Mechanical forces drive the remodeling of tissues during morphogenesis. This relies on the transmission of forces between cells by cadherin-based adherens junctions, which couple the force-generating actomyosin cytoskeletons of neighboring cells. Moreover, components of cadherin adhesions adopt force-dependent conformations that induce changes in the composition of adherens junctions, enabling transduction of mechanical forces into an intracellular response. Cadherin mechanotransduction can mediate reinforcement of cell–cell adhesions to withstand forces but also induce biochemical signaling to regulate cell behavior or direct remodeling of cell–cell adhesions to enable cell rearrangements. By transmission and transduction of mechanical forces, cadherin adhesions coordinate cellular behaviors underlying morphogenetic processes of collective cell migration, cell division, and cell intercalation. Here, we review recent advances in our understanding of this central role of cadherin adhesions in force-dependent regulation of morphogenesis. In addition, forces exerted on cells by each other and their surroundings serve as instructive signals to which cells adapt their behavior. This depends on the ability of cells to sense and convert mechanical forces into a biochemical intracellular response (termed mechanotransduction), which is mediated by force-induced changes in the conformation of proteins and the composition of molecular complexes4. Mechanical forces typically originate from contraction of the actomyosin cytoskeleton, which is anchored to sites of adhesion and can thereby be transmitted to neighboring cells and the extracellular environment. As such, focal adhesions (at the cell–matrix interface) and adherens junctions (AJs) (at the cell–cell interface) represent key force transmission and transduction complexes of the cell. In recent years, many additional components of the cell cortex, including growth factor receptors, ion channels, caveolae, and the glycocalyx, have been identified to be responsive to forces5. In this review, we will focus on AJs, as they have emerged as central mechano-responsive components in the regulation of morphogenesis by intercellular forces.Morphogenesis comprises the collection of processes that shape tissues and organisms, which relies on the coordinated regulation of cell behavior. Though most apparent throughout embryonic development, morphogenetic processes continue to play a role in adult tissues . Morphogenesis inherently is a mechanical process, as the coordinated generation of forces by cells is required to establish tissue shape. The cytosolic tail of cadherins compiles a large protein complex, which dynamically connects to the actomyosin cytoskeleton6. The core cadherin complex consists of p120-catenin, which controls cadherin membrane localization, and β-catenin and α-catenin, which provide a link with filamentous actin  or indirectly through other junctional components or the actin cytoskeleton . This large number of identified actin-associated proteins recruited to tensed cadherin junctions confers several layers of mechanical control of the AJ–actin link, potentially explaining the mild phenotype of selectively losing Vinculin in various tissues21.The AJ is composed of transmembrane cadherin proteins  that form homotypic dimers between neighboring cellss actin . Importas actin 7, whichinculin , which pkeleton . These b23. Moreover, the different mechanisms to transduce force into regulation of the organization of the actin cytoskeleton and its association with the cadherin complex strengthen cell–cell adhesions, enabling them to maintain integrity of tissues upon fluctuations in intercellular stresses. However, the cellular responses to junctional force reach much further, impacting, for instance, the cell cycle and cell division25, cell migration27, and cell metabolism28. This is mediated by force-regulated association of the cadherin complex with transcriptional activators , kinases , and interactors of microtubules and intermediate filaments  lamellipodia protruding forward and Rho-mediated adhesion disassembly at the rearllowers 16,27,27. Conformational opening of α-catenin induces local activation of Rho and downstream mDia-mediated actin remodeling, which strengthens anisotropy in junctional tension in migrating epithelia  through phosphorylation by LATS kinases71. As recently demonstrated in theDrosophila wing disc and cultured epithelia, increased tension on E-cadherin junctions  relieves this negative regulation, thereby promoting nuclear localization of YAP and cell cycle entry73. This is explained by force-induced opening of α-catenin that enables its interaction with proteins of the Ajuba/Zyxin family, which sequester LATS in an inactive state at AJs , specialized adhesions at sites where three (or more) cells meet, recruit microtubule force generators via the dynein-associated protein Mud. The distribution of TCJs aligns with cell shape and mechanical stress orientation within the tissue and this spatial information is retained upon mitotic rounding 118, an event that was recently shown to be directly regulated by tensile force82. A possible explanation of the opposite effects of myosin-generated tension on cadherin junctions, either junction shrinkage or junction reinforcement (discussed earlier), may lie in the force orientation with respect to the junction. During junction shrinkage, forces parallel to the cell–cell contact orientation result in shear stress on the junction complex, which decreases E-cadherin levels at the junction119. In contrast, force that is oriented perpendicular to anterior–posterior junctions was shown to increase junctional E-cadherin levels119. While E-cadherin levels decrease in shrinking junctions, the tension-sensitive recruitment of the actin regulators Vinculin, Ajuba, and AIP1 is increased, enabling the junction to cope with the elevated mechanical load121. After shrinkage of dorsoventral junctions, the germband extends upon resolving the intermediate multicellular vertices and elongation of anterior–posterior junctions. For AJs to withstand the high level of tension at multicellular vertices and for subsequent vertex resolution, the force-sensitive recruitment of Ajuba is essential121. Intriguingly, junction elongation does not happen merely as a result of tissue relaxation but instead is actively controlled by actomyosin activity as well123. Clearly, a dynamic interplay between mechanically controlled AJ turnover and strengthening is essential during intercalation, and future studies will likely provide more insights into the role of E-cadherin mechanotransduction in this.In addition to being transmitted by AJs and transduced into an intracellular response, mechanical forces can regulate tissue shape by remodeling of cell–cell contacts through force-dependent regulation of AJ turnover124. Several important questions remain just at the start of investigation, for instance how cells discriminate between forces of different magnitudes and timescales. Different timescales add the conundrum that the short-term responses of cells to intercellular forces  function to dissipate force and therefore potentially prevent long-term responses125. Moreover, it remains unanswered whether different force-dependent constituents of the AJ can be present simultaneously within the same cadherin complex, and mutual exclusivity is likely to exist . Understanding how the different components of cadherin mechanotransduction may be responsive to distinct magnitudes and types of force126 will further help to explain why cells may adopt different, sometimes opposing, mechano-responses through cadherin junctions .AJs have emerged as integral components in the regulation of morphogenesis by mechanical forces. In recent years, molecular details of cadherin mechanotransduction in morphogenetic processes of collective migration, cell division, and cell intercalation have started to be resolved. Future research will likely shed more light on how cadherin mechanotransduction impacts other processes that regulate tissue development and homeostasis, such as the control of cell shape, differentiation, and delamination127, adopt tension-sensitive changes in their composition128, and contribute to the transduction of intercellular forces39. Moreover, intercellular forces can be sensed by mechano-sensing complexes that respond to membrane deformations, a mechanism used by mechano-sensitive ion channels that adopt open and closed conformations depending on local force-induced membrane deformations130. So far, research on mechanotransduction has predominantly approached the various mechano-sensitive complexes as isolated units. To properly understand cellular responses to forces, it will be pivotal to know how they cooperate. Interplay further exists between AJs and mechano-sensitive complexes that are not localized at cell–cell contacts, such as focal adhesions that contain numerous mechano-sensitive components that are in part shared with the AJ6. Mechanotransduction by both adhesion complexes can converge on similar transcription factors and may cooperate in the activation of signaling pathways as has been shown for the activation of β-catenin77. In coming years, unraveling the interplay between these different mechano-sensitive complexes will be instrumental in understanding the variety of cellular behaviors that are controlled by mechanical force.Although we have focused primarily on AJs, cell–cell contacts contain additional adhesion complexes  that are exposed to external mechanical force132) or indirectly  regulated by mechanical force. Moreover, biochemical signaling pathways may feedback to modulate the cellular mechano-response134. A better comprehension of the dynamic interplay between mechanical and biochemical signaling will be imperative to understand the complexities of animal development.Finally, it is becoming increasingly apparent that mechanical signals that cells receive from their environment continuously crosstalk with biochemical signals, such as growth factors and hormones. Not only can both impinge on the same intracellular signaling cascades, but several growth factor receptors are also directly (for example, Notch


