowship.


The COVID-19 pandemic led to intensive research into finding new vaccines for human protection. On 21 December 2020, the European Commission granted conditional marketing authorisation for the messenger RNA vaccine ‘Comirnaty’, produced by Pfizer  and BioNTech . This happened only twelve months after the first identification of the virus, whereas the development and approval of vaccines usually take ten years. Through document analysis and interviewing key expert stakeholders, we examined whether the role of animal studies and alternatives in this fast approval process had changed and, if so, whether this could lead to using fewer animal studies and more alternatives in the future. It turned out that in this case, for vaccine development and production, the number of animal studies performed and required had indeed declined, more alternatives had been used and accepted, human studies started earlier and ran in parallel with  animal studies, and regulators accepted historical data from earlier vaccine research. The Pfizer/BioNTech vaccine case illustrates the tremendous progress in quickly producing and authorising reliable, safe and effective vaccines, using fewer animal studies and more alternatives. It is time to study the broader implementation of these new procedures on a larger scale to benefit animals and humans.n = 11 individuals representing five stakeholder groups in eight interviews and two written statements) and analysed relevant publications, policy documents and other grey literature (n = 171 documents). Interviewees observed significant changes in regulatory procedures and requirements for the ‘Comirnaty’ vaccine compared to vaccine approval in non-pandemic circumstances. Specifically, the European Medicines Agency (EMA) actively promoted changes by using an accelerated assessment and rolling review procedure for fast conditional marketing authorisation, requiring a reduced number of animal studies and accepting more alternatives, allowing pre-clinical in vivo animal experiments to run in parallel with clinical trials and allowing re-use of historical data from earlier vaccine research. Pharmaceutical companies, in turn, actively anticipated these changes and contributed data from non-animal alternative sources for the development phase. After approval, they could also use in vitro methods only for all batch releases due to the thorough characterisation of the mRNA vaccine. Pharmaceutical companies were optimistic about future change because of societal concerns surrounding the use of animals, adding that, in their view, non-animal alternatives generally obtain faster, better, and cheaper results. Regulators we interviewed were more hesitant to permanently implement these changes as they feared backlash regarding safety issues and uncertainty surrounding adverse effects. Our analysis shows how the EMA shortened its approval timeline in times of crisis by reducing the number of requested animal studies and promoting alternative methods. It also highlights the readiness of pharmaceutical companies to contribute to these changes. More research is warranted to investigate these promising possibilities toward further replacement in science and regulations, contributing to faster vaccine development.On 21 December 2020, the European Commission granted conditional marketing authorisation for the BNT162b2 COVID-19 vaccine ‘Comirnaty’, produced by Pfizer/BioNTech. This happened only twelve months after scientists first identified SARS-CoV-2. This stands in stark contrast with the usual ten years needed for vaccine development and approval. Many have suggested that the changes in required animal tests have sped up the development of Comirnaty and other vaccine candidates. However, few have provided an overview of the changes made and interviewed stakeholders on the potential of the pandemic’s pressure to achieve a lasting impact. Our research question is: how have stakeholders, including regulatory agencies and pharmaceutical companies, dealt with requirements concerning in vivo animal models in the expedited approval of vaccine candidates such as ‘Comirnaty’? We interviewed key stakeholders at the Dutch national and European levels ( By September 2021, the European Medicines Agency (EMA) had already approved four vaccines for SARS-CoV-2 ,4. This The public debate in the early months of the pandemic featured many claims regarding the use of in vivo animal tests, or the suspected lack thereof, in developing several SARS-CoV-2 vaccines. Many expressed worries that the pharmaceutical companies were cutting corners in the process ,8. In ren = 171) and interviewing a selection of key stakeholders directly involved in this process at the Dutch national level  and at the European level .We took the EMA’s December 2020 approval of the BNT162b2 SARS-CoV-2 vaccine ‘Comirnaty’ by Pfizer/BioNTech as our case study to investigate these changes and their potential broader impact. Our specific questions were: how did the developmental process and the regulatory conditional marketing authorisation approval of the SARS-CoV-2 vaccine BNT162b2 differ in required and submitted animal studies compared to the ‘normal’  situation? Moreover, how do stakeholders see the