NTR) and tropomyosin-related kinase (Trk) neurotrophin receptors in addition to cholinergic markers. It is known that the neurotrophins, particularly nerve growth factor (NGF), mediate cholinergic neuronal development and maintenance. However, the role of neurotrophin signalling in regulating adult cBF function is less clear, although in dementia, trophic signalling is reduced and p75NTR mediates neurodegeneration of cBF neurons. Here we review the current understanding of how cBF neurons are regulated by neurotrophins which activate p75NTR and TrkA, B or C to influence the critical role that these neurons play in normal cortical function, particularly higher order cognition. Specifically, we describe the current evidence that neurotrophins regulate the development of basal forebrain neurons and their role in maintaining and modifying mature basal forebrain synaptic and cortical microcircuit connectivity. Understanding the role neurotrophin signalling plays in regulating the precision of cholinergic connectivity will contribute to the understanding of normal cognitive processes and will likely provide additional ideas for designing improved therapies for the treatment of neurological disease in which cholinergic dysfunction has been demonstrated.Cholinergic basal forebrain (cBF) neurons are defined by their expression of the p75 neurotrophin receptor (p75 NTR) and tropomyosin-related kinase A (TrkA), TrkB and TrkC, the receptors for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4 (NT3/4).One of the defining features of the cholinergic basal forebrain (cBF) neurons is their expression of the neurotrophin receptors, p75 neurotrophin receptor  11 in the neuronal precursors of presumptive basal forebrain neurons  and preoptic area, zones mainly known as the origin of parvalbumin- and somatostatin-expressing interneurons that migrate tangentially into the cortex (reviewed in ). Howevese cells ,23, suchse cells ,25.NTR and all Trk receptors, indicating important functional roles for these receptors in both septal and magnocellular cBF neurons.Although the ventral telencephalon  is the birthplace of septal basal forebrain neurons ), some reports suggest that a different progenitor population forms the magnocellular nucleus  and the substantia innominata (SI)), and that this population may originate from progenitor zones in the ventral pallium ,26. ThisNTR receptors is regulated during embryogenesis is unclear. Although it is not unusual for TrkB and TrkC receptors to be co-expressed by neurons, the additional co-expression of TrkA, as occurs in the basal forebrain, is unusual in the brain [NTR, ChAT and acetylcholine synthesis both in vitro and in vivo [Exactly how the expression of Trk and p75he brain . Furtherhe brain , which p in vivo . Further in vivo ,30. Inde in vivo ,33.NTR- and Trk-dependent mechanism [In the magnocellular nuclei, ChAT expression is induced by P14, via a BDNF-induced, p75echanism . Interesechanism . In P14 echanism . Co-treaNTR and Trk receptors are expressed during the differentiation and/or specification of the cholinergic phenotype of developing cBF neurons, one might expect that mice in which individual neurotrophin receptor genes have been deleted would produce robust and obvious cBF phenotypes (see sections below). However, cBF neurons are unusual in co-expressing TrkA, B and/or C and have redundant mechanisms of development. Multiple trophic factors may act in concert to regulate the survival and growth of cBF neurons during the early postnatal critical period of development, with NGF being a particularly potent survival-promoting factor, and other cortically derived factors being more important for the growth, branching, and navigation of axons in the target regions [As p75 regions . AnotherAt completion of development, the basal forebrain consists of a heterogeneous cell population, including cholinergic, γ-aminobutyric acid-ergic (GABAergic) and glutamatergic neurons, which form complex networks that provide important modulation of cortical function ,37. As dMS/VDB neurons project to the hippocampus via the septohippocampal pathway . These The main afferent inputs into the basal forebrain are glutamatergic projections from the ventral tegmental area , GABAergIt is important to note that cBF axons can synapse on to specific neuronal targets (point to point neurotransmission) or evoke downstream responses via volume transmission, whereby the release of acetylcholine affects many neurons or synapses in the vicinity of the cBF terminal (see reference ). The prA recent series of studies revealed several organising principles of cBF neurons ,54,55. FConsistent with the anatomically derived connectivity maps ,47, receNTR-specific antibody was used to create a lesion that was specific to cBF neurons [cBF neurons, through their release of acetylcholine, have been implicated in a wide range of cognitive processes and behaviours, including attention, fear-related processes and reward-mediated behaviour, addiction, memory updating, extinction and renewal . Much of neurons . Dependi neurons  and fear neurons . Lesions neurons  and poor neurons  and Barn neurons , as well neurons . These l neurons .More recently, manipulating cBF neurotransmission in mice via optogenetics has further demonstrated cBF neuronal influences on cognitive processes. For example, selective activation of cBF neurons in different cBF nuclei has been reported to improve complex cortically dependent behaviours such as the encoding of natural scenes  and olfaNot all effects of the basal forebrain on behaviours and associative tasks require intact cBF circuits . Rather,Early work revealed that cBF-mediated neurotransmission also controls electrical oscillations in the hippocampus, as lesioning cBF neurons led to a decrease in low-frequency (6–12 Hz) theta oscillations . These fReal‐time monitoring of acetylcholine release in the cortex of rats performing attentional tasks has revealed that precisely timed  cholinergic signals in the prefrontal cortex coincide with the detection of behaviourally significant cues , such ascBF-mediated modulation of cortical activity also depends on the strength of concomitant inputs to the target neurons: cholinergic neurotransmission is inhibitory during quiescence, but excitatory when there is simultaneous post-synaptic activity . By reduElectrophysiological evidence points to the ability of the cBF-mediated cholinergic mechanisms to result in both (i) an