It is difficult to determinate the cause of death from exposure to fatal hypothermia and hyperthermia in forensic casework. Here, we present a state-of-the-art study that employs Fourier-transform infrared (FTIR) spectroscopy to investigate the hypothalamus tissues of fatal hypothermic, fatal hyperthermic and normothermic rats to determine forensically significant biomarkers related to fatal hypothermia and hyperthermia. Our results revealed that the spectral variations in the lipid, protein, carbohydrate and nucleic acid components are highly different for hypothalamuses after exposure to fatal hypothermic, fatal hyperthermic and normothermic conditions. In comparison with the normothermia group, the fatal hypothermia and hyperthermia groups contained higher total lipid amounts but were lower in unsaturated lipids. Additionally, their cell membranes were found to have less motional freedom. Among these three groups, the fatal hyperthermia group contained the lowest total proteins and carbohydrates and the highest aggregated and dysfunctional proteins, while the fatal hypothermia group contained the highest level of nucleic acids. In conclusion, this study demonstrates that FTIR spectroscopy has the potential to become a reliable method for the biochemical characterization of fatal hypothermia and hyperthermia hypothalamus tissues, and this could be used as a postmortem diagnostic feature in fatal hypothermia and hyperthermia deaths. Thermoregulation, as one important aspect of human homeostasis, keeps the body temperature of the organism within certain boundaries . The proThe hypothalamus is the main seat of thermoregulation, and its metabolic and functional activity changes as the core temperature of the body changes . Therefo−1), which includes the 3100–2800 cm−1 region, mainly representative of lipids, and the so-called fingerprint region (1800–900 cm−1), mainly representative of amide I/II, lipids, carbohydrates and nucleic acids ,53−1), wtressors ,82,83, ttressors . Our exp−1 (arising from DNA) illustrating the total nucleic acid levels was calculated (The sum of intensity at 1122 (arising from RNA) and 972 cmlculated A. As can−1) [−1) (−1/1151 cm−1) was also calculated to evaluate glycolysis progress    [−1) C. The raprogress D. We obs−1) −1/11 cm−1 wasIn this preliminary study, we demonstrate for the first time the potential of FTIR spectroscopy combined with advanced chemometrics as a method for easy and rapid assessment of the biomolecular variations seen in fatal hypothermic, fatal hyperthermic and normothermic hypothalamus tissues in rat models. Our FTIR-based results showed that the fatal hypothermic and hyperthermic hypothalamus tissues both have higher total lipid levels and lower lipid unsaturation levels, and their cell membranes demonstrated less motional freedom in comparison with the normothermic hypothalamus tissue. Additionally, the lowest total protein and carbohydrate levels and the highest protein aggregates and dysfunctions levels were observed in the fatal hyperthermic hypothalamus tissues, while the highest nucleic acid and glycolysis levels were observed in the hypothermic hypothalamus tissues.The brain often remains unexamined in fatal hypothermia/hyperthermia death investigations because it is thought to only indicate nonspecific histopathological findings. Nevertheless, our FTIR-based results show that the biochemical profiles of fatal hypothermic, fatal hyperthermic and normothermic hypothalamus tissues were significantly different. This study, supported by our previous studies ,90, demo


This retrospective chart review aims to address gaps in the literature regarding the efficacy and interaction of gonadotropin-releasing hormone agonists (GnRHa) and gender-affirming hormone therapies in medical transition regimens in transgender adolescents. We abstracted and reviewed data from 83 patients at our pediatric gender clinic, and found that patients who initiated treatment with GnRHa before gender-affirming hormones  required lower doses of those hormones than those who did not use GnRHa. The results of this preliminary research provide a foundation for future long-term prospective studies aimed to better understand these relationships. Barriers to such research include lack of Food and Drug Administration approval and reliable insurance coverage for GnRHa for this indication, as well as variations in prescribed hormone regimens. Our study attempts to address some of these unknowns by isolating descriptive statistics that may provide basis for further research. Specifically, we aimed to (1) determine whether dosages of gender-affirming hormones in those taking GnRHa differ from those not taking GnRHa; and (2) identify the frequency of associated side effects in both groups. It is posited that suppression of endogenous sex hormones via GnRHa may reduce necessary doses and associated side effects of gender-affirming hormones.Current clinical guidelines recommend the use of puberty suppressing hormones (gonadotropin-releasing hormone agonists [GnRHa]) in peri-pubertal patients diagnosed with gender dysphoria.T-tests, which are reported in the tables below.Institutional Review Board approval was granted based on the study design described below. Data from patients who began and were currently receiving gender-affirming hormone therapy at a pediatric gender clinic at a tertiary medical center before March 2016 were abstracted by a retrospective review of outpatient electronic medical records (EMR). Eighty-six patients were included in initial review, with one subject excluded due to nonbinary gender identification, as hormone doses for this individual differed from standard protocols so as to better address their specific transition goals. Data were extrapolated through January 2018 and included demographic information, health conditions and medications, GnRHa and gender-affirming hormone regimens, and reported side effects. Comorbid conditions were identified based on EMR Problem List as of the end of data collection, and recorded medications were limited to current prescriptions as of that date. Data regarding GnRHa and gender-affirming hormone dosing regimens were extrapolated from narrative and medication portions of the chart. Side effects were identified based on those recorded by care team physicians in narrative portions of EMR notes. In analyzing data regarding specific hormone regimens, we excluded two patients  who began GnRHa concurrently with gender-affirming hormones, to better distinguish between effects of GnRHa versus gender affirming hormones. Data from these subjects were included in demographic and health conditions/medication analyses. Statistical analysis included use of medians, ranges, and unpaired Of the 85 subjects included, 62 (73%) were male-identified, and 23 (27%) were female-identified.The majority of subjects (72%) had subjectively reported medical comorbidities as recorded in the problem list in the EMR. The severity or chronicity of these is unknown, but common conditions included depression and anxiety, asthma, and obesity.Prescriptions for psychiatric and nonpsychiatric medications were common, with 58/85 (68%) taking one or more medications aside from GnRHa or gender affirming medications.Of the 17 subjects taking GnRHa, 16 were taking leuprolide, and 1 patient had a histrelin implant (replaced yearly). Among these subjects, 11 (65%) experienced noted side effects, with hot flashes, mood swings, weight gain, and fatigue being most common. During the period of data collection, 10 (59%) patients discontinued use of GnRHa, most commonly due to loss of insurance coverage. Other subjects on GnRHa experienced lapses in insurance coverage for these medications, disrupting continuity of treatment. Female-affirmed subjects had a median duration of GnRHa use of 20.6 months, with a median 14.7 month overlap in GnRHa and hormone therapy treatment . Male-afp=0.0007 (2 Thirteen patients, all of whom were not taking GnRHa, were taking both spironolactone and estrogen at some point during the period of data collection.In the female-affirmed population, GnRHa use beginning before estrogen was associated with a significantly lower average dose of oral estradiol at the end of the data collection period, p=0.0007 . Rates op=0.0001 (Similarly, among male-affirmed subjects, GnRHa use correlated with lower doses of subcutaneous testosterone cypionate at the last data collection point, p=0.0001 . The maj2 This reiterates the importance of patient monitoring in gender-affirming hormone treatment, as is currently recommended by the Endocrine Society Guidelines. Lower doses of these hormones, as appears to be reasonable with concurrent GnRHa use, may lessen the frequency and severity of these laboratory changes. Furthermore, for female-affirmed patients, those not taking GnRHa were often placed on spironolactone in addition to estrogen, which reflects the need for adjunctive therapies to achieve suppression of testosterone levels to the female range.9 That those on GnRHa were not placed on spironolactone may reflect diminished need for supplemental medications due to endogenous testosterone suppression by GnRHa.GnRHa use was correlated with lower doses of gender-affirming hormones at the final point of data collection, suggesting that concurrent GnRHa may decrease doses of hormones needed to achieve desired physiologic changes. Frequency and type of side effects of gender-affirming hormones were similar regardless of whether GnRHa were prescribed, though severity of these effects was not assessed. In fact, the majority of subjects in all conditions experienced side effects of gender-affirming hormones, which appear to largely reflect changes commonly experienced in puberty, when these hormones are produced endogenously. As such, though uncomfortable, these “side effects” do not typically pose risk to the patient. More concerning are alterations in laboratory values, such as elevated liver enzymes or hematocrit, which if unaddressed can lead to organ damage.2 Though not inherently dangerous to the patient, such side effects can cause substantial discomfort and should be included in risk-benefit discussions with patients. Additionally, GnRHa have been postulated to have potential adverse effects on bone mineral density, though studies have largely been inconclusive on the true extent of this theoretical risk, and risks associated with prolonged use of GnRHa have not been examined.10 Though our chart review does not address this particular concern, we reiterate the importance of reviewing this possible risk with patients and of regular monitoring during treatment.Though these data support the use of GnRHa in adjunct to gender-affirming hormones, GnRHa are not without their own side effects, as noted in our findings. Side effects such as hot flashes, mood swings, and weight gain reflect the induction of hormone suppression in subjects.The aim of this retrospective chart review was to assess whether the use of GnRHa was associated with altered dosing and associated side effects of gender-affirming hormones; however, data review was notable for several additional findings that may impact care of this population.In this cohort, high rates of subjectively reported medical and psychiatric concerns and additional medication prescriptions indicate that care of these patients often requires coordination of multiple care teams, which may influence gender-affirming treatment decisions. Additionally, many patients taking GnRHa were noted to have lapses in insurance coverage of these medications, which prevented sustained use or resulted in discontinuation entirely. Without reliable insurance coverage, patients are often unable to access GnRHa in the first place, or are subject to suboptimal treatment due to early discontinuation or inconsistent use.Lack of standardized documentation, including specifically documented provider rationale for gender-affirming hormone prescription doses, combined with the already highly individualized nature of gender-affirming hormone regimens present a significant challenge to data collection and analysis in this study. Likewise, variation in timing of serum hormone level monitoring prevents precise correlation of serum hormones with dosage requirements. In addition, irregular patient follow-up and uncertainty of medication adherence complicate our discussion. Difficulties in acquisition and maintenance of consistent insurance coverage for GnRHa were an obstacle to our study and should be considered in the design of future research. Finally, given the retrospective nature of this study, it is impossible to isolate the effects of GnRHa therapy before versus concurrent with gender-affirming hormone therapies.Data from this study suggest the importance of conducting large, prospective studies with more standardized methods of data collection and clearly delineated hormone protocols. Such research may better elucidate the associations suggested by this research and establish causal relationships between use of GnRHa and doses and side effects of gender-affirming hormones. Additionally, such research may provide critical evidence necessary for Food and Drug Administration approval of GnRHa for this indication, as current lack thereof poses a significant obstacle to insurance coverage of these medications for many patients.