potential for lasting impact based on these changes, and what reasons do they give? We have answered these questions by analysing relevant documents  of Pfizer/BioNTech’s BNT162b2 SARS-CoV-2 vaccine ‘Comirnaty’, through document analysis and semi-structured interviews.As the first step, we performed a document analysis . We gathThe selected documents included guidelines and standards on the development of (COVID-19) vaccines by the World Health Organization (WHO) and EMA, policy documents at the European Union level, minutes from international stakeholder meetings, peer-reviewed articles on vaccine development, COVID-19 vaccines, animal studies (in vaccine development), alternative studies (in vaccine development), the role of stakeholders in determining the role of animal studies and alternatives in vaccine development, and international newspaper articles on the development of COVID-19 vaccines and the role of animal studies and alternatives in their development. Based on the documents, we produced an overview of the usual requirements in terms of animal studies in vaccine market authorisation. The document dataset also informed the questions for semi-structured interviews on the current role of animal studies and alternative methods in developing vaccines and their regulatory approval and focused on the Comirnaty vaccine in particular (more information below). We approached the following key stakeholder groups for a semi-structured interview: representatives from regulatory agencies at the national and European levels, vaccine developers, (bio)medical scientists/virologists working on animal models or alternative methods, animal rights organisations, and politicians at the national and European level. We identified the key stakeholder groups using a power-interest grid stakeholder analysis. Not all contacted representatives were available for an interview, citing time constraints, but two agreed to reply with a written statement. We conducted two ‘test’ interviews to develop the topic list, refine the questions, and determine which specific stakeholders to approach. We interviewed eleven representatives of five stakeholder groups . Under DOur topic list  includedWe transcribed the interviews verbatim and analysed the content of all transcribed interviews and documents through inductive thematic coding in ATLAS.ti . A list The development of a novel vaccine candidate takes, on average, ten to fifteen years from pathogen discovery to the first authorised vaccination . RegulatThe vaccine development process starts with an exploratory phase in which basic laboratory research studies obtain information about the pathogen . This knTo market a vaccine, one needs marketing authorisation. The requirements for complete marketing authorisation for vaccines in the European Union are outlined in Annex 1 of Directive 2001/83/EC [EUR-Lex]; . VaccineThe development process of the Comirnaty vaccine included all stages described above. However, our dataset highlights notable differences regarding the timeline, animal study requirements, and acceptance of alternative methods  stated there is no need for showing efficacy in animals before progressing to clinical trials. Moreover, the EMA allowed human-specific data to substitute for animal data when of sufficient quality. Our dataset highlighted several technical advances supporting this, such as the complete sequencing of the SARS-CoV-2 genome and, especially, in silico method development. Scientists used these latter methods to establish COVID-19 characteristics, e.g., how the virus infects the host , to discThird, we observed a difference in the number of animal studies performed during the pre-clinical phase of vaccine development . For theAccording to our interviewees, the process described above is a drastic reduction in the number of animal studies compared to ‘normal’ . To explain this reduction in animal studies in the pre-clinical step, interviewees highlighted the role of the thorough characterisation of mRNA vaccines like Comirnaty. In their view, this led to a better understanding of the product and optimised decision-making regarding safety and efficacy measurements . All available non-clinical data  were assessed in the first round of the rolling review procedure. The remainder of the non-clinical studies were performed later, during Phase 3 or even after vaccine approval  ,31.The thorough characterisation of mRNA vaccines like Comirnaty also positively affected the fourth element, batch release testing. In vaccine development, such batch release testing still often relies partly on animal testing due to the relatively poor characterisation of vaccines  and relaA fifth and final difference concerns the re-use of previous data throughout the approval process, which again benefited from the highly characterised nature of mRNA vaccines. The EMA did not consider it reliable or responsible to avoid animal studies entirely because of the need to demonstrate the product’s safety, immunogenicity, and efficacy. However, regulatory agencies accepted the submission of data previously acquired during research on other mRNA vaccines  . SpecifiAnimal studies take much time, which is one reason why developing and approving