Tal1. This study characterizes the behavioral and neurochemical changes caused by the absence of Tal1 function. The Tal1cko mutant mice are hyperactive, impulsive, hypersensitive to reward, have learning deficits and a habituation defect in a novel environment. Only minor changes in their dopaminergic system were detected. Amphetamine induced striatal dopamine release and amphetamine induced place preference were normal in Tal1cko mice. Increased dopamine signaling failed to stimulate the locomotor activity of the Tal1cko mice, but instead alleviated their hyperactivity. Altogether, the Tal1cko mice recapitulate many features of the attention and hyperactivity disorders, suggesting a role for Tal1 regulated developmental pathways and neural structures in the control of motivation and movement.The neural circuits regulating motivation and movement include midbrain dopaminergic neurons and associated inhibitory GABAergic and excitatory glutamatergic neurons in the anterior brainstem. Differentiation of specific subtypes of GABAergic and glutamatergic neurons in the mouse embryonic brainstem is controlled by a transcription factor  Altered dopaminergic neurotransmission has also been implicated as one of the mechanisms of hyperactivity disorders, including attention-deficit hyperactivity disorder (ADHD)2. Furthermore, the symptoms of ADHD are alleviated by drugs that evoke dopamine release, such as amphetamine.Brain functions behind movement, motivated behavior, attention, impulse control, and learning are regulated by neurons in the anterior brainstem. Extensive research has shown the roles of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and in the ventral tegmental area (VTA) in these behaviors4. Importantly, the inhibitory GABAergic and excitatory glutamatergic neurons in these nuclei not only regulate the dopaminergic neurons but also project to other brain structures. For example, the VTA GABAergic neuron projections to the prefrontal cortex (PFC) and the nucleus accumbens (NAc) are important for arousal and assessment of reward6, the SNpr GABAergic neuron projections to the superior colliculus and the thalamus provide the main output of the basal ganglia network regulating voluntary movement7, and the RMTg regulates both the dopaminergic and serotonergic systems, which have also been implicated in regulation of impulsivity8.Dopamine neurons receive both excitatory and inhibitory input from diverse brain regions. These regulatory neurons include GABAergic and glutamatergic neurons in the dopaminergic nuclei or in their vicinity, in particular in the substantia nigra pars reticulata (SNpr), ventral tegmental area (VTA), rostromedial tegmental nucleus (RMTg), and laterodorsal tegmental nucleus (LDTg)10. These neurons are born in a specific domain of the ventral rhombomere 1 of the hindbrain, where their differentiation depends on a transcription factor Tal110. In this region of the brain, Tal1 functions as a cell fate selector gene promoting GABAergic differentiation at the expense of the alternative glutamatergic neuron identities10. In conditional Tal1 mutant mice, where the Tal1 gene is inactivated in the midbrain and rhombomere 1 , all GABAergic neurons in the RMTg, VTA and most of the neurons in the posterior part of the SNpr fail to develop. In contrast to the posterior SNpr, the anterior SNpr, not derived from the rhombomere 1, as well as other midbrain GABAergic neurons are intact in the Tal1cko mice. In addition to the GABAergic neurons associated with the dopaminergic nuclei in the ventral midbrain, the Tal1cko mice lack specific brainstem GABAergic neuron subgroups, including the neurons of the ventral tegmental nucleus (VTg)11,12. Interestingly, the VTg was recently implicated in regulation of diencephalic medial mammillary bodies and the memory function in rats14. On the contrary to GABAergic neurons, in the Tal1cko mice, the number of glutamatergic neurons is increased in the interpeduncular nucleus and the LDTg, which provides excitatory projections to the VTA15.Most of the GABAergic neurons in the midbrain dopaminergic nuclei share developmental origins and regulatory mechanisms, which differ from the rest of the midbrain GABAergic neuronsTal1cko mice affects behavior and the function of the dopaminergic system. In these mice, we observed many behavioral features resembling clinical ADHD, including hyperactivity, increased motor impulsivity, altered response to reward, and impaired learning. Interestingly, the Tal1cko mice had no marked changes in their dopaminergic system but, similar to the ADHD patients, showed a paradoxical calming response to pharmacologically stimulated dopamine release.As the brainstem nuclei housing Tal1 dependent neurons have been implicated in the control of the dopamine neurons and in the regulation of movement, motivated behavior and learning, we studied how the altered development of the Methods are described briefly below, see En1Cre16 and Tal1flox11 alleles were crossed to generate En1Cre/+; Tal1flox/flox  mice. In the Tal1cko mice, the En1Cre allele drives recombination tissue-specifically both in the midbrain and rhombomere 1, but this results in a failure of brainstem GABAergic neurogenesis only in the embryonic rhombomere 118. Four cohorts of wild-type  and Tal1cko  mice were generated in outbred ICR background for behavioral analyses. The littermates of the Tal1cko mice, carrying different combinations of the Tal1flox and En1Cre alleles, were included in the wild-type group. The mice were analyzed at the age of 3–4 months. The body weight of the Tal1cko mice is lower than that of wild-type mice . Behavioral testing was approved by the National Animal Experiment Board of Finland  according to the EU legislation (Directive 2010/63/EU) harmonized with Finnish legislation.Mice carrying The following drugs were used: amphetamine (GlaxoSmithKline), GBR12909 dihydrochloride (Tocris), atomoxetine , SCH23390 hydrochloride (Sigma), and raclopride tartrate (Sigma) were dissolved in sterile saline. All drug solutions were injected at the volume of 10 ml/kg.Following behavioral analyses were performed: Open field, Elevated zero maze, Light-dark box, Social approach, Novel object recognition, 3-compartment test for sociability, Rotarod, Multiple static rods, Hot-plate, Pre-pulse inhibition, Forced swim test, Fear conditioning, T-maze, Water maze, Circadian activity, Nest construction, Burrowing, Marble burying test, Grooming, Stress-induced hyperthermia, IntelliCage for flexible sequencing task, motor impulsivity, saccharin preference and delay discounting. For the order of the behavioral tests and used cohorts of animals, see Tal1cko mice in an unbiased and counterbalanced manner as described19 with modifications20.Amphetamine-conditioned place preference was used to assess the rewarding properties of amphetamine in wild-type and 10.To analyze DA neuron number, the mouse brains were processed as previously described21. In addition, prefrontal cortex (PFC) was collected from in front of the striatal slice after removal of the olfactory bulbs. Dopamine and its metabolites were analyzed as described previously22 using HPLC with electrochemical detection.For brain monoamine measurements the samples were obtained from the relevant dissected brain regions using a mouse brain matrix as previously described21. Amphetamine  was added to the recording solution after a stable baseline of stimulated dopamine transients was reached, and the responses were recorded until the amphetamine-induced dopamine efflux through dopamine transporter was measured.Acute striatal mouse brain slices were prepared, dopamine elicited from axon terminals, and transient dopamine signals (release and uptake) were detected using fast-scan cyclic voltammetry as previously described21.Mice were surgically implanted with indwelling guide cannuli targeted into the NAc or dorsal striatum. After a recovery period, amphetamine was injected at 3 mg/kg, i.p., and thereafter dialysate was collected for dopamine measurement by HPLC with electrochemical detectionhttps://www.graphpad.com/quickcalcs/randMenu/). Blinding was applied at all steps during testing and analysis—experimenter was unaware of genotypes and treatments, the group codes were opened after collecting the data and running the analysis. Exclusion criteria for behavioral data were either sickness of the animal, verified measurement error, or technical failure. No data were excluded. Behavioral data were evaluated using an ANOVA model with genotype  and sex as between subject factors. Within subject factors were added as needed when exploring the dependence of genotype effects on place or time . Significant interactions and where necessary significant main effects were further explored by post-hoc tests or by splitting the ANOVA model, as appropriate. The data are presented as mean +/− standard error of the mean, and n indicates the number of biological replicates. Data between sexes were pooled for figures unless otherwise stated.The sample size was based on earlier experience on behavioral and other testing procedures. The experimental animals were subjected to the experimental groups in a random manner. Randomization was applied for the testing order in conventional tests  suggesting reduced depressive behavior. However, it is also possible that the reduced immobility of Tal1cko mice is simply caused by their hyperactivity. We observed unaltered behavior of the Tal1cko mice in tests of stress-induced hyperthermia and social behavior  suggested a hyperactivity phenotype, but the analysis was complicated by defects in locomotion, circling behavior and premature lethality11. Our approach to use the Tal1cko mice allowed a detailed behavioral characterization of mice, in which the neuronal defects due to Tal1 deficiency are restricted to specific regions in the anterior brainstem. The behavioral changes in the Tal1cko mice include hyperactivity, enhanced motor impulsivity, altered reward processing, deficits in learning, behavioral flexibility and task completion—all cardinal features of ADHD.An earlier study of mice with a global neuronal inactivation of the Tal1cko mice display a specific hyperactive phenotype comprising initial normoactivity in a novel environment that rapidly develops into hyperactivity23. The hyperactive phenotype is present already in a juvenile age, and persists into adulthood. Remarkably, the Tal1cko mice exhibit normal spontaneous locomotor activity during the light period of their circadian cycle, consistent with normal night-time activity by actigraphy observation in patients with ADHD24. This suggests that the observed hyperactivity in Tal1cko mice is not generalized, but instead shows specificity in tasks that require exploration and attention. Interestingly, different forms of impaired habituation have been reported in ADHD patients26 in addition to some other psychiatric disorders such as schizophrenia27. The prepulse inhibition of the startle response, used to measure defective sensorimotor gating in schizophrenia28, appears normal in ADHD patients29 (but see29 and30), and also was unchanged in the Tal1cko mice. In the analyses of habituation of the Tal1cko mice, we were able to distinguish deficiencies both in intra-session and inter-session habituation, which may provide means for refinement of the habituation deficit as an endophenotype in ADHD32. As with hyperactivity, initially normal impulsive behavior in ADHD patients manifests as motor impulsiveness as the test session progresses23. This pattern is also phenocopied in the Tal1cko mice. The Tal1cko mice are hypersensitive to rewarding effect of saccharin, which is of interest as ADHD and substance use disorder share comorbidity33. Normal social interaction was observed in the Tal1cko mice, unlike ADHD patients that have been reported to show changes in their social interaction34.The Tal1 dependent GABAergic neurons in the VTA, RMTg and SNpr regulate the adjacent dopaminergic neurons, but also have projections to other brainstem nuclei and more anterior regions of the brain, participating in the control of both motivated behavior and movement. For example, the targets of the VTA GABAergic neurons include the PFC, implicated in the control of impulsive behavior6, and the nucleus accumbens, which has been implicated in reward behavior4. PFC also receives neuromodulatory afferents from the brainstem nuclei which are innervated by the Tal1 dependent GABAergic neurons35. Of these, locus coeruleus noradrenergic and dorsal raphe serotonergic systems have been linked to impulsivity8. The RMTg controls avoidance behavior36, but