Apis sp.) and stingless bees (Meliponini sp.) and exhibits tremendous medicinal properties such as antimicrobial, anticarcinogen and antioxidant. However, it has not been included as a mainstream approach to disease management and has been disregarded in the modern pharmaceutical era. The stingless bee, which is known locally as lebah kelulut in Malaysia, is an important species that is well adapted for tropical countries and has emerged as an alternative source of honey. The reinventing honey quality (RHQ) project was introduced in 2012 to empower growth in the stingless bee industry, which has a direct impact on the production of high-quality honey. The objectives of the project include transforming the industry into a sustainable source of income for beekeepers, while simultaneously catalysing bee conservation activities for plant and crop pollination, thus becoming a new medium for targeting socio-economies and ecology.Honey is uniquely produced by honeybees ( Apis sp.) and stingless bees (Meliponini sp.) and exhibits tremendous medicinal properties such as anti-microbial, anti-carcinogen and antioxidant. It is specially highlighted as a natural remedy in the Holy Quran, as implied in Surah An-Nahl (‘the Bees’) in chapter 16, verse 69. Despite the medicinal properties, honey has never been included within the mainstream approach to disease management or classified under complementary medicine . The Tualang bees, which mainly nest in the jungle and far off the ground, limit the implementation of standard production procedures. Meanwhile, stingless bees (Meliponini sp.) or lebah kelulut which do not have stingers, build nests in already existing cavities or hollowed out areas of trees, buildings and hives. This nesting behaviour provides the opportunity for stingless bees to be cultivated in intensive farms with controlled environmental conditions or in homes in rural areas that implement standard operating procedures. Thus, empowerment of today’s stingless bee industry would have direct impact on production of high-quality honey, while also sustaining pollination of crops and other plants, particularly to maintain biodiversity. The stingless bee is well adapted to tropical and subtropical environments, such as Malaysia, Brazil, Mexico, Africa, Northern Australia and the Southeast Asian regions. Therefore, reinventing stingless bee honey industry in tropical countries could be a new medium for targeting socio-economies and ecology.In Malaysia, production of honey by honeybees, such as Geniotrigona thoracica, Heterotrigona itama, Lepidotrigona terminata and Tetragonula leviceps. The bees produce an average of 4 kg honey per colony each year. The major composition of stingless bee honey includes sugars (fructose and glucose) with nearly zero hydroxymethylfurfural (HMF). It also contains small amounts of other compounds, such as organic acids, phenolic compounds , proteins, amino acids , enzymes, vitamins and minerals  project (led by the corresponding author) has brought the stingless beekeeping field to light, transforming the stingless bee honey industry into a sustainable source of income for Malaysian beekeepers via premium honey production, while simultaneously catalysing bee conservation activities, thus ensuring sustainability of bees for plant pollination. The RHQ project, equipped with the artificial Mustafa-Hive , facilitates colony expansion and health screening, while simultaneously providing a habitat that can be adapted to changing climatic conditions. The Mustafa-Hive effectively promotes a more hygienic honey production practice via in-door harvest approach from systematically organised farms  in 2007 followed by a Universiti Sains Malaysia workshop for ed farms . The sysMalaysia’s tropical climate, coupled with its high humidity level and temperatures averaging between 28 °C–30 °C, results in honey with high moisture content, causing fermentation that produces a sour taste and foamy texture, which depreciates the value of Malaysian honey internationally. In response, the Honey Interlinked Dehydration and Dispenser Apparatus (HILDA) was invented under the RHQ project. Dehydrating the honey from 35% to 18% not only prevents fermentation but also raises both the quality and value of the final product. HILDA’s unique design represents an integrated platform for hygienic honey harvesting, water dehydration without a heating element and bottling.The stingless bee industry above has resulted in several spill-over impacts , such askelulut honey using neuromarketing tools is now important in rebranding traditional stingless bee honey at international market. Introduction of MS2683 and MS2679 2018 certification for a Malaysian standard of stingless beekeeping and honey content are among the big achievements in this field.Productive yields of stingless bee honey are expected to influence the supply and demand of the honey industry and becoming a new commodity for the country. Stingless beekeeping transformation programmes should increase investment towards strengthening honey product chains from beekeepers that range from small, traditional activities that are labour-intensive to huge, capital-intensive industrial processes. Hive identification and permit must be registered, and the honey source should be traceable. This will possibly involve initiation of a mobile-HILDA in order to reach the rural areas to ensure small-scale beekeepers’ participation in the transformation programme. Establishment of a national institute that centralises the transformation programme of stingless beekeeping is needed, with participation from all relevant communities, organisations, academia and governments. Planning should also be directed towards specific rearing guidelines, farm and honey certifications as well as upgrading marketing aspects, especially the facilitation phase, involving export requirement specifications. National positioning of kelulut factors and entomology signatures in honey are also crucial to improve the grade of stingless bee honey. Analysis of contents and efficacy test on honey should consider sampling of honey produced in an area of land with favourable tree species, such as coconut (Cocos nucifera), acacia (Acacia mangium), rubber (Hevea brasiliensis), durian (Durio zibethinus) and air mata pengantin (Antigonon leptopus) in the localities. Areas that are surrounded by these types of trees have shown notable achievements in honey yields, which can be introduced as plants of choice for ‘beescaping’ in the future.To support colony breeding, a genetic repository of stingless bee species should be assembled to preserve the genetic identification features of local stingless bees as well as for authentication purposes and queen bee production. Identification of local and unique Thus, upholding the stingless bee honey industry among communities serves as a holistic approach for a sustainable source of honey that can also benefit socio-economies, species survival and long-term ecological preservation.