vaccines are usually lengthy processes. The EMA’s conditional authorisation of the Comirnaty vaccine indicates that–next to regulatory flexibility, sufficient funding and significant international cooperation—using non-animal alternatives throughout the research, development, and registration processes aided in accelerating vaccine development and approval. These alternatives included, a.o., in silico data for the exploratory phase  and using previously acquired data in the pre-clinical phase. Moreover, the EMA allowed moving to in-human trials before completing the ‘essential’ animal studies still required. The use of mRNA technology, which one of our interviewees considered the most significant breakthrough of this pandemic, was vital to enabling many of these changes, though, notably, other vaccines using other technologies were developed and approved in similar timeframes. Interviewees also noted that the pandemic pressure led to more funding, knowledge sharing, and cooperation. As the pandemic pressure led to such widespread influence globally, with the involvement and cooperation of a variety of different stakeholders at multiple levels as well as efficient and fast data sharing and medical progress, it would be valuable to do further transition research into these processes in order to learn how we can apply these lessons more broadly ,37. COVINevertheless, this raises the critical question of how these ways of reasoning might hold in future non-pandemic situations, using both mRNA and other technologies. Pharmaceutical companies are ready to take the next step: they seem to consider non-animal alternatives better, cheaper, faster, and more societally salient. When asked about hurdles to implementation, interviewees from all stakeholder groups highlighted risk aversion among regulatory bodies as the primary reason for still requiring animal studies (the “belt and braces approach”), considering these necessary to prove that a product is safe and effective. The critical question is whether alternatives cause higher or lower translational risk than animal studies, as some approved vaccines based on animal studies have shown adverse effects on humans . Many of our interviewees consider continued dialogue with authorities essential for promoting future acceptance of non-animal alternatives to tackle these risk perception issues. We suggest further quantitative and qualitative research to continue and accelerate these promising processes in obtaining new, better vaccines faster using modern technologies to benefit animals and humans. It will also need further research on how these expedited processes for the mRNA Comirnaty vaccine could work for other conventional vaccine technologies or whether the mRNA technology could help to replace these. Open science, giving complete transparency on methodologies and data, is essential for better decision-making.The pressure of the COVID-19 pandemic has shown significant acceleration of the approval process of allowing new vaccines to come to market. The Pfizer/BioNTech vaccine Comirnaty was developed and approved in twelve months, whereas under ‘normal’ conditions, vaccine development and approval take about ten years. In our case study on this fast approval process, we performed document analysis and expert interviews, revealing that regulatory agencies requested fewer animal studies and accepted more alternatives. This also included moving to human trials faster  and accepting historical data from earlier vaccine research. As far as we know, until now, the marketed COVID-19 vaccines have shown promising efficacy and safety. These are all auspicious developments necessitating more research on how to continue using fewer animal studies and more alternatives in the developmental chain towards new, faster and better future vaccines for humans.


Gene therapy makes it possible to engineer chimeric antigen receptors (CARs) to create T cells that target specific diseases. However, current approaches require elaborate and expensive protocols to manufacture engineered T cells ex vivo, putting this therapy beyond the reach of many patients who might benefit. A solution could be to program T cells in vivo. Here, we evaluate the clinical need for in situ CAR T cell programming, compare competing technologies, review current progress, and provide a perspective on the long-term impact of this emerging and rapidly flourishing biotechnology field. However, an annual course of treatment comes at a steep price: $475,000 for Kymriah and $373,000 for Yescarta. Consequently, many patients cannot afford these treatments. The high costs and complexity involved in manufacturing a bespoke T cell product for each patient limit the wider use of adoptive T cell therapies and their competitiveness with frontline therapeutic options, such as small molecule drugs or monoclonal antibodies, which can be produced in bulk quantities as conventional pharmaceuticals can (a) leukapheresis to extract T cells from a patient who is connected by two intravenous lines to an apheresis machine for several hours (b) activation and transduction of T cells; (c) expansion of transduced T cells in cytokine-supplemented tissue culture medium; (d) washing and concentrating the T cells prior to administration ; and (e) quality control release assays, which are necessary for each batch of CAR T cell product. The entire production process has to be conducted according to strict, environmentally controlled good manufacturing practices (GMP) at a central, accredited location, which may be far from the medical center where the patient is located. Because each bespoke CAR T cell product is made from material (T cells) obtained from the patient to be treated, there are no economies of scale . Ho. Ho23). 