a lesion or inhibition of the RMTg also results in increased locomotor activity in the rat40. The SNpr GABAergic neurons provide output from the basal ganglia inhibiting the motor nuclei in the thalamus. Therefore, loss of SNpr neurons may contribute to the hyperactivity of the Tal1cko mice. The neurons of the SNpr fall into two broad categories, the anterior SNpr and the posterior SNpr neurons, which differ by their embryonic origins and gene expression10. In the Tal1cko mice, only the posterior-type SNpr neurons are affected. Future studies should address the projection patterns and functions of the distinct SNpr subgroups, including projections to the thalamic nuclei. In addition to GABAergic neurons associated with the dopaminergic nuclei, Tal1 dependent GABAergic neurons are located in the ventral tegmental nucleus of Gudden (VTg)11,12. These neurons project to the medial mammillary bodies, regulate hippocampal theta waves, and are important for memory13. Interestingly, lesions of the VTg in rodents also cause hyperactivity42.Tal1cko mice, glutamatergic neurons in specific brainstem nuclei, including the interpeduncular nucleus, LDTg and SNpc, are increased in their numbers10. In contrast to the inhibitory RMTg projections, the LDTg sends excitatory glutamatergic projection to the VTA to regulate reward behavior15. The increased numbers of the glutamatergic neurons may enhance excitatory drive to their target structures. However, although changes in excitation-inhibition balance have been associated with hyperactivity and ADHD46, it remains unclear how the imbalance in the brainstem GABAergic and glutamatergic neuron differentiation is reflected in the synaptic control of the multiple target cell types of the GABAergic and glutamatergic brainstem neurons in the Tal1cko mice.Whereas brainstem GABAergic nuclei are defective in the 48. Further studies should address if this reduction is due to changes in the local tegmental nuclei, axonal tracts, or both.It is unclear if the development or function of these tegmental nuclei is altered in the human ADHD patients. Very interestingly, however, a reduction in the anterior brainstem size has been shown to distinguish the ADHD patients from the controlsTal1 dependent anterior brainstem neurons are thought to regulate the dopaminergic neurons and basal ganglia circuits, whereas altered basal ganglia activity and dopaminergic signaling has been associated with ADHD. However, the exact roles of these pathways have remained elusive and it has been debated whether ADHD is a hypodopaminergic or hyperdopaminergic defect2.Many of the Tal1cko mice, an increase in the dopamine neuron activity could be predicted. However, our neurochemical analyses of the Tal1cko mice suggest only minor changes in the dopaminergic neurotransmission. Moreover, in the target tissues, the dopamine and dopamine metabolite levels are decreased rather than increased. Our results are consistent with the conclusion that ADHD-like symptoms can correlate with apparently reduced dopaminergic signaling. It is possible that the dampening of dopamine signaling in the Tal1cko mice is due to developmental feedback regulation or functional compensation.Based on the neuroanatomical defects in the 49. Hyperactivity in the Tal1cko mice was rescued by amphetamine treatment, a finding that further supports the putative ADHD-like phenotype. Amphetamine did not induce stereotypy by the moderately low dose used, which is in line with earlier reports51. Striatal dopamine system may be underactive in the ADHD patients, and stimulant medication restores the deficit52. Similarly, amphetamine increases extracellular dopamine in the striatum, and at the behavioral level normalizes the hyperactive state in the Tal1cko mice. The release mechanisms for dopamine also remain functional in the Tal1cko mice both in vitro and in vivo, as shown by voltammetry and microdialysis data, respectively.Stimulant drugs such as amphetamine provide the mainstay of the ADHD pharmacotherapyTal1cko mice, amphetamine may act through increasing both dopamine and noradrenaline tone. Dopaminergic tone can be increased directly by stimulants both in prefrontal and striatal regions52. However, noradrenaline very sparsely innervates striatum, but instead the brainstem noradrenergic neurons send projections to the prefrontal regions53, and consequently prefrontal cortex has been suggested as a target for the atomoxetine effect54. Dopamine receptor antagonists failed to rescue the hyperactivity of the Tal1cko mice, which supports a conclusion that Tal1cko mice have a hypofunctional dopamine system. Taken together, stimulant-sensitive hypodopaminergia of the Tal1cko mice points towards a view of co-existence of an ADHD-like phenotype with attenuated dopamine signaling.The mechanism of action of amphetamine in relieving ADHD symptoms remains elusive, partly because the pathophysiological basis of ADHD has not been confirmed, and partly because amphetamine’s effects are mediated by multiple molecular targets. Our data using more selective pharmacology suggests that, at least in the Tal1cko mice, yet is reward-inducing for both of them, further suggests that the hyperactivity and reward are independent of each other and mediated by distinct circuits. Interestingly, it has been proposed that tonic and phasic dopamine signaling are differentially affected in the ADHD patients55.Our observation that amphetamine can have dramatically different effect on the locomotor activity and its sensitization in the wild-type and 2. Dopamine transporter deficient mouse line, coloboma mutant mice, and spontaneously hypertensive rats show behavioral hyperactivity similar to the Tal1cko mice, but there are also specific differences between these models56. In Tal1cko mice the task-related hyperactivity develops over time, which is similar in spontaneously hypertensive rats, but this is not seen in dopamine transporter deficient mouse line which are highly hyperactive from the beginning of the task. Coloboma mutant mice harbor a selection of neurological and other symptoms, which downplay their utilization as an animal model of ADHD56. The hyperactivity of Tal1cko mice shows sensitivity to stimulants, which is similar to dopamine transporter deficient mouse model and coloboma mouse model58, but spontaneously hypertensive rats have shown variable responses62. Atomoxetine reduces hyperactivity in Tal1cko mice and in spontaneously hypertensive rats, but not in dopamine transporter deficient mouse line64. Differences between the models exist in prepulse inhibition, which is deficient in spontaneously hypertensive rats65 and in the dopamine transporter deficient mouse line66, while Tal1cko mice are normal in this aspect. Social interaction was found to be normal in Tal1cko mice, while dopamine transporter deficient mice show decreased social investigation and increased reactivity in response to social investigation67. In spontaneously hypertensive rats, an unchanged68, reduced68, or increased69 social interaction has been reported. Impulsiveness is a shared phenotypic property in Tal1cko mice and spontaneously hypertensive rats. Both of these animal models have hypodopaminergic neurochemical phenotype70. It is not clear how well any of these animal models satisfy the construct validity of human ADHD. Multiple preclinical models may be needed to fully understand this behavioral disorder.ADHD is likely a heterogeneous deficit with different neurobiological bases. Similarly, clinical symptoms of ADHD have been observed in a variety of gene-modified animal strains, which have been adopted as animal models of ADHD2. Therefore, it is possible that defects in the other components of the basal ganglia circuitry are behind the etiology of this disorder. Although Tal1 gene variants have not been associated with the etiology of ADHD, our results show that a defect in a specific developmental event dependent on a single transcription factor, namely balanced differentiation of GABAergic and glutamatergic neurons in the developing anterior brainstem, leads to several endophenotypes of ADHD. Studies of these neurons, and the circuits they participate in, can lead to better understanding of the normal and abnormal control of activity, attention and reward.ADHD is a highly heritable neurodevelopmental disorder, but its genetic components remain poorly understood. Although work with genetically modified mice has demonstrated the causal relationship between the altered dopaminergic neurotransmission and hyperactivity, the candidate gene studies have failed to reveal a substantial role for variation in genes involved in dopaminergic neurotransmission as the genetic basis for ADHDSupplemental material


The durability of a cement-based material is mainly dependent on its permeability. Modifications of porosity, pore-structure and pore-connectivity could have significant impacts on permeability improvement, which eventually leads to more durable materials. One of the most efficient solutions in this regard is to use permeability reducing admixtures (PRA). Among these admixtures for those structures exposed to hydro-static pressure, crystalline waterproofing admixtures (CWA) have been serving in the construction industries for decades and according to ACI 212—chemical admixtures’ report, it has proven its capability in permeability reduction and durability-enhancement. However, there is substantial research being done on its durability properties at the macro level but very limited information available regarding its microstructural features and chemical characteristics at the micro level. Hence, this paper presents one of the first reported attempts to characterize microstructural and chemical elements of hydration products for cementitious composites with CWA called K, P and X using Scanning Electron Microscopy (SEM). Backscattered SEM images taken from a polished-section of one CWA type—K—admixture were analyzed in ImageJ to obtain paste matrix porosity, indicating a lower value for the CWA-K mixture. X-ray analysis and SEM micrographs of polished sections were examined to identify chemical compositions based on atomic ratio plots and brightness differences in backscatter-SEM images. To detect chemical elements and the nature of formed crystals, the fractured surfaces of three different CWA mixtures were examined. Cementitious composites with K admixture indicated needle-like crystal formation—though different from ettringite; X and P admixtures showed sulfur peaks in Energy Dispersive Spectrum (EDS) spectra, like ettringite. SEM images and X-ray analyses of mixtures incorporating Portland Limestone Cement (PLC) indicated lower-than-average porosity but showed different Si/Ca and Al/Ca atomic ratios. The porous nature of cementitious composites can be one of the main sources of their proneness to degradation. Due to such porous structures, aggressive agents  can be carried by water and penetrate through pores and micro-cracks, which endangers the composite’s microstructure, thereby leading to a reduction in its overall durability. In the aforementioned framework, there is an increasing interest in innovative and sustainable approaches to inherently reduce the permeability of cementitious materials as a promising, direct way to improve its durability at different scales. In real applications for cementitious materials, chemical admixtures are widely used ,2,3,4 to2. An important parameter that influences the effectiveness of the CWA is the curing conditions during the first 28 days of ageing. They also concluded that CWA has an influence on the increase of the amount of portlandite (CH) [2CO3 and organic calcium ions showed better self-healing performance than the mixture incorporating only CWA. Li et al.’s [As a smart construction material, crystalline products have been used for over three decades in the construction industry and their effects on concrete self-healing, durability and shrinkage behaviour have been investigated in some respects. The visual closure of cracks in mortar specimens incorporating fly ash, expansive admixtures, silica fume, CWA and limestone powder under water immersion conditions was investigated by Jaroenratanapirom and Sahamitmongkol ,7. Self-ite (CH) . SEM samite (CH) . SEM obsite (CH)  confirmeite (CH)  that theite (CH)  showed tet al.’s  study deet al.’s  study inTo better understand the influence of innovative materials like CWA in terms of improving durability and strength of concrete, it is essential to first obtain information about CWA’s microstructure which provides useful physical and chemical characteristics of the