Traumatic brain injury (TBI) is a significant cause of disability worldwide and can lead to post-traumatic epilepsy. Multiple molecular, cellular, and network pathologies occur following injury which may contribute to epileptogenesis. Efforts to identify mechanisms of disease progression and biomarkers which predict clinical outcomes have focused heavily on metabolic changes. Advances in imaging approaches, combined with well-established biochemical methodologies, have revealed a complex landscape of metabolic changes that occur acutely after TBI and then evolve in the days to weeks after. Based on this rich clinical and preclinical data, combined with the success of metabolic therapies like the ketogenic diet in treating epilepsy, interest has grown in determining whether manipulating metabolic activity following TBI may have therapeutic value to prevent post-traumatic epileptogenesis. Here, we focus on changes in glucose utilization and glycolytic activity in the brain following TBI and during seizures. We review relevant literature and outline potential paths forward to utilize glycolytic inhibitors as a disease-modifying therapy for post-traumatic epilepsy. While accounting for only 2% of the body’s weight, the human brain accounts for 20% of its energy utilization . In pathThe brain requires energy in the form of ATP to power its cellular processes. The ability of the brain to conduct electrical signals between cells requires a steep electrochemical gradient to be maintained across cellular membranes. Reestablishing the electrochemical gradient following synaptic activity accounts for ≈80% of the total brain energy costs , with acFigure 1). The systemic delivery of multiple kinds of fuel  results in an increase in extracellular glucose in the brain . While oxidative phosphorylation ultimately generates the majority of the ATP in aerobic respiration, it also requires oxygen and is much slower than the glycolytic production of ATP. Thus, these two processes might fuel different aspects of brain function. As an alternative to glycolysis, glucose can be shuttled into the pentose phosphate pathway, which generates reduced NADPH and supports the production of glutathione. Glutathione provides an important defense mechanism against oxidative stress, as it serves as an electron donor in the detoxification of reactive oxygen species.Once a glucose molecule leaves the vasculature and enters the brain parenchyma, it is transported into cells for metabolism (through GLUT3 in neurons or GLUT1 in astrocytes). Canonically, glycolysis converts glucose to pyruvate, which is then converted to acetyl-CoA to enter the citric acid (TCA) cycle and provide substrates for oxidative phosphorylation . Lactate is then shuttled to neurons through monocarboxylate transporters (MCTs), converted back to pyruvate, and further metabolized through oxidative phosphorylation  or positron emission tomography (PET) scanning. fMRI relies on the signal of oxy- versus deoxy-hemoglobin, where changes in the “BOLD” signal can occur with blood flow or metabolic flux. PET scanning, on the other hand, utilizes tracers to label blood, glucose, or oxygen. For example, using 18F-labeled 2-deoxyglucose (F-DG), one can visualize the accumulation of “glucose” in areas of the brain which require more fuel, and are thus transporting more glucose and F-DG into the brain parenchyma. We can also look at metabolic flux in brain slices from animal models or in culture, using metabolomic or molecular imaging of pH, NADH , ATP, orFigure 2).A better understanding of brain fuel utilization at baseline and in pathological states may allow us to harness the therapeutic potential of metabolic targets. Our motivation for this approach is based on the robust anticonvulsant and neuroprotective effects of the ketogenic diet in humans , where vin vitro experiments can aid in elucidating the mechanisms by which activity results in metabolic changes.As the energy requirements of the brain change, fuel utilization can be upregulated to accommodate these requirements. For example, fMRI studies have shown that visual tasks result in increased cerebral blood flow and increased oxidative metabolism in the visual cortex to meet the metabolic needs of the region . HoweverFigure 1, changes in network activity can affect a broad range of metabolic processes in both astrocytes and neurons. Astrocytes are poised to detect local changes in neuronal activity, and thus energetic need, in the brain. These cells have close interactions with synapses, and dynamically change their morphology in response to activity , where 2-NBD-glucose uptake parallels the spatial movement and kinetics of sodium transport into astrocytes (It has been previously shown that glutamate uptake requires re-establishment of the sodium gradient across the membrane (as glutamate is co-transported with sodium), which results in a decrease of ATP in astrocytes . Alongsitrocytes . The neutrocytes . This po+/HCO3- cotransporter NBCe1  result in increased deoxyglucose accumulation  and glycer NBCe1 . Additioer NBCe1 .Astrocytes are also able to mobilize energy for utilization through glycogenolysis, where astrocytic glycogen stores are catabolized into single glucose units. Extracellular potassium has been shown to induce glycogenolysis in mouse cortical slices in a calcium-dependent manner . The brein vivo glucose uptake in neurons, but not astrocytes, suggesting that neuronal glucose utilization is dynamic with activity state. Additionally, data mining of previously published transcriptomic and proteomic results show increased protein levels for many of the enzymes required for glycolysis in neurons relative to astrocytes  is positively correlated with NMDA receptor-mediated activity in neurons . This fiWhile some studies have suggested that neurons rely on oxidative metabolism , glycolyATP channels conduct potassium in response to decreased ATP concentration, and can thus directly modulate neuronal activity by altering membrane resistance   induced As with clinical studies, however, many of these assays monitor glucose uptake as a measure of metabolic activity. In the brain, glucose utilization is intimately linked with astrocytic metabolism, where astrocytes can mobilize glucose from glycogen stores or perform glycolysis to provide lactate to neurons for further metabolism. In a rat model of sustained seizures, glycogen concentrations were diminished after 20 min and 2 h of status epilepticus. Interestingly, lactate levels were elevated after 20 min of SE, but were decreased after 2 h of SE . This su+ imaging alongside recording of oxygen levels, in vitro models of seizure-like activity. In a zero-magnesium model, oxidative phosphorylation increased strongly during ictal activity and then decreased abruptly before the ictal activity ceased. Using a bicuculline model, oxidative phosphorylation increased in brief transients and then decreased during ictal activity , the enzyme which converts pyruvate into acetyl-CoA for entry into the TCA cycle . The acceleration/deceleration and shearing forces associated with TBI disrupt cellular membranes and result in massive neurotransmitter release, blood-brain-barrier compromise , fluid percussion injury (FPI), and weight drop. Each of these models recapitulates aspects of human TBI, including cellular losses , behavioSome animal models have recapitulated the early hypermetabolism and prolonged hypometabolism observed in humans. For example, after FPI, there are local increases in glucose utilization at early time points , which tInterestingly, the use of excitatory neurotransmitter antagonists, particularly APV to block NMDA receptors, prevented the increase in glucose utilization after FPI , suggestA key advantage of utilizing animal models is the ability to harvest brain tissue after injury to assess changes in expression patterns. These changes provide insight into which processes are up- or down-regulated following injury. Examination of the transcriptome after CCI in mice shows increased expression of glycolytic enzymes at 6 h after injury, with later decreases in glycolytic enzyme expression at later time points . SimilarAdditionally, animal studies can begin to elucidate the cell type-specific changes of metabolism after injury. For example, GLUT3 expression (which is responsible for glucose transport into neurons) is increased by 300% from 4 to 48 h after injury, while glial transporter GLUT1 is not changed . This fic release, Bax upregulation, and apoptotic cell death after weight drop in rats  are more vulnerable than others to PTE, and without good biomarkers for epileptogenesis to stratify study subjects, it is difficult to power a preclinical drug study with our available models. However, since glucose metabolism is clearly dysregulated in both epileptic brains and following TBI, this may be an exciting area to look for possible biomarkers. Additionally, attempts to maintain physiologic glucose utilization (avoiding acute hyperglycolytic and chronic hypometabolic states) may constitute a valuable therapeutic approach that should be investigated.As described above, brain metabolism (particularly glucose utilization and glycolysis) plays a key role in maintaining network activity in both physiologic and pathologic states. There has been increasing interest in manipulating brain metabolism in order to control aberrant activity states. The rationale behind this approach is well-founded, based on the clinical success of the ketogenic diet (KD) in humans suffering from intractable epilepsy. This diet is very low in carbohydrates and high in fats, which is thought to cause a shift in brain fuel utilization from glucose/glycolysis to ketone bodies/ketosis. Many molecular mechanisms have been proposed to modulate the antiepileptic effects of the KD, in an effort to find a small molecule mimic [reviewed in  and othein vitro and in vivo models , including pharmacologic hexokinase inhibition with lonidamine , or bicuculline . They showed an increase in the after-discharge threshold in the perforant path kindling model with 2-DG  in reducing epileptiform activity is abolished through genetic or pharmacologic blockade of KATP channels , BAD/Kpyruvate . The Yelchannels . Finallychannels , has beechannels  and protchannels .anticonvulsant effects and have not explored the possible disease-modifying, anti-epileptogenic effects of the treatment. In addition to 2-DG’s anticonvulsant effects, glycolytic inhibition following TBI may result in other benefits that prevent the development of post-traumatic epilepsy. Metabolic manipulation as a therapeutic target is a rational approach based on the body of evidence presented above. First, diverse