11 CD8-targeted lentiviral particles encoding a CD19-specific CAR (targeting B cells) substantially reduced the tumor burden in B cell lymphoma-bearing immunodeficient NOD scid gamma (NSG) mice that had been engrafted with human T cells prior to virus infusion . Instead, as one would hope, the CAR-transduced cells subsequently underwent selective clonal proliferation and differentiation into effector T cells following antigen encounters. To confirm the utility of this platform in a more physiological test system, Buchholz and colleagues  of the infused viral vectors were not assessed, the study established an important proof of concept: that injection of T cell–targeting lentivirus can deliver CAR transgenes into enough host T cells to induce a therapeutic response.To restrict the tropism of CAR-encoding lentiviral vector to T cells, Buchholz and colleagues , 29 pseuinfusion . Tumor rinfusion , these hlleagues  infused The success of established CAR T cell therapy has been heavily dependent on ex vivo lentivirus-mediated gene transfer. Thus, the development of lentiviral vectors as an injectable reagent for in situ reprogramming of T cells was an obvious next step for the reasons outlined below.Lentiviruses are becoming the gene-transfer vehicle of choice for use in the treatment of a variety of genetic and acquired human diseases . LentiviLentiviral vectors are complex bioproducts with an ordered architecture that would be challenging to engineer using synthetic material. For example, the surface of the virus particle displays all the targeting ligands in the desired orientation and density to achieve efficient cell targeting. Furthermore, after binding to the receptor on the target cell, lentiviruses are inherently efficient at becoming internalized into the cytoplasm through a variety of endocytic pathways . These sDespite the advantages outlined above, systemic delivery of lentiviruses presents major drawbacks.Producing a GMP-grade lentiviral vector preparation for human use requires rigorous testing of the final product to establish its purity, potency, and safety , 38. In 3 per cell , or imaging agents at tumor sites, in an effort to minimize off-target toxicities –52. Withphocytes .Surface-anchored targeting ligands that selectively bind the nanocarriers to T cells and initiate rapid receptor-induced endocytosis to internalize them: In our studies, we used anti-CD3ε F(ab′)2 fragments as well as full-length anti-CD8 antibodies with deactivated constant regions (Fc).A negatively charged coating that shields the nanocarriers to minimize off-target binding by reducing the net surface charge of the nanocarriers: We used polyglutamic acid to accomplish this in our experiments.A carrier matrix that condenses and protects the nucleic acids from enzymatic degradation while they are in the endosome but releases them once the particles are transported into the cytoplasm, thereby enabling transient transcription of the encoded protein: Our group tested a panel of cationic materials, including hyperbranched star polymers , polyethNucleic acids that are encapsulated within the carrier and produce expression of a disease-specific CAR: To achieve persistent CAR expression in actively dividing T cells, we loaded each nanoparticle with two DNA plasmids: one encoding a mouse version of a CAR that confers specificity for CD19 [the target expressed by leukemia cells and normal B cells ] and a sOur group adapted these platforms to design injectable DNA nanocarriers that choreograph robust CAR production in host T cells . To crea+ leukemia and found the therapeutic efficacy to be similar  in mice with established CD19 similar . A signiIn a follow-up study , we explTargeted nanoparticle-based gene modification systems exhibit some key features that could help steer this field toward clinical applications.Synthetic nanoparticles are designed in a modular fashion, which facilitates switching out of individual modules and incorporating custom modalities without interference from the biological constraints that viral vectors face. Depending on the desired characteristics, nanoparticles can be fabricated with organic materials such as protein, polymer or lipid, or inorganic compounds . Also, the therapeutic cargo encoding the CAR can be customized; the cargo can range from IVT mRNA for transient in situ gene expression to constructs that deliver a transgene into the nucleus for persistent gene expression, such as linear, closed-ended DNA  or minicFor injectable CAR-programming reagents to be competitive with lentiviral products, it will be vital to create sufficient quantities for clinical use at substantially lower costs than those entailed in producing