material. Microstructural examination of such new construction materials can also lead to better implementation and development of these materials for the construction and building industries. CWA has been utilized for (decades) in different arenas of the construction industry; only a few recent studies have been identified that address the effect of CWA in the cement-based materials as a promoter of permeability and self-healing ,34,35,36This paper aims to investigate the role of the addition of different CWA and PLC on the development of microstructure, nature of hydration phases, porosity reduction of paste matrix and morphological properties of cementitious composite. This study has been organized into three different phases. Phase I covers detailed information about paste’s pore network, determined by implementing an image analysis technique on backscattered mode scanning electron microscopy (BSEM) images, taken from polished sections of cementitious composite mixtures containing only one CWA type  and Portland limestone cement (PLC). Phase II, using BSEM micrographs and the energy dispersive X-ray spectroscopy (EDS) technique, microstructural features, the development of pore-blocking crystals and the chemical components of similar cementitious composite mixtures are investigated. This phase was also designed to identify differences in hydration products and mineral formation while CWA was present in the mix. Although hydration products and morphology were successfully identified in Phase II, formation of crystals inside the matrix was not observed, mostly due to damage induced during harsh surface preparation required for conventional polished surface preparation method. Hence, the detrimental effect of surface preparation in Phase II led to finding an alternative surface preparation technique that supports crystal growth as claimed by CWA’s manufacturer. One of the main goals of this study was to demonstrate whether or not CWA form similar microstructures  or share the same chemical hydration products. In order to investigate the nature of pore-blocking crystals and their chemical compositions, Phase III was initiated, which delved into SEM and EDS results taken from fractured surfaces of cementitious composites treated with different CWA types . For all treated CWA mixtures, the observed needle shape crystals were quite similar to ettringite crystals, indicating the need for further analysis of their EDS spectra. Chemical analysis of these crystals showed a high sulfur peak in the EDS graphs for X and P admixtures similar to the ettringite spectra, but this was not the case for K admixture. Ultimately, the last section in this paper draws final conclusions and summarizes the authors findings obtained from all phases. A previous study by the authors  investig2 (X and P admixtures) as described by the American Concrete Institute (ACI) Committee 212 [Generally, a CWA consists of a proprietary mix of active chemicals, implanted in a carrier of cement and sand, reacting with cement compounds (K admixture) or by-products of cement hydration, most likely Ca(OH)ttee 212 . In the ttee 212 , it is sttee 212 . The chettee 212 , Quikretttee 212 ) was usettee 212  requiremttee 212 . In the first phase, polished and flat surfaces of cementitious composite specimens were used to systematically analyze porosity of the treated system using BSEM micrographs and ImageJ software (2018), National Institutes of Health, Bethesda, Maryland, USA. To keep the scope of this project manageable and due to failure in detecting needle-like crystals after harsh surface preparation procedure, only one type of CWA (K admixture) and two cement types (Ordinary Portland Cement (OPC) and Portland Limestone Cement (PLC)) were used in this experiment. A typical dosage (2% weight of cement) was used in this experiment to understand actual behavior of CWA in blocking pores or cracks. 3 cubic molds. Promptly after the casting samples, they were sealed with a plastic sheet to keep almost constant moisture inside for 24 h at laboratory environment (room temperature) during concrete stiffing and strength development. After 24 h of hardening and prior to testing, samples were continuously cured in a water bath at 23 ± 2 °C.The mortar samples were prepared following the procedure reported in ASTM C305 . First, 3) from each mix were cut into thin slices using a diamond saw so that edging is maintained. These 10 mm thick slices were then embedded in low viscosity epoxy resin using vacuum impregnation in a vacuum desiccator. Once the resin was hardened, a few millimeters from the surface were removed using wet-and-dry abrasive sheet to reveal surface to be examined. Later, the specimen surface was ground to a flat surface using a polishing/lapping machine. The time spent on each grinding and polishing stage was about 10–15 min. The flat surface was cleaned in acetone, dried and made ready for carbon coating. After applying carbon-coating by an evaporative coater, the coated sections were ready to be studied by Hitachi S-4800 SEM, Hitachi, Chiyoda City, Tokyo, Japan, with a backscatter electron detector and Energy Dispersive Spectrum (EDS) analysis. The microscope was operated at an accelerating voltage of 15 kV and working distance 15 mm. The EDS system had been calibrated with oxide standards suitable for cement analyses. X-ray spectra and elemental mappings were collected for elements O, Na, Mg, Al, Si, S, K, Ca, Ti, Mn and Fe, which are the typical cementitious materials’ hydration products [To prepare polished sections, three cubes  of various mixtures of cementitious composite  with two cement types (OPC and PLC) were mixed together to make cubic samples  due to the conventional harsh surface preparation procedure, including grinding, lapping and polishing, which can abrade crystals from the surface . Hence, To investigate the morphologies of the hydration products or the shapes of the formed crystals, the third phase in this study included three CWA types, called K, P and X admixtures, as summarized in In Phase III, specimens were broken into the small pieces with fresh fracture surfaces after 56 days of immersion in water in separate containers for further investigations using SEM. The specimens with approximate dimensions of 2 × 2 cm were randomly selected; covered to minimize chance of carbonation (or contamination) of the surface; left at ambient temperature for 7 days; and later placed into a vacuum desiccator to dry. Next, the surfaces of three samples from each mix were coated with carbon to dissipate excess charge from the specimen during imaging.From a BSEM image of a polished section of concrete, an approximate idea of the concrete porosity, degree of cement hydration and cement type used in the mix can be gained all by looking at the image. With less reliability and longer analysis time, this information can be also obtained by SEM images taken from fractured surface of the specimen. In backscatter mode when the surface is smooth, electrons are collected at a very high angle close to 180°) and bounced back from a sample in a straight line, making it less sensitive to small topographic features. In contrast, secondary electrons are collected at a low angle in order to enhance topographic features. BSE detector is for collecting scattered primary electrons and SE detector is for collecting the electrons “jumping” out samples. Back scattered electrons have high energy and can deliver a high contrast of different phases. The path length of X-rays generated at depth, which is dependent on the X-ray take-off angle, plays an important role in X-ray travelling time and eventually element peaks in the EDS spectrum . Figure 0° and boTo study morphological aspects and hydration products of polished cementitious composite sections, backscatter mode of SEM along with X-ray detector was used. Fracture surfaces from specimen are typically useful to study morphology of the hydration products or the shapes of the clinker mineral crystals while polished sections are generally much more beneficial in evaluating concrete defects . Figure Atomic ratio plot is one of the methods to analyze the EDS data and understand more about the hydrated compositions . To examIn this phase, SEM analysis was carried out on fragments collected from cementitious composite specimens treated with three CWA admixtures in order to obtain useful morphological information and identify formation of any pore-blocking crystals. SEM micrographs of both admixture-treated and control samples, taken from different random locations on the specimen surface is illustrated in Although differences in nature of these crystals’ and their geometry/morphology might not be obvious from SEM micrographs in 2O3∙3CaSO4∙32(H2O), and is composed of 19.16% Ca, 4.3% Al, 7.66% S and 63% O in atomic percentahes [Based on the EDS analysis of needle shaped crystals, the main difference is the sulphur peak. Both P and X have shown signs of sulphur content b,c, but centahes . To diffFrom the preliminary analysis, it appears that the composition of the “needle-like” crystals in the CWA specimens with K admixture are different from ettringite due to low peak of sulfur (S) in the EDS graph which is one of the major elements in the ettringite chemical composition. Lower peak of an element in the EDS plot indicates less availability of that element in the matrix where in this case shows that sulfur amount is very low. Hence, needle-shaped crystals observed in the K mix are not ettringite. The EDS spectrum of CWA samples in  study waThe results obtained from the EDS spectra for X and P admixtures indicated that not all admixtures can possibly develop needle-like crystals—acting as a physical membrane—which have different compositions from ettringite. Ettringite’s X-ray spectrum typically has the strongest peaks due to calcium, sulphur and aluminium according to the spectrum plotted in Winter’s book . In admiSimilar procedures, described for This paper aimed to draw some insights about the microstructural features and durability improvement of cementitious composites modified by adding three different crystalline waterproofing admixtures (CWA) called K, P and X. These CWA types were examined by SEM to confirm previous durability/permeability findings and understand their effects on modifying microstructure, forming pore blocking crystals and hydration phases. Using backscatter-mode SEM images and EDS spectra, examination of polished cementitious composite sections with and without CWA was conducted for the first time. The results showed typical hydration phases, including CSH and CH with different Si/Ca and Al/Ca forming in the control system. Excepting crystal formations in the matrix, CWA-added mixtures indicated noticeably similar morphologies to those without an admixture and showed similar hydration phases. Image processing using ImageJ software indicated a decrease in total average porosity of K treated cementitious composites (about 15% lower) compared tp those with only OPC or PLC cement. Inclusion of PLC in the mix resulted in a slight decrease (around 5%) in average porosity as compared to OPC, while adding CWA into PLC mix led to a ~7% decrease when compared with control PLC mix. It should be noted that the calculated porosity reduction (~15%) in the treated samples is referred to cementitious composite specimens, while in the case of concrete, as previously studied by the authors  showed aObservation of crystals using conventional SEM surface preparation is not practical due to possible mechanical damage that occurs through the polishing process. Due to the limitation of conventional SEM surface preparation, the fractured surfaces of cementitious composite specimens were analyzed to investigate the shapes and chemistries of crystals that were generated as a result of adding CWA admixtures. The SEM micrographs taken from specimens’ fractured surfaces showed formation of pore-blocking crystals for all treated mixtures, while similar spots in untreated sections did not indicate such crystal growth. Although needle-shaped crystals were observed in the treated cementitious composite specimens showing similar shape, the chemical analysis of these crystals proves that they have different chemistry and chemical elements. For both P and X treated samples, a peak of sulphur has been observed, which is a sign of ettringite presence in the hydration products, but K sample does not show sulphur peak on EDS spectrum (no sign of ettringite). This study successfully sheds light on the microstructural development  of cementitious system treated with crystalline waterproofing technology.