and robust metabolic changes occur following TBI. At a basic level, this implicates metabolism as a part of disease pathology, and therefore a potential avenue for therapeutic intervention. Second, inhibiting metabolic systems offers the opportunity to affect a wide range of energy-dependent processes that may contribute to disease pathology. Neurotransmission, maintenance of ionic gradients, and inflammatory processes (among others) all have significant energy demands and may contribute to disease progression. While disrupting metabolism may lack specificity, its breadth of effects may contribute to a truly disease-modifying approach. Additionally, this approach may be more specific than first appreciated, as it aims to decrease the activity of only the most active cells. Third, increased metabolic activity acutely following brain injury may drive secondary injury processes that contribute to epileptogenesis. To date, no data exists to support or refute a direct link between them, but a deeper understanding of how different pathophysiological processes interact to result in post-traumatic epileptogenesis will help drive novel therapeutic approaches. If intervening in metabolic pathways after injury is protective, it is likely not via acute anticonvulsant effects, as other traditional anticonvulsant drugs have failed to prevent PTE. Instead, it may be acting by preventing other epileptogenic processes. Finally, manipulating metabolism, in some ways, is reminiscent of TBI treatment with hypothermia. While hypothermia has many effects, it slows metabolic activity via its effects on enzyme kinetics. A recent meta-analysis of therapeutic hypothermia for TBI suggests it may improve neurological outcomes for adults following focal TBI  and glucocorticoids have been examined in clinical trials to prevent PTE, but have not shown significant long-term beneficial effects . Could mocal TBI . BecauseFigure 4). Clinical literature supports that TBI-induced hypermetabolism occurs only in the short term after TBI. The injury itself causes cells to rupture and release their intracellular contents (including pro-excitatory species like glutamate), breaches the blood-brain barrier, activates immune and inflammatory cascades, and reduces local blood flow and oxygenation. All of these events could favor the use of glycolysis to power neuronal activity and inflammatory activity, especially in a local low-oxygen environment. If any of these early events, and their immediate downstream effects, contribute to the development of PTE and rely on increased glycolysis, then attenuating excessive glycolytic activity may be beneficial. The time window in which hypermetabolism occurs may be extremely short, perhaps lasting only minutes to hours following injury  to reduce tissue loss and improve behavioral function after TBI (There are several conceptual concerns that arise when considering glycolytic inhibition as a treatment to prevent PTE following TBI. First, why would one consider reducing energy production in a brain that desperately needs energy to restore cellular and network function after injury? This becomes even more relevant when considering the long-term hypometabolic state that exists following TBI. In fact, there is significant controversy among clinicians as to whether glucose should actually be supplemented for post-TBI patients . Additiofter TBI . Furtherfter TBI . Again, The challenges to treating TBI and preventing PTE are extensive. Heterogeneous injury categories, diverse metabolic responses to injury, and difficulty in powering both preclinical and clinical trials for PTE all make this a daunting translational problem. Based on the compelling evidence of metabolic changes following TBI, strong neuroprotective and anticonvulsant properties of the ketogenic diet, and multiple small molecule approaches to manipulate metabolism, we believe that therapeutic opportunities exist to harness metabolic systems to reduce PTE. There are many outstanding questions the field must address, including identifying the cellular sites and types of glucose utilization during normal brain function and after TBI, developing diagnostic tools that provide molecular insight into brain metabolism, understanding how metabolism contributes to post-traumatic epileptogenesis, and identifying biomarkers to stratify the patients at highest risk of developing PTE. We would recommend prioritizing the development of a quantitative assay to assess changes in glucose utilization in the brain following TBI. This assay could serve as both a prognostic and a predictive biomarker for PTE. Increased glucose utilization may be prognostic as it is likely associated with uncontrolled network activity, consistent with epileptogenesis. It may also serve as a predictive biomarker of which patients would respond best to a metabolically targeted therapy, such as glycolytic inhibition. This is especially important as hyper-glycolysis may only occur in a brief temporal window and/or in a subset of patients following TBI. Developing new tools and biomarkers is critical, as the clinical needs of TBI patients are clear: to improve long-term quality-of-life and to prevent devastating TBI complications such as PTE. Continued collaboration between basic scientists and clinicians will allow for better understanding of post-TBI pathophysiology and will ultimately advance novel interventional strategies to help these patients.JK and CD conceived of and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A recent study identifies a locus controlling seed dormancy – a key trait of the ‘domestication syndrome’ – that has been selected for in parallel across multiple crop families. Ten to twelve thousand years ago, in different regions around the world, humans launched an intriguing ‘co-evolutionary experiment’ – the domestication of crop plants and farm animals. In the case of crops, domestication was expected to generate more-productive plants, better adapted to different agro-ecosystems and easier to harvest and/or producing edible products that were safer to consume. Although the species and locations vary, domestication events are typically associated with the series of morphological changes that collectively are known as the ‘domestication syndrome’. This encompasses cultivars with large non-dispersing seeds, uniform germination, photoperiod insensitivity, reduced branching and several nutrition-related phenotypes. The emergence of such phenotypic traits of morpho-agronomic relevance shared by different crops prompts a question regarding whether similarities in human demands regarding cultivation, harvest and consumption led to widespread convergent artificial selection of genes Fig. . A recenTraditional genetic screenings and large-scale genomic studies have allowed the identification of a number of domestication-related genes that, in many cases, are particular to each domestication history, even within the same plant genus –4. More Shattering1 in cereals [Domestication has occurred across varied cultures and geography. Even though there has been a strong convergence in terms of the evolution of domestication-related traits in different crop families, there are few examples where one single gene has been equally affected by artificial selection in distantly related species . More o cereals . This sc cereals  found noSeed dormancy delays and desynchronizes germination under unsuitable environmental conditions for plant growth. In the wild, this is a useful strategy that promotes seed dispersal and warrants that at least some seeds will germinate and survive when conditions are favourable. In domesticated cultivars, however, it is desirable to have uniform germination to ensure maturity and crop management by farmers. Thus, the loss of seed dormancy has been a crucial trait selected for during crop domestication. Different studies have associated dozens of QTLs to seed dormancy in cereals and legumes, which explain a rather discrete amount of phenotypic variation.G gene (GmG) encoding a CAAX amino-terminal protease and controlling the pigmentation of the seed coat, was found to be strongly associated with the loss of seed dormancy in soybean. Not only does this gene display signatures of strong selection in soybean, but its orthologs in rice (OsG) and tomato (SolyG) are also encoded within genomic regions with signatures of selective sweeps, as seen by tests of FST, cross-population composite likelihood ratio (XP-CLR) and extended haplotype homozygosity (EHH). Based on genome resequencing of 176 rice accessions and a large available collection of wild and domesticated cultivars, it was observed that two groups of haplotypes in the population differentiate wild (group I) from domesticated Indica and Japonica accessions (group II). Similarly, in a large collection of 360 resequenced accessions, it was demonstrated that cherry (CER) and big-fruited (BIG) tomato cultivars displayed a non-synonymous single-nucleotide polymorphism  that differentiated them from the G allele present in the wild Solanum pimpinellifolium.In a recent report by Wang and collaborators , a stay-G locus in seed dormancy was achieved using transgenic soybean and rice lines – that is, GmG introduced into a g allele soybean cultivar (DN50), OsG introduced into a g allele cultivated rice line (ZH11), a CRISPR knockout of Osg in ZH11, overexpression of two OsG alleles from Oryza rufipogon and Osg in ZH11. In all these combinations, the G allele was confirmed to cause a strong dormancy phenotype compared with the g form of the locus. Furthermore, GmG introduced in Arabidopsis atg mutants complemented the fast-germinating phenotype, showing that this gene is also involved in seed dormancy in Arabidopsis.Experimental validation of the role of the Arabidopsis AtG was found to interact with two key enzymes of the abscisic acid (ABA) biosynthetic pathway – AtNCED3 and AtPSY. Both ABA and several carotenoids were observed to be more abundant in AtG than atg lines, as well as in transgenic soybean lines overexpressing GmG compared with Gmg cultivars.The function and possible interactors of the G protein were evaluated by means of yeast two-hybrid assays. Arabidopsis.The results of this study confirm the role of ABA in seed dormancy as previously reported, but, more importantly, they show for the first time that the same protein-coding gene has been targeted by artificial selection in three distant crop families, Leguminosae, Poaceae and Solanaceae Fig. , and posAs plant breeding has traditionally been based on phenotype-targeted selection, it becomes crucial to dissect the genetic elements behind relevant morpho-agronomic traits to improve crops with great precision and efficiency. Furthermore, crop breeding does not rely uniquely on direct selection, but also on the effects of linked loci that hitchhike during introgression events, such as metabolic genes hitchhiking with fruit weight genes as large tomato fruits were selected, or metabolic genes from inedible green-fruited species dragged by resistance genes from wild relatives .G/g alleles identified in distant plant families could potentially decrease the seed dormancy phenotype in other crops. Thus, high-throughput molecular breeding combined with precise genome editing should accelerate crop improvement and even, perhaps, domestication of plant species capable of growing under unforeseen environmental conditions, which could be crucial in the near future when facing changeable climatic conditions.In the past few years, our understanding of crop domestication has evolved from the view of a few large-effect loci to polygenic interactions that alter complex networks. In this context, the results reported by Wang and colleagues provide an excellent candidate for targeted breeding, as the introduction of a limited number of