conventional ex vivo engineered CAR T cells. Several continuous-flow microfluidics platforms designed for scalable manufacturing of nanoparticles under GMP conditions are now available , 78. TheWhile injectable nanoreagents could enable physicians to rapidly program circulating T cells with disease-specific CARs, translation of this nanomedicine into the clinical setting may still be challenging for the reasons outlined below.The gene-transfer efficiency of nonviral systems is usually less than that of viral systems , which mIn contrast to therapeutics that are already established in the clinic, such as small molecules or antibodies, CAR-programming nanoparticles are multicomponent three-dimensional constructs that require a reproducible manufacturing process to reliably achieve the intended physicochemical characteristics, biological behaviors, and pharmacological profiles. The safety and efficacy of such nanomedicines can be influenced by minor variations in many parameters and need to be carefully monitored, particularly in the context of targeting to unintended sites and potential toxicities. Furthermore, nanomedicines require additional developmental and regulatory considerations compared with conventional medicines. Only a few facilities with the requisite degree of expertise are currently operational in the United States.Researchers at Moderna Therapeutics are pursuing a nontargeted approach to program CAR T cells by repeated infusion of untargeted mRNA nanoparticles . The comAnother interesting approach to help constrain nanoparticle transfection to T cells in vivo without using targeting ligands was described by Dahlman’s group . This teBiotechnology companies developing conventional nanomedicines generally try to avoid surface functionalization of the nanocarriers, as it complicates scale-up manufacturing and quality control and adds cost. Manufacturing T cell–specific targeting ligands in a GMP facility is expensive and time consuming. To avoid immunogenicity, the chosen constructs must be fully humanized. Furthermore, to maximize targeting efficiency, a GMP-compliant coupling strategy must be developed to tether the targeting ligands to the surface of the nanoparticles while ensuring they are displayed in the appropriate orientation to allow the ligand to bind its receptor on T cells. In contrast, simple methods are already established for mass production of untargeted liposomes , 88.Intravenous infusion of untargeted nanoparticle formulations could have significant disadvantages over lymphocyte-targeted delivery systems.Exploiting receptor-mediated endocytosis is advantageous for achieving targeted gene delivery as it improves both the efficacy and the side-effect profile of CAR-programming nanomedicines. Through the targeting of nanoparticles to specific surface receptor proteins on T cells that are actively internalized, the nanoparticles can be selectively shuttled into endosomes of T cells , 90. NonThe consequences of expression of a CAR protein in non-T cells are unknown at this point. The untargeted nanoreagents described by Moderna Therapeutics and Dahlman’s group  deliver Following the success of the patient-derived ex vivo engineered CAR T cell products Kymriah (tisagenlecleucel) and Yescarta (axicabtagene ciloleucel), CAR T cell developers are looking for technologies to provide better scale and lower costs similar to those of biologics. The ability to benefit a large patient population at an affordable price will also be key to move CAR T cell therapy beyond cancer applications. Preclinical studies showing the utility of CAR T cells outside of cancer are ramping up, and developers have taken notice. Autoimmunity is seeing the most activity, followed by infectious disease and transplant settings. Academics are pushing the technology even further, into less obvious indications such as heart failure. The adoption of CAR T cells as a mainstream therapy is still some years away, as the biotechnology industry currently lacks off-the-shelf injectable reagents that do not require complex manufacturing. Instead, the technology is currently limited to allogeneic platforms such as those described below.In the hope that allogeneic cells can broaden the reach of CAR T cells to more patients and more types of cancer, most companies involved in clinical-stage autologous T cell therapy are also now developing T cell products from allogeneic healthy donors, rather than from the patient ,92. The 6 transduced viable T cells per kilogram of body weight have been shown to be effective  but also reduces the viability of the lymphocytes and their yield . Allogenffective , 96. In ffective , 98. Sinffective .Nature Biotechnology, Sadelain and colleagues at Memorial Sloan Kettering Cancer Center (a) Complex genetic cell engineering  needs to be done only once and gives a renewable engineered iPSC line that can be validated and banked, and (b) clones can be screened from a pool of cells and only the top-performing clones that meet the standards for overall quality and safety  can be selected to generate a master engineered iPSC bank for GMP production of the CAR product. Whether this platform can address the commercial scalability challenges of conventional CAR T cell