The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing all around the world and it may become the primary cause of terminal liver disease in adults and children in the next few decades. However, the pathogenesis of NAFLD is complex, and the Food and Drug Administration (FDA) has not approved any drugs for its treatment. Kupffer cells are the key cells regulating immunity in the liver, and the effect of their unique polarization on NAFLD has received increasing attention. Kupffer cells mainly reside in the lumen of hepatic sinusoids and account for 80% to 90% of colonized macrophages in the human body. They are phagocytic cells with the capacity for self-renewal that rarely migrate from their niche in the liver, and play a crucial role in regulating and maintaining homeostasis. Upon liver damage, Kupffer cells will be activated, releasing a good deal of inflammatory cytokines and chemokines. This review summarizes the multiple roles of Kupffer cells in the pathogenesis of NAFLD, the role of infiltrating macrophages in the pathogenesis of NAFLD is also briefly discussed, and aims to provide a theoretical basis for designing an NAFLD treatment strategy with Kupffer cells as the therapeutic target. Non-alcoholic fatty liver disease (NAFLD) is a public health problem worldwide, and 25% to 30% of the global population suffers from different degrees of it Figure . Moreover, NAFLD is also a systemic disease related to obesity, type 2 diabetes mellitus (T2DM), insulin resistance (IR), cardiovascular disease (CVD), and metabolic syndrome (MetS) As the name implies, NAFLD occurs without excessive alcohol consumption, which is defined using the widely used high alcohol intake thresholds of 20 g/day for women and 30 g/day for men In the past 20 years, numerous studies of basic pathology and clinical trials have been conducted worldwide to better understand and prevent the development of NAFLD, but the Food and Drug Administration (FDA) has been approved no drugs. Currently, the general therapeutic strategies for NAFLD are as follows: (1) implementation of lifestyle interventions, including exercise and a proper diet; (2) active treatment of MetS to improve IR, weight loss, blood pressure and dyslipidemia; (3) treatment of liver injury to inhibit oxidative stress and improve liver fibrosis; and (4) perform liver transplantation in patients with advanced end-stage liver disease. Considering the increasing clinical burden and poor quality of life of patients with NAFLD, studies aiming to identify effective strategies that slow the increase in the number of NAFLD cases and improve the therapeutic options are urgently needed.Hepatic macrophages mainly consist of two types: Kupffer cells, which originate from yolk sac-derived red bone marrow progenitor cells and reside in the liver, and monocyte-derived macrophages, which are derived from bone marrow hematopoietic stem cells and transplanted through the blood circulation to the liver In addition to the above-mentioned hepatic macrophages, other macrophages also play more and more roles in the pathogenesis of NAFLD. For example, adipose tissue macrophages (ATM) promote the recruitment of blood mononuclear macrophages to the liver during the occurrence of NAFLD, and the activation of adipocyte death-related macrophage may promote liver injury by stimulating neutrophil recruitment, thereby exacerbating lipolysis-mediated lipotoxicity and liver injury + monocyte-derived macrophages replacing the resident Kupffer cells as the dominant macrophage population Kupffer cells are the resident liver macrophages and account for 80-90% of all macrophages in the body. They are the largest component of the reticular endothelial system Historically, macrophages have been considered to undergo phenotypic differentiation in response to different microenvironmental signals, and the phenotypes have been traditionally oversimplified as classically activated macrophages (M1) and alternatively activated macrophages (M2) Figure 2). Through in vivo and in vitro experiments, J. Wan and colleagues showed that the selective induction of M1 Kupffer cells apoptosis enabled M2-polarized Kupffer cells to protect against alcoholic liver injury by neutralizing the remaining M1 Kupffer cells; these researchers concluded that limiting the number of M1 Kupffer cells while promoting M2 Kupffer cells polarization may be a valuable therapeutic strategy for ALD In individuals with alcoholic liver disease (ALD) and NAFLD, the balance between classic pro-inflammatory M1 Kupffer cells and selectively activated anti-inflammatory M2 Kupffer cells is crucial to strictly regulate the occurrence and development of liver injury Kupffer cells, with their high heterogeneity and plasticity, are excellent at maintaining homeostasis and defense functions. In the body, factors such as lipids, lipids metabolites, and LPS, activate Kupffer cells. Once activated, Kupffer cells increase the expression of inflammatory cytokines, exacerbate the degree of necrotic inflammation among liver cells, and alter the expression of genes associated with fibrosis and oxidative damage, leading to the development of NAFLD. The role of Kupffer cells in NAFLD, as elucidated in recent years, is summarized in four pathological processes, including inflammation, steatosis, oxidative stress, and fibrosis, which are discussed to provide a theoretical basis for designing treatment strategies for NAFLD with Kupffer cells as the therapeutic target.The intestinal barrier acts as the body's first line of defense to protect against damage caused by bacteria, food antigens, toxins and other factors in the intestinal cavity LPS binds to CD14 on the surface of Kupffer cells through the TLR4 signaling pathway, which subsequently recruits T lymphocytes, B lymphocytes and other leukocytes, resulting in the release of inflammatory cytokines and chemokines, such as TNF-α, IL-1β, IL-6, IL-12 and interferon-γ (IFN-γ) Based on accumulating evidence, the liver is exposed to various microbial molecules produced in the digestive tract through the local circulation and can be infected by various hepadnaviruses, leading to the activation of inflammatory bodies and pyroptosis, which mediates the inflammatory defense mechanism against pathogens. During the pathogenesis of NASH, the levels of NLRP3 inflammatory bodies in the intestine are reduced, which weakens the intestinal barrier function against bacterial translocation, allowing a large amount of LPS to enter the liver and exacerbating the development of NASH Figure 3). As shown in the study by Shi and colleagues, caspase-11 exposes the N-terminal fragment of gasdermin D (GSDMD), which triggers an inflammatory cell death response called pyroptosis in response to cytoplasmic LPS that is essential for endotoxic shock in mice One study revealed a new mechanism by which macrophages are activated by cytoplasmic LPS via a TLR4-independent pathway, thereby activating the inflammatory enzyme caspase-11 The liver has a high capacity for fatty acid uptake and plays a significant role in the metabolism of lipids and lipoproteins + macrophages and promoting lipid synthesis, which increases the transcription of activating protein 1 (AP-1) and pro-inflammatory cytokines As an early stage of NAFLD, hepatic steatosis occurs when an imbalance exists between the inward and outward fluxes of free fatty acids (FFAs) in the liver and is defined as triglyceride accumulation in more than 5% of hepatocytes Interestingly, increasing experimental and clinical evidence suggests an association between the pathogenesis of NASH and changes in intrahepatic cholesterol homeostasis and the accumulation of free cholesterol A common feature of chronic liver disease is oxidative stress, which affects the development of NASH Oxidases in different cellular compartments, such as mitochondrial, NADPH oxidase and cytoplasmic xanthine oxidase, inhibit ROS production and damage the antigenicity of Kupffer cells to a similar extent During steatohepatitis, activated Kupffer cells secrete a large number of pro-inflammatory cytokines, such as TNF-α and IL-6, to recruit T lymphocytes, natural killer T (NKT) cells and other inflammatory cells, indirectly participating in inflammation and the immune response Table 1). During the inflammatory response, LPS binds to CD14 on the membrane and signals through the TLR4 pathway to activate Kupffer cells and induce the production of various inflammatory factors; LPS also directly binds to Kupffer cells and activates the inflammatory enzyme caspase-11, causing pyroptosis and the release of inflammatory factors, highlighting the multiple roles of this molecule. During steatosis, FFAs mediate Kupffer cells activation and increased MCP-1 expression to promote lipid accumulation. During oxidative stress, Kupffer cell activation and ROS production form a positive feedback loop. In the process of fibrosis, Kupffer cells produce cytokines such as TGF-β1 and IL-6 to promote fibrosis.The above description clearly highlights the multiple functions and various regulatory activities of Kupffer cells in the proregression of NAFLD and their important roles in pathological processes .Kupffer cells are phagocytes with the capacity for self-renewal that rarely migrate from their niche and targeting Kupffer cells for the treatment of NAFLD has great significance. In an experimental NASH model established in mice, Rivera et al., found that disrupting Kupffer cells reduces histological manifestations in the liver, such as steatosis, inflammation, and necrosis Kupffer cells activation: As mentioned above, activated Kupffer cells show increased expression of inflammatory cytokines and thereby exacerbate the degree of necrotic inflammation of liver cells, leading to the occurrence of NAFLD. The inhibition of Kupffer cell overactivation may reverse the course of this disease. Benzyl isothiocyanate (BITC) is an organic sulfur compound that is abundant in cruciferous vegetables. Recently, Chen et al. showed that BITC ameliorates the effects of a high-fat/high-cholesterol diet by inhibiting cholesterol crystal-activated NLRP3 inflammasomes in Kupffer cells, thereby preventing the development of diet-induced NASH M1 and M2 Kupffer cells polarization: Kupffer cells are the key cells regulating hepatic immune responses, and their specific polarization has received increasing attention in research on NAFLD. In one experiment of mice fed the antioxidant β-cryptoxanthin, the total population of hepatic macrophages and T cells was reduced, but the population of M2 macrophages was increased. β-Cryptoxanthin may at least partially prevent and reverse IR and steatohepatitis by activating M2 macrophages/Kupffer cells In the past few years, our basic understanding of Kupffer cells has substantially improved Kupffer cells are a “double-edged sword”; they promote inflammation that leads to liver cell damage and activate hepatic stellate cells, causing fibrosis, angiogenesis and tumor development, but they also stimulate fibrosis regression and limit disease progression. Important issues to consider in future research include controlling the ratio of M1 to M2 Kupffer cells and therapeutically interfering with the M2 polarization of Kupffer cells. Although pyroptosis has been shown to be closely related to the progression of liver disease, many details remain unknown, and further research is required. For example, researchers have not clearly determined how pyroptosis causes liver damage and affects disease progression. Currently, more systematic research is needed to clarify the function of Kupffer cells in NAFLD and resolve the controversy. These studies will not only facilitate the further elaboration of NAFLD pathogenesis but also provide new research ideas and targets for NAFLD in the near future.In recent years, research on the use of natural drugs in the treatment of NAFLD has expanded. Some herbs also have potential value as treatment for NAFLD. According to modern pharmacological studies, Salvia miltiorrhiza promotes blood circulation, improves the microcirculation, inhibits collagen fiber hyperplasia, and exerts anti-fibrotic, anti-free radical and anti-peroxidation activities In general, the therapeutic effects of certain drugs mentioned above on patients with NAFLD are controversial or unclear. The prevention and treatment of NAFLD, a metabolic disorder caused by an unhealthy lifestyle, must not rely solely on medication. A reasonable recommendation is to improve detrimental lifestyles and poor eating habits, increase aerobic exercise, and control body weight. The combination of lifestyle interventions and drug therapy is the basis for the treatment of NAFLD, and the efficacy of therapeutic drugs will depend on the success of lifestyle interventions.