In August 2017, the National Institute of Allergy and Infectious Diseases convened a meeting, entitled “Understanding the Liver-Stage Biology of Malaria Parasites to Enable and Accelerate the Development of a Highly Efficacious Vaccine,” to discuss the needs and strategies to develop a highly efficacious, whole organism–based vaccine targeting the liver stage of malaria parasites. It was concluded that attenuated sporozoite platforms have proven to be promising approaches, and that late-arresting sporozoites could potentially offer greater vaccine performance than early-arresting sporozoites against malaria. New knowledge and emerging technologies have made the development of late-arresting sporozoites feasible. Highly integrated approaches involving liver-stage research, “omics” studies, and cutting-edge genetic editing technologies, combined with in vitro culture systems or unique animal models, are needed to accelerate the discovery of candidates for a late-arresting, genetically attenuated parasite vaccine. In addition, vaccines that reduce transmission and target both Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) will become increasingly important, as global malaria control and elimination efforts approach their end game.Development of a highly efficacious malaria vaccine with at least 75% protective efficacy is one of the research and development (R&D) targets identified by both the Global Vaccine Action PlanPf and Pv. In recent years, significant progress has been made in the development of whole sporozoite vaccine strategies targeting the liver stage of Pf parasites. These include radiation-attenuated sporozoites (RAS), genetically attenuated sporozoites (GAP), chloroquine prophylaxis with (live) sporozoites (CPS), and the use of murine parasite Plasmodium berghei (Pb) as a platform to deliver human Pf parasite antigens (Ags) (Pb/Pf).4 Evaluation of these strategies in a controlled human malaria infection (CHMI) setting indicated that CPS immunization using live sporozoites was much more effective at eliciting protection (> 75%) compared with RAS immunization; protective immunity elicited by CPS is believed to be directed against preerythrocytic (most likely liver-stage) parasites.7 This increased potency is thought to reflect the greater biomass and diverse Ag expression of live Pf parasites that can replicate in the liver for at least 6–7 days before being killed by drug treatment, in contrast to RAS which only develop in the liver for approximately 2–3 days.The malaria liver stage represents a population bottleneck and, as such, is an attractive vaccine target for both Pf, especially GAPs that can support longer liver-stage development. To date, however, only mutations that arrest parasite development at the early-liver stage have been made, and the development of a stable Pf GAP with late liver-stage replication comparable with that of CPS has not been successful. Undoubtedly, there remain knowledge gaps in the liver-stage biology for both Pf and Pv in human infections as compared with what has been observed in rodent malaria infections in animal models. To address these, a workshop was convened to bring together investigators from different research areas, including sporozoite vaccine R&D, malaria immunology, malaria liver-stage biology, and molecular parasitology to discuss the current state of whole sporozoite vaccines, the liver-stage biology and immunology of malaria parasite infections, and to brainstorm about scientific and technical approaches to accelerate research in priority areas. The goal was to identify targets and strategies to assist in the design and development of a safe, efficacious GAP vaccine against malaria.Genetically attenuated sporozoites approaches offer opportunities to arrest sporozoites at certain times of their liver-stage development. There are ongoing efforts in multiple laboratories to develop and test several such approaches in 10 In a natural exposure setting in Mali, where the parasite attack rate is more than 90%, vaccination conferred 29% protection by proportional analysis but 52% protection by time-to-event analysis.11 Of note, in a Phase IIb trial in Kenya where the attack rate was 49%, the RTS,S/AS01 vaccine achieved only 29% protection in a similar time-to-event analysis.12 Currently, the RAS vaccine is being tested in 5- to 12-month-old infants in Tanzania. Clinical trials to optimize the regimen and address other vaccine-related issues needed to bring the RAS product to licensure and deployment are underway or in the planning stage.During the workshop, progress on the development of whole parasite/sporozoite-based vaccines targeting liver stages was summarized. Various forms of attenuated sporozoite vaccines delivered via direct venous inoculation (DVI) have been demonstrated to be safe, tolerable, and efficacious. Both RAS and CPS immunizations showed a significant level of short-term sterile protection against both homologous and heterologous parasite strains under CHMI in malaria-naïve adults in the United States. Protection remained significantly high against a homologous strain of parasite 1 year later but diminished significantly against a heterologous strain.4 live sporozoites  under chloroquine coverage (CPS) provided 100% protection for 10 weeks,7 three doses of 4.5 × 105 sporozoites were needed for 87% protection with RAS immunization.9 Therefore, it appears that at least more than 9-fold more RAS sporozoites are needed to achieve similar levels of protection with live sporozoites immunization. Although live infectious sporozoites seem to be highly efficacious, they will not be an ideal vaccine candidate because of their pathologic potential and suboptimal manufacturing consistency. The GAP approach, on the other hand, introduces stably arrested sporozoites at defined liver-stage timepoints and presents an attractive alternative for sporozoite vaccine development. The greater efficacy of live sporozoites has been attributed to their prolonged liver-stage development, enabling them to replicate and form a larger biomass that expresses greater amounts of diverse protein Ags and, thus, induces potentially broader and longer lasting immune stimulation. In fact, in mouse models, a Pb late-arresting GAP (LAGAP) vaccine showed not only a much superior level of protection as compared with an early-arresting GAP (EAGAP) sporozoite vaccine, but also cross-species and cross-stage protection. The Pb LAGAP also expressed a much larger repertoire of parasite proteins and higher levels of protective CD8 T cells.13 Therefore, a Pf LAGAP resembling the Pb LAGAP would potentially represent an ideal vaccine candidate for malaria control and elimination.Quantitatively, although immunization with three doses of 5 × 10Pf LAGAP remains a great challenge, however. The biggest hurdle is to ensure safety, that is, no leakage of erythrocyte-infective merozoite progeny into the blood when parasite development is approaching the end of the liver stage. Two different Pf GAP sporozoites have been tested in humans via mosquito bite delivery, all of which arrested early in the liver stage (days 2–3).15 Unfortunately, due to attenuation “leakage,” the P53-P36- double-knockout GAP produced parasitemia in one vaccinated subject. In a recent Phase I trial, however, the P53-p36-Sal1- triple-knockout GAP produced no such “leakage.” Several laboratories are attempting to generate a LAGAP but thus far have not reported success. It was pointed out that basic research to investigate the mechanism of P53-/P36- GAP attenuation leakage, or parasite liver-stage arrest or “breakthrough” in general, may help guide future GAP development.Generation of a Pv whole sporozoite vaccines was also reviewed. Importantly, whole Pv sporozoite vaccine development and Pv liver-stage basic research have both been significantly hampered by the lack of a continuous, long-term, in vitro liver-stage culture system, and have relied on primary human hepatocytes or nonhuman primate (NHP) malaria liver models. Plasmodium cynomolgi exhibits relapse features in NHPs similar to that of Pv in humans, and has been used to study relapse biology. The extent of its relevance to Pv relapse biology remains unknown. As for Pv whole sporozoite vaccine development, the current strategies are limited to the following: 1). using specific pathogen–free NHP as a “factory” to grow live Pv sporozoites for radiation attenuation; 2). exploring Pf RAS for potential cross-species protection in clinics; or 3). using Pb or Pf sporozoites as a backbone to genetically introduce select Pv Ags. To facilitate Pv whole sporozoite vaccine development, the following Pv research priorities were strongly emphasized: establishing an in vitro culture system; discovering infectivity genes in the Pv genome; and understanding liver-stage hypnozoite biology and relapse control.The latest research progress on In summary, it was unanimously agreed that attenuated sporozoite platforms provide a promising approach for generating a highly efficacious vaccine for malaria, and an LAGAP, if made, could have the potential to provide a far superior performance over current RAS or GAP vaccine candidates. Although current attenuated sporozoite vaccine development efforts should not be slowed down, priority should be placed on using existing technologies and knowledge to produce a potentially highly efficacious LAGAP. Understanding the mechanisms of liver-stage “arrest” and “breakthrough” are important for designing future GAP mutants. Of note, it was pointed out that several definitions have