therapy remains to be seen. Fate Therapeutics, one of the developers of iPSC-derived immunotherapies, chose to test their allogeneic NK cell product (FT-516) in patients first , possibly because generating NK cells from iPSCs has proven to be easier than producing antigen-specific cytotoxic T cells  understanding the mechanism by which the physicochemical properties of the material impact its biological effects ; (b) understanding the in vivo release mechanism based on the material’s physicochemical properties; (c) predictability of in vivo release based on established in vitro release methods; (d) physical and chemical stability; (e) maturity of the nanotechnology ; (f) potential impact of manufacturing changes, including in-process controls and the robustness of the control strategy, on critical quality attributes of the drug product; and (g) dissolution, bioavailability, distribution, biodegradation, and accumulation data and their predictability based on physicochemical parameters and animal studies.Efficient and selective in situ reprogramming of patient T cells requires highly engineered products containing multiple functional components, such as a lymphocyte-targeting moiety, nucleic acid encoding the disease-specific CAR, and some sort of carrier/condensation matrix, which can be a biopolymer in the case of synthetic nanoparticles or the capsid and envelope for lentiviral vectors. Characterization of the final product containing all components can be daunting, even for a seasoned scientist with expertise in analytical chemistry. At a minimum, characterization should include the following: size (and size distribution), zeta potential, particle concentration, targeting quantification, nucleic acid loading efficiency, coating integrity and quantification, purity , stability, batch-to-batch consistency, and sterility and endotoxin levels . In addiAs alluded to above, the highly competitive (and litigious) intellectual property landscape could further complicate clinical translation of multicomponent nanomedicines. There have been a large number of patent filings with claims to various aspects of nanoparticle delivery of nucleic acid and polymer compositions. Moreover, the CAR T cell patent landscape is becoming increasingly crowded, and this will inevitably lead to narrower claims being granted (if any) in many cases. Ultimately, translating the concept of programming disease-fighting lymphocytes in situ into novel therapeutics for cancer patients will likely require joint efforts involving industry leaders in gene therapy, bioengineering/nanotechnology, and T cell therapy. Ongoing legal wrangles over intellectual property rights covering CAR T cell technology  and inte


This Research Topic endorses the embodied view of language processing, which claims that, rather than being abstract, amodal, and arbitrary, the body plays a central role in shaping it  to reveal that, in the verb processing stage, verb-object metaphors trigger simulation, whereas subject-verb metaphors trigger abstraction. This finding suggests that verbal metaphors are processed using both simulation and abstraction, and that metaphorical meaning is integrated in real-time during metaphor comprehension. Relatedly, Chen et al. investigated whether processing of metonymy, like metaphor, entails embodied simulation. Using eye-tracking, they found that readers take longer to arrive at a literal interpretation than a metonymic one when preceding information is weakly related to target words but not when it is strongly related to target words. Moreover, they found that preceding and spillover contextual information contributes to metonymy processing when spillover affects metonymy more than literal meaning. These findings provide insight into how sentential components contribute to metonymic processing of target words in Chinese, contributing to a model of metonymy processing.Several of the papers in this Research Topic focus on metaphor and closely related topics, an important theme in research on language embodiment Gibbs, . Li et aSun et al. used ERPs and event-related spectral perturbations (ERSPs) to investigate the processing of literary metaphors in modern Chinese poetry in comparison to non-literary conventional metaphors and literal expressions outside literary texts. They found more positive P200 and more negative N400 fronto-central components for literary metaphors than for other stimuli as well as increases in the delta and theta frequency bands during different time windows for literary metaphors. Together, these findings indicate that literary metaphor processing has distinct EEG spectral patterns and that it is characterized by early allocation of attention and conscious experience. Similarly, Tang et al. examined comprehension of scientific metaphors in first  and second  language using ERPs. Relative to L1 Chinese scientific metaphors, L2 English scientific metaphors elicited more negative N400 and less positive late positive components (LPCs) in the parietal region as well as larger late negativities encompassing smaller areas of brain. These findings indicate that non-native and non-dominant language processing entails increased effort, decreased automaticity, and decreased