OFD1 and associated with cilia dysfunction, display excessive autophagy and we demonstrated that autophagy inhibition significantly ameliorates the renal cystic phenotype in a conditional mouse model recapitulating the features of the disease . We speculate that abnormal autophagy may underlie some of the clinical manifestations observed in the disorders ascribed to cilia dysfunction.The autophagy-lysosomal pathway is one of the main degradative routes which cells use to balance sources of energy. A number of proteins orchestrate the formation of autophagosomes, membranous organelles instrumental in autophagy. Selective autophagy, involving the recognition and removal of specific targets, is mediated by autophagy receptors, which recognize cargos and the autophagosomal membrane protein LC3 for lysosomal degradation. Recently, bidirectional crosstalk has emerged between autophagy and primary cilia, microtubule-based sensory organelles extending from cells and anchored by the basal body, derived from the mother centriole of the centrosome. The molecular mechanisms underlying the direct role of autophagic proteins in cilia biology and, conversely, the impact of this organelle in autophagy remains elusive. Recently, we uncovered the molecular mechanism by which the centrosomal/basal body protein OFD1 controls the LC3-mediated autophagic cascade. In particular, we demonstrated that OFD1 acts as a selective autophagy receptor by regulating the turnover of unc-51-like kinase (ULK1) complex, which plays a crucial role in the initiation steps of autophagosome biogenesis. Moreover, we showed that patients with a genetic condition caused by mutations in  Autophagy is an evolutionary conserved and highly regulated cellular degradation pathway which entails the engulfment of cellular components into double-membrane vesicles, called autophagosomes, which then fuse with lysosomes to degrade their content and recycle nutrients back into the cytoplasm. Autophagy is active at basal levels in most cell types where it plays a housekeeping role in maintaining the integrity of intracellular organelles and proteins. It is also, however, strongly induced by nutrient depletion to promote survival until nutrients become available again. Bulk autophagy is non-selective, while selective autophagy involves the recognition and removal of specific targets through autophagy receptors. Autophagy receptors link their cargo to autophagosomes through LC3B-interacting regions (LIR) that mediate binding to autophagosomal membrane proteins LC3 and/or GABARAP, thus enabling cargo sequestration and degradation together with their own receptors.Autophagy, both basal and selective, plays a variety of roles in physiological and pathological conditions. Both insufficient and excessive levels of autophagy can have detrimental effects. Indeed, this process has a protective effect in neurodegeneration and lysosomal storage disorders; conversely, excessive activation of autophagy can contribute to cancer.Recent studies have shown that primary cilia and ciliary signaling pathways control autophagy and that, conversely, autophagy is one of the main players in the regulation of ciliogenesis. Primary cilia are microtubule-based organelles protruding from the cell surface of mammalian cells and display sensory functions. In post-mitotic cells, the centrosome, composed of two centrioles and of centriolar satellites, moves to the apical cell surface where the mother centriole docks with the plasma membrane and subtends axonemal microtubules of primary cilia. Centrioles participating in ciliogenesis and located under the ciliary axoneme are called basal bodies. Ciliary dysfunction has been implicated in disorders called “ciliopathies” which present overlapping phenotypes, such as primary ciliary dyskinesia, retinal degeneration, cystic disease, skeletal defects, situs inversus, obesity, mental retardation, and central nervous system (CNS) malformations.Pampliega and colleagues described that both mature autophagosome markers and autophagy proteins implicated in the initial steps of autophagosome formation localize to cilia or periciliary regions. The authors proposed that the ciliary membrane could act as a nucleation site for pre-autophagosome formation. Moreover, their report and others showed that autophagy activation requires the presence of a functional primary cilium. On the other hand, it has been shown that autophagy influences ciliogenesis in a cellular context dependent manner. In particular, selective autophagic degradation of the satellite pool of OFD1 that physiologically acts as a cilia inhibitor, promotes ciliogenesis. OFD1 is a centrosomal/basal body protein whose centriolar fraction is instead required for primary cilia formation. However, different laboratories have reported contrasting results, suggesting that the role played by this degradative pathway in ciliogenesis might be cell context dependent.The impact of this sensing organelle in autophagy and conversely, the molecular mechanisms underlying the direct role of core autophagic proteins in cilia biology, remains elusive.Figure 1). We found that OFD1 controls the turnover of the unc-51-like kinase (ULK1) complex, which plays a crucial role in the initiation steps of autophagosome biogenesis and comprises the protein kinase ULK1 and the regulatory components ATG13, FIP200 and ATG101. In particular we demonstrated that OFD1 interacts at centrosomes with LC3B and GABARAP-L1 through a classical LIR motif and with ATG13 thus promoting its LIR-mediated autophagic degradation. We demonstrated through pharmacological and genetic approaches that ATG13 is an autophagy target, and that ATG13 molecules are less abundant in the lumen of swollen lysosomes in human kidney knock out (KO)-OFD1 cells compared to controls. In addition, we confirmed that OFD1 is degraded through autophagy and demonstrated that the autophagy-mediated degradation of OFD1 is dependent on its LIR. Altogether, our data demonstrate that OFD1 is a novel autophagy receptor, which selectively promotes the autophagic degradation of ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. These observations raise the question of the physiological role of OFD1 as an autophagy receptor. We tested the ULK1 kinase activity in OFD1 depleted cells and observed that the ULK1 complex enhanced protein stability was associated with increased ULK1 kinase activity. Consistent with this model, analysis of autophagy confirmed that loss of OFD1 increased the number of autophagosome vesicles. We also showed, by monitoring lysosome-mediated autophagosome clearance and the levels of the autophagic substrate p62 that OFD1 depletion enhances overall autophagic flux. Furthermore, we verified that the increased autophagy observed in KO-OFD1 cells is independent of the mammalian target of rapamycin kinase pathway, the main negative regulator of autophagy. Conversely, OFD1 overexpression resulted in reduced autophagosomes biogenesis, which was dependent on OFD1-LC3/GABARAP binding through LIR, confirming its role in selective autophagy-mediated inhibition of autophagosome biogenesis.In our recent study we revealed the molecular mechanisms underlying the direct functional role of the centrosomal/basal body protein OFD1, in LC3-mediated autophagy control, and the far-reaching implications for ciliopathies. We demonstrated that OFD1 is a novel selective autophagy receptor which controls the early phases of the autophagic cascade (OFD1 cells that cannot form primary cilia reproduced the same autophagic phenotype in conditions promoting ciliogenesis (i.e. mitotic quiescent and confluent cells), and in those not allowing cilia formation (i.e. cycling and subconfluent cells). This observation allows us to speculate that the OFD1 protein could be regarded as a novel noncanonical autophagic player controlling autophagy, independently from its role in cilia biology. On the other hand, we cannot exclude that the autophagy receptor OFD1 could selectively recognize other cargoes besides ATG13. We speculate that OFD1 could act as a novel autophagic receptor for ciliary proteins exerting either a positive or negative effect on ciliogenesis and mediate their selective degradation to promote or inhibit ciliogenesis in a cell context dependent fashion. Recent evidence reporting that different cilia components such as IFT88, ARL13, centrin 1, pericentrin, PCM1 and CEP131 are degraded through autophagy, support this hypothesis. OFD1 could be one of the players coordinating the deep and intricate bidirectional crosstalk between autophagy and cilia that could explain the need for the cell to compartmentalize autophagic and ciliary proteins to the same cellular area.In addition, we also demonstrated that OFD1 controls autophagy independently from cilia: KO-However, the mechanisms underlying OFD1-mediated selective autophagic degradation of the effectors of ciliogenesis are yet to be determined and could involve more autophagy receptors. To validate this hypothesis, studies exploring the interaction of OFD1 with LC3 and other OFD1-interacting proteins are required to identify and characterize the potential substrate/s of the autophagy pathway that use/s OFD1 as receptor. Experimental evidence which shows that different cilia components, such as centriolar satellites proteins PCM1 and CEP131, are autophagic substrates and physically interact with LC3 and GABARAP support this hypothesis.OFD1 gene are responsible for Oral-Facial-Digital type I syndrome , a dominant male lethal X-linked ciliopathy characterized by the presence of renal cystic disease in 50% of cases. We showed that patients with OFD type I syndrome display excessive autophagy and that genetic inhibition of autophagy in a mouse model recapitulating the features of the disease significantly ameliorates the renal phenotype, implicating excessive autophagy in the pathogenesis of renal cysts. Nevertheless, we cannot exclude that abnormal autophagy could have a role not only in cystic kidney but also in other clinical manifestations, such as skeletal abnormalities and CNS involvement, and that ciliopathies should be considered autophagic diseases. This consideration implies that other genes encoding centrosomal/basal body and ciliary proteins could have a role in autophagy and that their mutations could be associated with abnormalities of autophagic flux. Further studies on the characterization of autophagy in other ciliopathy models will be necessary to prove if abnormal autophagy could underlie some of the clinical manifestations observed in disorders ascribed to cilia dysfunction. It is tempting to speculate that abnormal function of centrosomes/centriolar satellites/basal body/ciliary proteins could be associated with autophagy-related disorders such as cancer, neurodegenerative, muscle, and liver diseases.Mutations in the Further understanding of the molecular mechanisms underlying the functional interactions between OFD1 and autophagy will be of utmost importance not only from a basic science point of view but also for the possible therapeutic implications for ciliopathies and other conditions.