been used to define preerythrocytic vaccine efficacy for different trials or different vaccine candidates, including sterile protection, delayed patency , or clinical malaria incidence. Great emphasis was placed on the need to harmonize the definition of vaccine efficacy to ensure accurate assessments and comparisons of vaccine performance.Pf sporozoite vaccine protection. The level of CD8 T cells in blood following immunization, however, has been very low, perhaps due to inefficient or suboptimal assays to measure CD8 T cells ex vivo or in vitro. As such, the exact role of human CD8 T cells for vaccine protection has not been confirmed. A better assay to measure sporozoite-specific CD8 T cells is needed, and the relationship between CD8 T cells in the blood and CD8 T cells in the liver needs further exploration. In addition, the level of Ab response was consistently observed to be much lower in African adults as compared with malaria-naïve adults in the United States, and many CD8 T cells in individuals in endemic areas recognize blood-stage Ags unrelated to preerythrocytic immunity. Efforts should be directed at gaining a better understanding of the underlying immunological reasons for the decreased vaccine efficacy observed in endemic areas as compared with the United States.The immunological mechanism underlying sporozoite vaccine protection is much more complex than originally thought. Accumulated clinical data have demonstrated that protective immunity is a combined immune signature rather than a single immune parameter in a human setting. The specific immune profile of the recognized Ags could be quite heterogeneous among individuals; therefore, whole sporozoite vaccines have a much higher chance of success than subunit vaccines with a narrow Ag focus. It was shown that trained innate immunity could also reduce liver-stage parasite infection and development but only for a short duration. Antibody (Ab) and effectors of cellular immunity, including CD4, CD8, natural killer cells, γδ T cells, or other polyclonal T cells, are involved in Pf, data obtained from NHP studies appeared to be predictive of human vaccine efficacy. Direct venous innoculation of RAS, unlike intradermal (ID) or subcutaneous (SC) immunization, proved to be efficacious in humans and also induced significantly higher levels CD4 and CD8 T cells in the liver of NHPs as compared with ID or SC routes. In mice, besides Ab and CD4 T cells, CD8 T cells were consistently shown to be the most important protective immune factor, functioning as the most specific effectors through cognate hepatocyte recognition, Ag presentation, and secretion of interferon-γ, tumor necrosis factor, or perforin. An extremely high frequency of circulating CD8 T cells is required for sterile protection. Recently, rather than the conventional CD8 central memory TCM or CD8 effector memory TEM cells, CD8 tissue resident memory TRM cells were observed to constantly patrol sinusoids, exert a strong effector function, and be essential for sterile protection.16 TRM cells have been shown to form spontaneously on RAS immunization and can be enhanced by inflammation  or Ags in tissues. TRM cells can also recruit TCM and TEM cells to the liver as a backup strategy. Most importantly, initial priming was thought to occur in the spleen and liver-draining lymph nodes but not in the liver on RAS immunization by DVI; however, the liver is still an important site because it may generate Ags for priming to occur. Subsequent boosting may convert primed cells into TRM cells. Further knowledge about TRM formation, function, durability, and maintenance would help improve whole sporozoite vaccine design and vaccination strategies. Nevertheless, it was noted that there are still gaps in translating findings from mouse models to human settings. Research on human TRM cells in general is lacking and studies on the anatomic sites and cellular/molecular events for priming and boosting TRM cells during sporozoite immunization would be very helpful in designing a better sporozoite vaccine and relevant vaccination strategy.Protective immunity in animal models, on the other hand, has been rather well characterized. Even though NHPs are not a natural host for There were active discussions that LAGAP may generate some degree of blood-stage immunity which might skew host immune responses toward those that are more strain specific; no evidence of this was found in mouse models of infection, however. The consensus was that rather than being detrimental to preerythrocytic stage immunity, the blood-stage immunity elicited by LAGAP, if any, could be an added benefit. Understanding the link between liver-stage biology and liver-stage immunity, and the difference between EAGAP-specific and LAGAP-specific T cells, including their Ag specificity, phenotype, and functionality, were identified as key areas of investigation. It was also suggested that defining vaccine efficacy using time-to-event analysis could be meaningful in measuring immunological benefit because delayed infection/parasitemia could reflect more late liver stage Ag expression for immune priming or more time for specific immunity to develop and mature. In addition, the immunological mechanisms associated with differential vaccine efficacy between malaria-naïve adults in the United States compared with African adults with a history of prior malaria exposure should be further explored. Finally, developing assays to measure human CD8 T cell responses with better specificity and sensitivity, identifying CD8 T cell epitopes and protective Ags for Ab recognition, are needed not only to improve our understanding of sporozoite vaccine protection mechanism, but also to better select more informative immunological endpoints for clinical trial evaluation.Plasmodium parasites have evolved a complex life cycle with the sporozoite constituting the most versatile and enigmatic form of the parasite, being the stage formed in the mosquito vector and then undergoing further differentiation in human liver cells. Following the bite of an infected mosquito, highly motile sporozoites leave the bite site, travel through the circulation until they find and invade hepatocytes. Once hepatocyte invasion is complete and infection is established, the complex process of parasite liver-stage development is initiated, transforming the parasite from an invasive extracellular form to an intracellular trophozoite within a parasitophorous vacuolar membrane (PVM), culminating in the production of invasive exoerythrocytic merozoites. Along the liver-stage journey, the parasite encounters many different cell types and conditions, coupled with commensurate changes in parasite gene expression and morphology. For Pf, this process takes 6–7 days. Plasmodium vivax sporozoites can also differentiate into a dormant form, the hypnozoite, which can remain latent for months to years before reactivating, developing further in the liver, and causing blood-stage infection, thus, accounting for the periodic relapses seen in vivax malaria.Pf and Pv liver-stage development becomes extremely important in designing an attenuated sporozoite vaccine that arrests precisely at a desirable time point of liver-stage development. Since the discovery many years ago of the exoerythrocytic development of mammalian malaria parasites in the liver, however, much of our knowledge of this stage has been gained from studies performed with rodent malaria in animal models. The panel discussed the surprisingly limited knowledge of Pf and Pv liver-stage biology and noted that studies on molecular mechanisms surrounding merozoite invasion, development, and egress in red blood cells (erythrocytic phase) have been far ahead of those investigating sporozoite invasion of hepatocytes, intrahepatic development, and subsequent egress. This is in part because liver infections are asymptomatic and cannot be readily observed during natural human infections. In addition, Pf and Pv have a narrow host preference , and a natural sporozoite infection of human hepatocytes usually results in the invasion of only a small number of hepatocytes. It is anticipated that the recent development of in vitro human hepatocyte platforms and liver-humanized mice would greatly aid in liver-stage Pf and Pv basic research.Understanding the molecular and cellular events during Some rodent parasite proteins involved in PVM formation were described to be essential for liver-stage formation and development, including proteins that transport lipids and iron across the PVM to support parasite growth. A more comprehensive understanding of the identity and function of all such proteins is still needed. Inside hepatocytes, there is a massive increase in parasite biomass, suggesting active anabolic pathways in addition to nutrients acquired from the metabolically active host cell. One of these pathways has been demonstrated to involve de novo fatty acid synthesis, although the exact role of these fatty acids remains largely unknown.Pf and Pv liver-stage parasite differentiation, gene transcription, protein expression, and functional identification. At the cellular level, knowledge of Pf and Pv parasites’ interplay with host hepatocytes is also limited. Better transcriptomic and proteomic data for both Pf and Pv are needed. Given the current availability of human hepatocyte lines in 2D or 3D culture systems, primary human hepatocyte cultures, and severe combined immunodeficiency mice engrafted with human hepatocytes, identification of genes controlling parasite arrest in human liver should be possible. Suggested approaches included studying Pf gene transcription or expression profiles across life cycle stages to identify those that are highly expressed in the liver stage but less or not at all in other life cycle stages and detailed time course studies of parasite gene expression in infected hepatocytes to dissect gene expression regulation. The panel noted, however, that the most abundant proteins are not always the most important or the best targets. The egress pathway was also suggested as a priority target because drugs targeting egress allow parasites to leave the vacuole but remain in the host cell, and the egress process in liver-stage parasites seems distinct from that in blood-stage parasites. Exploring compounds that specifically interfere with hepatic stage development to extract information for genetic arrest was also proposed. Another suggestion was to study “master regulatory genes” and consider HTP deletion methods to identify late-arresting phenotypes, or use comparative biology approaches to identify similar/conserved genes/pathways that control parasite proliferation, differentiation, or