sensitivity to the conventionality of metaphoric meanings. Finally, Huang et al. used hemifield presentation to investigate functional laterality during processing of scientific and conventional metaphors. The results support the fine-coarse coding hypothesis, which posits that the left hemisphere supports integration of the loosely associated domains of scientific metaphor. Moreover, compared to literal word pairs, conventional metaphors elicited higher LPCs during right visual field presentation, whereas scientific metaphors elicited lower LPCs during left visual field presentation. These findings suggest that processing mechanisms differ between novel and conventional metaphors and that right hemisphere plays a special role in novel metaphoric processing during the mapping stage.Metaphor is pervasive in literary and scientific texts, so it is important to consider how it relates to embodied views of language processing within these contexts. Zang et al. examined how L1–L2 language switching influences negation processing using a story reading and question verification paradigm in which the language was either kept the same or changed across tasks. They found that switching from L1 to L2 facilitated negative compared to affirmative responses, suggesting that domain-general mechanisms of inhibitory control are recruited simultaneously for L1–L2 language switching and negation. Seeking to reconcile conflicting results concerning the effect of multilingualism on executive function, Kim et al. examined the impact of task modality and found that it yields different patterns of performance between monolingual and multilingual participants, suggesting that distance of transfer from everyday language use may provide a viable explanation for these discrepancies. Yao R. et al. used a Stroop-like task to examine gesture-speech integration in deaf and hard of hearing adolescents. The results revealed that deaf and hard of hearing adolescents showed stronger effects of gesture-speech integration as well as semantic congruency than their hearing peers, revealing how sensory experience can influence language processing.Another important theme in this Research Topic is the relationship between domain- general and domain-specific processing. Zhou et al. examined the effects of musical primes with and without tempo changes on different classes of words using ERPs. When primed with tempo changes, state verbs and inanimate nouns elicited larger N400 amplitudes than action verbs and animate nouns, suggesting that such priming facilitates processing of action verbs and animate nouns due to the shared concept of motion across music and language. In a conceptually similar vein, Yao Z. et al. used picture-word semantic priming to examine the impact of social experience on processing of social abstract  and emotional abstract  words. Pictures of positive social scenes facilitated processing of positive social abstract words, and pictures of corresponding facial expressions and gestures facilitated processing of positive emotional abstract words, whereas no facilitatory priming was observed for negative pictures and social or emotional abstract words.One method that has been instrumental in illuminating the relationship between domain- general and domain-specific processing in this Research Topic is priming, in which a  prime influences processing of a (domain-specific) target. These findings provide evidence that social experience selectively affects processing of positive social and emotional language, providing a mechanism for the grounding of these concepts.Li and Guan examined the effects of hand writing and drawing on development of orthographic representations. Behavioral and ERP data indicated that drawing facilitates visual word recognition in Chinese compared to viewing. Moreover, N170 amplitude correlated positively with N400 amplitude in the drawing condition, suggesting that hand movement may facilitate the neural correlates between early word recognition and later comprehension. Finally, Guan and Meng examined the effect of embodied morphological training by hand writing roots, dragging roots, and gesturing roots on children's L2 word learning. The results revealed that hand writing roots facilitated sound-meaning and word-sound form integration, whereas dragging and gesturing roots facilitated word form-meaning association, providing evidence that embodied morphological training contributes to children's L2 vocabulary acquisition.In addition to illuminating the mechanisms of embodied language processing, some papers in this Research Topic provide insight into how they can be leveraged to promote language comprehension in instructional settings. In summary, the articles in this Research Topic reveal the inextricable relationship between language processing and embodied experience from several perspectives using cutting- edge behavioral and neuroscience methods. Importantly, they demonstrate how the embodied grounding of language can be leveraged to enhance learning in typically and atypically developing individuals, complementing other recent efforts in this regard (Boieblan, The author confirms being the sole contributor of this work and has approved it for publication.