Cancer cells in solid tumors often experience lack of oxygen (hypoxia), which they overcome with the help of hypoxia inducible transcription factors (HIFs). When HIFs are activated, they stimulate the expression of many genes and cause the production of proteins that help cancer cells grow and migrate even in the presence of very little oxygen. Many experiments have shown that agents that block the activity of HIFs (HIF inhibitors) can prevent growth of cancer cells under hypoxia and, subsequently, hinder formation of malignant tumors or metastases. Most small chemical HIF inhibitors lack the selectivity required for development of safe anticancer drugs. On the other hand, peptides derived from HIFs themselves can be very selective HIF inhibitors by disrupting specific associations of HIFs with cellular components that are essential for HIF activation. This review discusses the nature of available peptide HIF inhibitors and their prospects as effective pharmaceuticals against cancer.Reduced oxygen availability (hypoxia) is a characteristic of many disorders including cancer. Central components of the systemic and cellular response to hypoxia are the Hypoxia Inducible Factors (HIFs), a small family of heterodimeric transcription factors that directly or indirectly regulate the expression of hundreds of genes, the products of which mediate adaptive changes in processes that include metabolism, erythropoiesis, and angiogenesis. The overexpression of HIFs has been linked to the pathogenesis and progression of cancer. Moreover, evidence from cellular and animal models have convincingly shown that targeting HIFs represents a valid approach to treat hypoxia-related disorders. However, targeting transcription factors with small molecules is a very demanding task and development of HIF inhibitors with specificity and therapeutic potential has largely remained an unattainable challenge. Another promising approach to inhibit HIFs is to use peptides modelled after HIF subunit domains known to be involved in protein–protein interactions that are critical for HIF function. Introduction of these peptides into cells can inhibit, through competition, the activity of endogenous HIFs in a sequence and, therefore also isoform, specific manner. This review summarizes the involvement of HIFs in cancer and the approaches for targeting them, with a special focus on the development of peptide HIF inhibitors and their prospects as highly-specific pharmacological agents. Oxygen is indispensable for cellular metabolism, as it is essential for aerobic respiration and energy production. Moreover, oxygen has a vital role as a substrate in many enzymatic reactions that regulate diverse biological processes. Mammals use oxygen sensing and delivery mechanisms to match available oxygen to their tissue demands so as to fulfill their metabolic needs but also prevent toxicity caused by excess oxygen . Lack ofAt the cellular level, hypoxia triggers the stabilization of the hypoxia inducible factors (known as HIFs) . HIFs arHere, we review the different strategies that directly or indirectly target control points of the hypoxic signaling pathway and their application in disease models. We specifically highlight the use of peptides as effectors of HIF activity and discuss their future perspectives and clinical significance.EPO gene. Unlike HIF-1α that can be expressed in all types of cells, HIF-2α, encoded by the EPAS1 gene, is expressed in a few tissues such as placenta, lungs, liver, and heart, and holds a central role in angiogenesis and erythropoiesis . AR. AR17]. The C-terminal parts of both HIF-1α and HIF-2α are also critical for function as they contain their oxygen dependent degradation domain (ODDD) as well as two distinct transactivation domains, N-TAD (overlapping with ODDD) and C-TAD (at the very C-terminus), which is responsible for the interaction between HIFα and the transcriptional coactivator proteins CBP/p300  21]. HI. HI21]. 2 levels as well as the production of ROS by oxygen-starving mitochondria inhibit hydroxylation and HIFαs escape degradation, accumulate, and translocate inside the nucleus where they assemble with ARNT into transcriptionally active heterodimers . Their breakthrough experiments revealed that the HIFα subunits are subjected to hydroxylation in two specific prolyl residues when cells are grown under atmospheric oxygen concentrations (normoxia) . This porodimers  31]..2 levelsOver the past few years, it has become clear that HIFs can also be regulated by mechanisms not directly affected by oxygen concentration. This regulation can occur at multiple levels including transcription, translation, post-translational modification, stabilization, nuclear translocation and activation of HIFαs ,31,32,332 concentration as both in vivo and in vitro hypercapnia decrease HIFα protein levels independently of the PHD/pVHL-mediated degradation pathway and, most likely, via lysosomal proteolysis . I. I106]. In conclusion, peptides as modulators of PPIs involving HIFs are not only important for in vitro experimentation and the elucidation of regulatory mechanisms but can also lead the way for the design and development of novel peptide analogues or synthetic peptidomimetics that can target exclusively HIF-overexpressing cells with great selectivity, low toxicity and pharmacological properties similar to those of small molecule chemical inhibitors. The effectiveness and advantages of HIF peptide inhibitors summarized herein, certainly provide proof-of-principle for the development of novel therapeutic options based on the interruption of PPIs that are essential for the adaptation of cancer cells to the hypoxic tumor micro-environment and urge for better and high resolution structural characterization of the corresponding HIFα PPI domains.


Primary ovarian insufficiency (POI) is an uncommon yet devastating occurrence that results from a premature depletion of the ovarian pool of primordial follicles. Our understanding of both putative and plausible mechanisms underlying POI, previously considered to be largely “idiopathic”, has been furthered over the past several years, largely due to advances in the field of genetics and through expansion of translational models for experimental research. In this review, our goal is to familiarize the multidisciplinary readers of the F1000 platform with the strides made in the field of reproductive medicine that hold both preventative and therapeutic implications for those women who are at risk for or who have POI. The purpose of the present review is to familiarize readers with the recent advances that have furthered our understanding of POI and which, in the foreseeable future, may even offer women with POI hope for biological parenting in every sense of the expression.Despite the resounding progress that the field of reproductive endocrinology has witnessed over the past three decades, our understanding of the myriad of mechanisms causative to the entity of primary ovarian insufficiency (POI) remains lagging. Consequences of POI range from psychological devastation relating to the diagnosis to symptom burden, of which loss of fertility dominates the spectrum, to the long-term consequences of premature loss of ovarian function which include an increased lifetime risk for skeletal fragility and cardiovascular and neurocognitive disorders. Many gaps remain in our understanding of the processes regulating ovarian follicle quantity and quality, and what causes these processes to go awry, as occurs in POI. Until recently, the treatment modalities available to patients with POI were limited to the use of hormone therapy to mitigate symptom burden and minimize long-term risks of estrogen deprivation as well as the use of donor eggs as the only viable option for biological parenting. We recently reviewed the clinical presentation and diagnosis of POI2. The diagnosis of POI is dependent on evidence of hypergonadotropic hypogonadism, namely elevated serum levels of the pituitary gonadotropin follicle-stimulating hormone (FSH) with low serum levels of estradiol (E2) in a patient who has irregular or absent menses and is younger than 40 years. When a POI diagnosis is suspected, serum levels of FSH and E2 should be measured twice, at least one month apart; persistently elevated FSH levels greater than 25 IU/L are consistent with POI4.The prevalence of POI in the overall population is about 1%, although contributing to this background prevalence are the increasing rates of premenopausal cancer survivors with iatrogenic POI caused by gonadotoxic therapy5. Once POI diagnosis is suspected, further workup is warranted to elucidate the etiology and identify covert comorbidities .The clinical presentation of POI is highly variable. Changes in menstrual cyclicity , symptoms of hypoestrogenism , and issues of subfertility or infertility are common presentations of POI. A finite spectrum of known causes of POI is summarized inidities . The var6. These genetic mutations (>60 identified thus far) demonstrate the complexity of the genetic architecture leading to this condition and offer targets for future genetic screening panels and possibly treatment modalities for women with idiopathic, sporadic POI. In efforts to detect novel POI genes, genotyping via genome-wide association studies (GWASs) and genome-wide sequencing through next-generation sequencing (NGS) have been used to identify genetic variations associated with POI. GWASs identify associations between genetic variation to a particular phenotype using many individuals with the disease or trait of interest. This evaluation is achieved by comparing common genetic variations known as single-nucleotide polymorphisms (SNPs), the most common genetic variations in the human genome in patients with POI versus unaffected controls. These SNPs may thus be used to identify genetic profiles that can be used to assess for causal relationships between phenotypes, many of which have been validated throughin vivo orin vitro functional studies or both.Certain karyotypic abnormalities and single-gene mutations have long been recognized as causes of POI . In addiWhereas GWAS uses traditional Sanger sequencing, which can analyze only 700 base pairs per reaction, NGS is a sequencing technique that enables the analysis of millions or billions of DNA strands in parallel. Whole exome sequencing (WES), a format of NGS, allows simultaneous analysis of base pairs across the entire exome and has been used in multigenerational POI families to identify single-nucleotide variants within genes known to be involved in ovarian function. From these studies, many potentially relevant variants from multiple family members can be rapidly investigated via gene panels using WES/NGS. Whereas GWAS is a more cost-effective method for common genetic variants, NGS allows a determination of the exact DNA sequence and can reveal information about all genetic variants, including those that are rare.in vitro andin vivo techniques can be used for functional validation. One recent example of this is RNA polymerase III subunit H (POLR3H), a gene that is highly expressed in developing spermatogonia, oocytes, and ovarian granulosa cells 17. In this study, two of eleven unrelated families with idiopathic POI were found to have a missense mutation c149.A>G via WES. In this setting, mouse models can be used for functional validation using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system 9 (Cas9) technology to demonstrate functional effects of a particular mutation, which can readily investigate a particular gene’s dosage on phenotype, rather than complete loss of function via gene expression modulation. In that study,D50GPolr3h knockout mice exhibited delayed puberty and decreased litter size and decreased expression of the ovarian transcription factor forkhead box O3 (FOXO3) and fewer