egress.Creating LAGAPs for clinical use as a vaccine requires the identification of candidate genes that, when knocked out, will completely block late-stage parasite development. Several “omics-based approaches” have previously been used to identify liver-stage targets, including cDNA sequencing, RNA sequencing , proteomics and other bioinformatics approaches, and high-throughput (HTP) reverse genetic screens. Again, such studies have largely been performed in rodent malaria parasites and are not completely predictive of human malaria parasites. Thus, there remains an extensive knowledge gap regarding Pf sporozoites is now technically feasible, these should be made readily available to the research community to enable and facilitate worldwide basic research, especially on the Pf liver stage. In addition, although green fluorescent protein-labeled Pf is routinely used for microscopy, it is highly problematic for the sorting of infected hepatocytes for downstream omics studies because of a high signal to noise ratio. As such, transgenic parasites expressing a lower noise fluorescent protein for such studies would be a valuable resource for the research community. Further, systematically obtaining Pf liver-stage transcriptomic information, facilitated by substantially reduced costs of transcriptomic analysis and availability of single-cell transcriptomic analyses, would greatly enable liver-stage research. Finally, research on Pf liver-stage biology has been hampered in part by low hepatocyte infection rates and incomplete development in current in vitro models. Although parasite development is more complete using a humanized mouse model, high costs and technical challenges remain issues. As such, an improved, more easily accessible system, supporting the growth and full development of the liver-stage parasite is needed.Several immediate tasks were repeatedly emphasized by the panel. Because the production of vialed, live Pf and Pv. Empirical approaches for identifying targeted arrest genes may consider the egress pathway, “master regulatory genes,” chemically validated lethal genes, or other genes and pathways identified using various model organisms.In summary, a better understanding of liver-stage biology and sporozoite gene function is needed for targeted gene deletion strategies. There is a current need to create better liver stage transcriptomes and proteomes for Pf liver-stage–“omics-based studies” and the less tractable features of human malaria parasites, the use of functional genetic screens in various model organisms to gain coordinated insight into the identity of appropriate target genes would be useful in predicting and down-selecting targets for further investigation. Although rodent malaria models, and the ready availability of reagents and screening tools associated with them, provide the most convenient approach to gain useful biological information, limitations in translating findings in rodents to humans were noted. For example, development of rodent Plasmodium yoelii sporozoites with gene deletions in fatty acid biosynthesis pathway was arrested late in the liver stage, whereas gene ortholog deletions in Pf turned out to be lethal for the parasite. The use of model organisms, such as the more genetically tractable apicomplexan, Toxoplasma, could also be informative for several reasons. Chiefly, there appears to be a high correlation between expression profiles of essential genes in Toxoplasma and those of human malaria parasites,17 suggesting the potential predictive value of Toxoplasma transcriptomics. In addition, Toxoplasma is more genetically tractable than Plasmodium, in part, due to its higher transformation efficiency and higher germinal center content, which allows for pooled genetic screens and genome-wide phenotypic analyses following reverse genetic engineering. In fact, many dispensable and essential genes of Toxoplasma have been identified, and the corresponding complex phenotypes have been further dissected using conditional knockout strategies and conditional functional screens. Another apicomplexan genus, Cryptosporidium, is considerably less tractable than Toxoplasma and has been difficult to culture in vitro, hampering genetic manipulation. Despite this limited tractability, advances have been made in genetic manipulation and in understanding the biology of this parasite. Although not as directly predictive as studies in Toxoplasma, lessons learned from Cryptosporidium may inform efforts for Pf and Pv culture and manipulation. Comparative biology research using knowledge gained from the study of these model organisms would likely yield useful information that may guide relevant research efforts in human malaria parasites.Given the limited availability of Pf to create deletion mutant parasites and is expected to be a valuable tool in LAGAP sporozoite generation, due to increased efficiency and HTP potential, as compared with homologous recombination alone. It is generally thought that repair of double-stranded breaks in Pf only occurs through homologous recombination with the addition of a repair template, due to the lack of nonhomologous end-joining. The requirement for this template further decreases the likelihood of off-target effects in Pf. Regardless of the mechanism used, more efficient transfection and gene deletion/alteration strategies, and better molecular tools and cellular systems, would significantly improve the HTP efficiency of the genetic manipulation of Plasmodium parasites.Various methodologies have been used in reverse genetic approaches, including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. Recently, CRISPR/Cas9 editing has been used successfully in Pf strains other than NF54 is an important topic and should be carefully considered. A major advantage of NF54 sporozoites is that manufacturing and production data for future development are readily available, yet it is worth considering whether other Pf strains may offer better genetic manipulation characteristics and potentially better vaccine candidacy. The panel also emphasized that one gene deletion may be insufficient and two or more gene disruptions  may be required to produce the most effective product. Finally, it was highly recommended that once generated, all mutant cell lines be systematically cataloged, deposited into a repository, and preserved for the research community at large, similar to the malaria box for drug development. This should also include down-selected or unpublished mutants, such as those not leading to protection, or those producing an undesirable phenotype or no discernible phenotypic changes.It was highlighted that the technical process chosen for generation of mutant parasites must consider safety and regulatory requirements for future downstream vaccine development. Certainly, the clean deletions of candidate genes that are selection marker-free is an important consideration. Additional features to enhance safety include kill switches or built-in auxotrophy for biocontainment. Furthermore, the selection of To ensure sufficient parasite biomass for efficient priming of host immunity, an LAGAP should ideally arrest in the liver at days 4–5 or beyond. Initial assessment of liver-stage genetic mutants should include the abilities to complete blood- and mosquito-stage development, productively infect hepatocytes, and produce gametocytes to ensure production of sporozoites. Once confirmed, the most critical features of these mutants are safety and potency, which can be evaluated using humanized mouse models or microculture systems.Pv liver-stage anti-hypnozoite drug screening, Pf sporozoite infectivity has been high enough (5%) for an initial HTP effort of LAGAP screening.18 Apparently, further optimization and refinement are still needed. Other in vitro culture systems that need improvement include the hepatocyte cell line HC-04, which thus far has only supported early sporozoite growth, and hepatocyte culture, which allows only suboptimal sporozoite infectivity. Regardless, tissue culture screening can only be used as a HTP approach for initial down-selection of candidate mutants. Breakthroughs observed with cultured cell lines may not resemble the in vivo situation. Further confirmatory studies need to be carried out, for example, with a humanized mouse model to confirm lack of breakthrough before clinical trials. Some potential cost-saving approaches, such as multiplex infection using genotype barcoding, were suggested. In addition, the cost of the humanized mouse model may soon be significantly reduced by improved technology to generate copious quantities of human primary hepatocytes for mouse engraftment. As such, direct testing in the humanized mouse model at a relatively large scale without any initial tissue culture screening may soon be possible.For rodent sporozoites, mouse models provide the most convenient approach to evaluate breakthrough and protection efficacy of LAGAP. However, it was highlighted that data need to be interpreted carefully because mouse models and parasites are intrinsically variable and can show various levels of attenuation depending on the mouse or parasite strains, which could easily create misleading data and safety concerns. For human GAP mutants, HTP approaches using culture systems to down-select genetic mutants is now considered possible. Long-term primary human and NHP hepatocyte cultures have been successfully established. In these cultures, which were originally developed for Regardless of assays or models used for candidate screening or validation, several experimental parameters need to be further fine-tuned or even standardized; for example, the quantification of liver-stage development using quantitative polymerase chain reaction and in vivo imaging, the definition of late liver-stage arrest , and, as culture systems allow, the evaluation of merosome formation or merozoite emergence (breakthrough). Finally, the potency of selected mutants could be evaluated by immunogenicity in relevant animal models or Phase I clinical trials. In conclusion, critical assays or models are available, although further optimization is needed to fully characterize LAGAPs and ensure absence of breakthrough events before testing them in human clinical trials.Given the large genome of malaria parasites, sporozoites possess a large Ag repertoire which changes and increases as liver-stage development progresses and the corresponding parasite biomass increases. As such, an LAGAP can potentially induce a much higher level and better quality of protective immunity leading to better vaccine efficacy as compared with the current EAGAP. To reach this goal, a series of key challenges and opportunities  were ide