antral and primary follicles17. In a study of a family with four generations of women affected by POI, WGS identified a heterozygous nonsense mutation in an RNA polymerase II subunit, POLR2C21; subsequent POLR2C knockdown in an embryonic carcinoma cell line resulted in decreased protein production and impaired cell proliferation. These data support a role for RNA polymerase II and III mutations as candidates in the etiology of POI. However, despite efforts to identify single genes causative for POI, it is likely that in many POI cases the disorder is more polygenic in nature. In a retrospective cohort study of 69 women affected by POI, WES identified 55 coding variants in 49 genes potentially related to POI; in 42% of the patients studied, at least two mutations in distinct genes were identified28.Once potential genetic candidates for POI have been identified,29. As a clinical screening tool, genetic testing (beyond karyotype and FMR1 testing via WES/NGS) may help to associate a diagnosis of POI with a specific syndrome, cancer predisposition, or neurodegeneration. However, broad use of genetic testing (via WES/NGS) is controversial given the poorly understood phenotypic variation associated with each genetic mutation; if implemented, it is likely best performed in conjunction with a multidisciplinary team, including a genetic counselor and experts across multiple medical fields . Furthermore, genetic testing can be more closely tailored via candidate gene sequencing if a monogenic syndromic condition is suspected on the basis of whether the patient is suspected to have a syndromic or non-syndromic phenotype30. An ultimate clinical goal is the design of a targeted panel that can best describe a patient’s risk or even provide a diagnosis, allowing a more timely intervention.As the increasing use of GWAS and NGS has led to the identification of many genes potentially increasing an individual’s POI risk, the next step will be sifting through the data to better understand the synergy and specific relevance for these genes32. Because the supply of available follicles is finite, the ability to detect and repair DNA damage is crucial for survival32. Aging oocytes and follicles are less able to detect and repair DNA damage34. DNA damage is considered a “hallmark of aging”—a process that manifests during normal aging, accelerates the aging process when induced, and retards normal aging when blocked35. DSB repair is critical in oocyte survival given the high frequency of DNA DSB which occurs during the meiosis portion of gametogenesis. Impaired DNA DSB repair, such as that observed in oocytes with deficiencies in RAD51 and ATM, blocks mouse oocyte maturation and results in oocyte death11. Loss of oocyte DNA damage checkpoints also appears to be an important mechanism for the drastic increase in oocyte aneuploidy seen with aging36.Mutations within the DNA damage repair (DDR) system represent intriguing candidate regulators of ovarian function . DNA dou30. Perhaps among the most well-known DSB repair genes are breast cancer type susceptibility protein 1 and 2 (BRCA1 andBRCA2). Mutations of bothBRCA1 andBRCA2 have been associated with decreased ovarian reserve11, failed follicular development and lower oocyte yield after ovarian stimulation in women undergoing assisted reproductive technology, and with an earlier age at natural menopause; conversely, in an experimental model, meiosis was restored with recovered levels of BRCA2 protein37. Existing data are, however, limited by the heterogeneneity of the control groups, observational study designs, and small sample sizes38. Other DDR genetic mutations  have been shown to cause a premature depletion of the follicle pool, decreased primordial follicles, and increased recruitment and subsequent atresia/destruction of the growing follicles40. Many other genes involved in the same DBS repair process have been discovered as potential gene candidates underlying POI. Other potentially pathogenic variants recently identified via WES of patients with POI include minichromosome maintenance 8/9 (MCM8 [20p12.3] and MCM9 [6q22.31])41. In particular,MCM8, a DNA DSB repair gene, has been implicated in the timing of menopause onset; a GWAS of about 70,000 women implicated MCM8 as well as HELB and SLC04A1 in the process of ovarian aging38. Mutations inPSMC3IP, a gene regulating meiosis of germ cells and DSB repair, have been associated with the phenotype of primary amenorrhea and POI in a consanguineous family of four sisters with ovarian dysgenesis and a brother with azoospermia42. Another interesting finding that overlaps two categories of POI—syndromic and non-syndromic  in non-syndromic patients. FA genes are involved in DNA DSB repair14 and are implicated in the category of syndromic POI; half of the patients with FA were reported to be infertile8, and FA genes have been described as relevant variants of known causative gene mutations of gonadal dysregulation.It has been hypothesized that owing to the inheritance patterns of early menopause and POI within the same families, these two conditions are possibly manifestations of the same underlying genetic susceptibility with variable expressivities43 but these factors remain poorly understood. A detailed understanding of the processes regulating timing of follicular activation would potentially allow the development of therapies designed to suppress this process and preserve ovarian function, which would be transformative for women with POI.A crucial question in ovarian biology is why some primordial follicles are maintained in dormancy for many years whereas others are activated for growth. Unlike in menopause, which occurs upon gradual depletion of the follicular pool via atresia or activation, residual primordial follicles unresponsive to standard gonadotropic signals remain in the ovaries. What is the mechanism by which adjacent follicles can be earmarked for such different fates? Current evidence suggests that whether an individual dormant follicle stays in the resting pool or initiates growth, transitioning out of a growth-arrested phase and developing into a mature peri-ovulatory follicle may depend on the balance of stimulatory and inhibitory factors at a particular point in time44. Live births have been achieved for POI patients with bone marrow transplant; this is thought to be due to replenishing factors necessary for an environment that facilitates growth within the ovary. One study injected bone marrow–derived stem cells (BMDSCs) and peripheral blood mononuclear cells into two groups of chemo-induced POI mice and immunodeficient mice with xenografted ovarian cortical fragments from poor-responder patients45. The study showed many promising findings for both rodent and human ovarian tissue after BMDSC injection, including higher ovarian weight, higher ovulatory follicles, metaphase II oocytes, two-cell embryos, live births, estradiol secretion, and ovarian vascularization45. Another recent study used an alternative stem cell source: endometrial mesenchymal stem cells (MSCs) injected into mice with chemo-induced POI resulted in higher circulating levels of anti-Mullerian hormone , greater number of developing follicles, and higher ovulation and live birth rates46. Furthermore, the therapeutic benefit of stem cells from other tissues, including adipose, umbilical cord blood, and amniotic epithelial cells, has been explored50. Recent research has begun to explore how stem cells specifically affect the ovarian microenvironment and restore ovarian function through the use of exosomes derived from stem cells in chemotherapy-induced POI models52. However, it is unclear at this point whether therapies aimed at prevention of POI in the oncofertility population will be broadly applicable to the larger POI population, including women with autoimmune and idiopathic POI and women in whom the onset of POI occurs early in life. One exciting advance in POI is the exploration of stem cell therapy via residual follicle rescue53. Growth factors that are thought to be delivered via PRP include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor b-I (TGFb-I), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and basic fibroblast growth factor (bFGF)54. This PRP-based tissue regenerative approach has been attempted across several injured human tissue types  with the goal of improved healing55 primarily through the promotion of neoangiogenesis. In a case report, the use of PRP with direct gonadotropin intraovarian injection resulted in successful conception in a POI patient who subsequently achieved a live birth56. Another approach that has been explored aims at increasing the concentration of local growth factors within the ovary utilizing intra-ovarian injection of platelet-rich plasma (PRP). Prepared from autologous plasma, intraovarian PRP has been shown to support the viability and growth of preantral follicles and to increase the number of retrieved oocytes58, especially for those with cancer diagnosed before puberty (and thus before controlled ovarian stimulation for oocyte cryopreservation can feasibly be achieved) . However, it is unclear whether OTC studies largely consisting of patients with cancer can be extrapolated to patients with or at risk for POI relating to other etiologies, such as an abnormal genotype65. A retrospective case-controlled study looking at ovarian biopsies in 15 patients with TS after OTC showed follicles in only nine of the ovaries (eight of these nine in Turner mosaic karyotype), along with markedly lower follicular fluid testosterone and estradiol concentrations65, suggesting that a careful and patient-specific approach is essential when identifying TS patients who may benefit from OTC66. Ovarian tissue cryopreservation (OTC), in which a portion or the entire ovary is removed and frozen with the intention of autotransplantation, is a promising preventative option for fertility preservation in women planning initiation of chemotherapy or radiationet al., blockade of mTOR pathways with small-molecule inhibitors preserved ovarian reserve, primordial follicles, AMH levels, and fertility in a cyclophosphamide-treated mouse model67. AMH, as a critical “gatekeeper” hormone that regulates follicular quantity by inhibiting recruitment and growth, is a particularly interesting molecule in the regulation of this follicular “clock”73. The use of exogenous AMH has been explored as an innovative new preventative strategy and treatment option for gonadotoxin-induced follicle loss74. In two recent studies, co-treatment with exogenous delivery of AMH in chemotherapy-exposed mice resulted in an inhibition of primordial follicle growth activation, although the protective effect varied depending upon the agent used76. Because the PI3K signaling pathway is not activated by AMH, FOXO3 phosphorylation has been proposed as a causative mechanism for the protective effects of AMH75  pathway, which modulate activity of protein kinase A (Akt) and mechanistic target of rapamycin (mTOR) and FOXO3 , similar increases in follicle counts were observed. Others have also demonstrated that primordial follicle activation  is regulated in part by AKT79. In humans, fragmentation of human ovarian tissue cubes followed by Akt stimulation also resulted in antral follicle growth when tissue strips were transplanted into immune-deficient mice. Subsequently, 27 patients with POI underwent ovarian tissue harvesting and fragmentation, and tissue was subjected to Akt treatmentin vitro for 2 days, followed by auto transplantation beneath Fallopian tube serosa. Follicle growth was subsequently observed in eight patients and mature oocytes were retrieved from five patients; one woman achieved a live birth78. In light of the understanding of the Hippo signaling pathway and its relevance for ovarian follicular development, it is plausible to consider that any benefits of intraovarian PRP injection on ovarian physiology may be secondary to a modulation of the Hippo pathway from physical disruption of the ovarian tissue (from injection), and not from intraovarian growth factors, as is hypothesized56.ith POI , (reviewith POI . Disrupt65 and FMR1 premutation carriers82), pediatric and reproductive age cancer survivors80, or women with autoimmune conditions83.POI has historically been considered a poorly understood and catastrophic condition. Although the etiology of POI has largely been considered idiopathic, recent advances in the field of genetics have begun to unravel a complex network of molecular pathways that are critical to normal ovarian biology and that may be involved in the pathophysiology of this heterogeneous entity. Many potentially promising diagnostic and treatment modalities are already being explored. The overarching goal for the emerging tests and tools is to allow opportunities for an earlier diagnosis in those at risk of POI. Timely interventions may offer salvage of residual reproductive potential in the earlier stages of the POI processes. Elective freezing of oocytes and ovarian tissue are tried and tested interventions that allow for a preemptive approach to fertility preservation in those deemed at risk for POI, such as young girls and women with genetic predispositions (for example TS


Intramuscular lipid accumulation has been associated with insulin resistance (IR), aging, diabetes, dyslipidemia, and obesity. A substantial body of evidence has implicated ceramides, a sphingolipid intermediate, as potent antagonists of insulin action that drive insulin resistance. Indeed, genetic mouse studies that lower ceramides are potently insulin sensitizing. Surprisingly less is known about how physical activity  regulates ceramides, especially in light that muscle contraction regulates insulin sensitivity. The purpose of this review is to critically evaluate studies (rodent and human) concerning the relationship between skeletal muscle ceramides and IR in response to increased physical activity. Our review of the literature indicates that chronic exercise reduces ceramide levels in individuals with obesity, diabetes, or hyperlipidemia. However, metabolically healthy individuals engaged in increased physical activity can improve insulin sensitivity independent of changes in skeletal muscle ceramide content. Herein we discuss these studies and provide context regarding the technical limitations  and a lack of mechanistic studies that prevent a more sophisticated assessment of the ceramide pathway during increased contractile activity that lead to divergences in skeletal muscle insulin sensitivity. Obesity is a mounting health problem and the percentage of those who meet the classification is anticipated to double or triple in the next 40 years ,2. ObesiOf those suffering from these metabolic derangements, older adults make the largest proportion and as of 2017 ~48% of older adults are considered pre-diabetic . Since iThe focus of this review is to examine the effects of physical activity in aging on a class of lipid intermediates, ceramides, that have been implicated in obesity and inflammation-induced skeletal muscle insulin resistance and impair insulin signaling downstream of the receptor .Can ceramides, a dominant mechanism of diet-induced IR, also be a viable candidate by which physical activity (exercise) induces major swings in insulin sensitivity? To address this question, we evaluated the literature comprising rodent and human investigations of chronic exercise in the context of aging. We considered whether the robust changes in insulin sensitivity tied to exercise habituation correspond with the levels of skeletal muscle ceramides, as well as with the expression or activity of enzymes governing ceramide metabolism. Although, we will discuss all phenotypes, for the purpose of this review, our primary focus was on healthy phenotypes , across the lifespan in order to remove the confounding effects of current metabolic dysfunction and understand the extent to which the pathways related to overnutrition-induced muscle dysfunction relate to the pathways resulting from physical activity.A hallmark of diabetes is obesity and the ectopic deposit of fat in many tissues, including skeletal muscle . An overCeramide production relies on the availability of fatty acids and serine, and the overabundance of these nutrient precursors drives accumulation of the sphingolipid in models of overnutrition ,21,30. NThe diversity of ceramide species is a reflection of the activity of the six ceramide synthase (CERS) enzyme isoforms, which differ by tissue distribution and substrate specificity (for more detailed review see ). CERS1,Of the thousands of sphingolipids present in cells, ceramides are a particularly intriguing class of molecules. In addition to serving as common precursors for all other highly abundant sphingolipids, they have distinct bioactivities in cellular stress responses . Ceramidtrans-double bond. At this point ceramide can be metabolized to (1) glycosphingolipids through glucosylceramide synthase, (2) ceramide-1 phosphate with a ceramide kinase, or (3) sphingomyelin through sphingomyelin synthases.De novo biosynthesis of ceramide starts with the rate limiting condensation of palmitoyl-CoA with L-serine through serine palmitoyltransferase (SPT), producing the transient intermediate 3-ketosphinganine. 3-ketosphinganine is then reduced to sphinganine by 3-ketosphinganine reductase. The (dihydro)ceramide synthase (CerS 1–6) enzymes acylate sphinganine to generate dihydroceramide, which is further modified by dihydroceramide desaturase (DEGS 1–2) through the placement of a 4,5-++ dependent sphingomyelinase (nSMase) or acid sphingomyelinase (aSMase), transfer the choline residue of SM to DAG to generate phosphatidylcholine. The rate of ceramide degradation is also controlled by three isoforms of ceramidase: acid (aCDase), neutral (nCDase) and alkaline . These enzymes hydrolyze the lipid to generate sphingosine and a FFA. Recent studies have found that adiponectin receptors are another class of ceramidases, degrading the lipid in response to the binding of the insulin-sensitizing adipokine [The salvage and sphingomyelinase pathways also influence ceramide levels. Hydrolysis of the far more abundant sphingomyelin can occur in plasma membranes or intracellular organelles. The various SMases, including neutral Mgdipokine ,44,45,46Theoretically, ceramide accumulation in skeletal muscle could arise through several potential routes: (1) increased availability of substrate for ceramide biosynthesis through mass increases in circulating FFA ; (2) red2peak: 30–40 mL∙kg−1∙min−1) in both the old and young groups compared to the cohort in their next report demonstrating a greater content with aging [2peak: 20–30 mL∙kg−1∙min−1) [Skeletal muscle IR is a common feature of aging ,53, but th aging . In that1∙min−1) . Future The literature on exercise training and muscle ceramide content in rodent is varied . The firA following study from Dr. Jan Gorski’s group using a more robust form of endurance exercise in male rats  demonstrated increased insulin sensitivity (HOMA-IR) and sphinganine in the soleus and red gastrocnemius similar to the previous report  but no c2 peak data in When examining cross-sectional studies of exercise-trained individuals compared to those that are sedentary, results when evaluating muscle ceramides are fairly ambiguous . The varThe improved insulin sensitivity following exercise training is well established . LongituSkeletal muscle ceramides decrease in obese ,82,83,86In summary, changes in ceramide content after exercise training may occur in individuals with obesity or T2DM, likely do not change in healthy individuals and these factors drive the impact of age. These improvements following exercise training in metabolically compromised individuals may occasionally be associated with the improved insulin sensitivity following exercise training. A major limitation appears to be the reliance on whole cell lysate ceramide content/composition, which may not be as precise as studies assessing subcellular localization or ceramide flux.A panoply of studies with rodents clearly identifies a role for skeletal muscle ceramides in mediating insulin sensitivity under conditions with pronounced derangements in metabolic function ,87,91. YIf the effect of ceramide on IR in healthy humans is subtle then the assessment of ceramide content and composition needs to be specific to capture the relationship. More recent reports have revealed that skeletal muscle subsarcolemma C16:0 ,89 and sAltogether, this review of the literature points to a dynamic response of skeletal muscle ceramide metabolism to physical activity, the significance of which is not fully understood. Aging itself does not appear to impact skeletal muscle ceramide content independent of physical inactivity, obesity and metabolic disruption that typically are associated with the aging process. Cross-sectional comparison participants by activity level and chronic exercise training does not support a relationship between skeletal muscle ceramide content and insulin sensitivity in healthy individuals. In agreement with this finding, chronic exercise training does not consistently reduce ceramide content linked to improved insulin sensitivity—a relationship that is occasionally observed in participants with robust metabolic dysfunction. Future considerations include more frequent assessments and longer durations of physical activity and inclusion of sex as a biological variable. Thus, the role of ceramide lipotoxicity in the early development of aging-induced IR is yet to be determined and resolution of this question would greatly benefit from the modern comprehensive lipidomic assessments of sphingolipids, in specific myocellular localizations and certainly with addition of ceramide flux assays. The possibility of subtle subcellular redistribution of skeletal muscle ceramide not detectable in whole muscle homogenate cannot yet be discounted in the etiology of age-induced skeletal muscle IR. If skeletal muscle ceramide intramyocelluar distribution and content is not involved in the development of age-induced IR, it is probable that ceramides may serve as a likely mechanism to worsen the progression of metabolic dysfunction into metabolic disease during longer durations of inactivity and during aging.Clearly, a divergence in the rodent ,103 and Though lipid/obesity induced insulin resistance is well examined, the mechanism(s) linking activity to insulin sensitivity is largely unknown, particularly in aging.Aging is only linked to increased skeletal muscle ceramide content in obese individuals.Cross-sectional studies of exercise-trained adults reveal a range of insulin sensitivities related to physical activity level that are independent of skeletal muscle ceramide content and species. Indeed, acute exercise in healthy humans may actually increase ceramide content within skeletal muscle.By comparison, longitudinal studies indicate that exercise may not impact ceramides in skeletal muscle of healthy individuals but is rather more likely to decrease skeletal muscle ceramides in individuals with previously elevated ceramides resulting from obesity or T2D.Clarity of the role of skeletal muscle ceramides on insulin sensitivity during physical activity may be enhanced with improved methodologies to evaluate distinct ceramide species , their subcellular distribution, and their turnover. Additionally, there is a need for mechanistic genetic studies of ceramide metabolism examining the interaction between physical activity level and insulin sensitivity. Greater attention to these nuances of ceramide action could unveil relationships which may be masked in prior literature.