enhanced probability of propagating cortical output in response to previously associated information, and alternatively, (ii) regulating new learning in response to salient or novel stimuli by releasing dendritic inhibition while suppressing low threshold pyramidal neuronal transmission . EvidenccBF neurons have axonal collaterals within all layers of a cortical column, but the modulation is layer specific, and can also vary according to cortical area and species. Examples are provided below and in Taken together, the above data indicate that cBF neuron-mediated neurotransmission requires temporally precise modulation of post-synaptic targets by these cells. There is also a requirement for a high degree of anatomical organisation of (i) cBF axonal innervation to dendrites, neurons and microcircuits within cortical columns, (ii) cBF neurons within the same basal forebrain nuclei innervating distinct cortical regions, and (iii) coincident transmission to downstream targets by cBF neurons located in different nuclei. It is as yet unclear how cBF neurons are able to establish and maintain such a dynamic range of connections. This also raises the question of whether directed modulation of post-synaptic targets by cBF neurons is achieved through a high degree of plasticity of their connections in the adult brain. We propose that neurotrophins provide a key mechanism that could provide a cBF-cortical neuron organisational structure within target areas. However, how the cBF neurons receive incoming information from cortical or subcortical relays in an organised way, and innervate the cortex in a rostral caudal manner are topics outside the scope of this review.NTR in both rodents and primates [The neurotrophin receptors are co-expressed to differing extents in developing cBF neurons ,47,115 aprimates ,117. In primates . The proNTR leads to an increase in the number of cBF neurons, a finding that is proposed to be due to a lack of developmentally regulated apoptosis [NTR developmental programmed cell death in a range of neuronal types, particularly peripheral neurons [NTR in mediating cBF neuronal death during the early postnatal period (but not in age-related decline in cBF cell number) based on a conditional knockout of p75NTR from P4 in the mouse cBF, using ChAT-cre-mediated deletion [NTR in cBF neurons at birth using a TrkA-cre reduced the number of cBF neurons  and cholinergic-specific p75NTR knockout mice are changes in behaviour in cognitively complex tasks, with little change in baseline behaviour. In comparison with littermate controls, mice lacking p75NTR have generally been reported to have improved spatial learning and/or memory performance in an egocentric passive place avoidance task, as well as in cued, hippocampal-dependent Barnes and Morris water mazes. The mice also display changes in the amount of time spent in light–dark compartments, and reduced cued fear conditioning and fear extinction consolidation [NTR in adult animals attempted in order to reverse the observed phenotype. The study demonstrated that acute p75NTR signalling, rather than the increased number of neurons , is important for cBF neuron synaptic innervation and fear extinction consolidation [One of the most consistent observations of p75lidation ,128,131.lidation . Howeverexon III−/− and p75exon IV−/− mice have been shown to have reduced hippocampal LTD, without an accompanying change in LTP [exon III−/− mice have been reported to have enhanced LTP in the hippocampus and amygdala [NTR promotes the persistence of transmission between neurons, albeit by different synaptic mechanisms. Increased complexity of the dendritic spines of CA1 hippocampal pyramidal neurons, changes to glutamatergic receptors, and increased cholinergic transferase activity in p75exonIII−/− mice have also been demonstrated [NTR, the excitability and persistent firing of pyramidal neurons in layer 5 of the entorhinal cortex is unregulated due to a lack of inhibition [NTR in the entorhinal cortex occurs in the innervating cBF axons, the role of p75NTR could be to regulate cBF neuron structure or function thereby indirectly mediating the inhibition or activation of post-synaptic pyramidal cell activity.Juvenile (2–3 weeks old) p75e in LTP ,133. Intamygdala ,131. In nstrated . In addihibition . As the NTR-deficient mice, whether they are due to p75NTR signalling in cBF neurons during development or in the adult is difficult to determine. Experiments carried out using brain slices of developing animals have provided evidence of post-synaptic p75NTR expression , but caications ,136–138,ications . TherefoNTR signalling in cBF neurons, and whether p75NTR acts to facilitate Trk receptor signalling as well as acting independently to facilitate synaptic pruning, driven by the developmental stage or expression levels of co-receptors. The current dogma would suggest that proneurotrophins, acting together with the sortilin/SorCS family of co-receptors, is likely to be responsible for the pruning of synapses and axons via p75NTR . Althoupulation , suggestNTR-deficient mice, it has often been difficult to differentiate the adult role from the developmental functions. Despite current dogma, target-derived neurotrophins are not essential for the survival of cBF neurons [Neurotrophin-mediated Trk receptor signalling appears to regulate both the development and adult function of cBF neurons. However, as with the analysis of p75 neurons , but ratNTR-) positive cell number was due to the increased developmental death of cholinergic cells observed at P7. Although it is unclear whether the innervation phenotype represents loss of axons/cells or merely down-regulation of ChAT/p75NTR expression, the authors reported that the expression of ChAT in the remaining neurons and the density of the remaining cholinergic varicosities were unchanged, suggesting that there was no difference in the connectivity of the surviving neurons. Therefore it is possible that a subpopulation of neurons was affected by the loss of NGF-TrkA signalling. Surprisingly, these TrkA-deficient animals displayed normal function across a large number of assays of cognitive domains, which included attention performance, anxiety, exploration and working and spatial memory. The authors concluded that deficits in TrkA signalling are not sufficient to induce cognitive impairments in adult animals, despite the mice displaying a significant cellular phenotype.The first study of TrkA-deficient mice revealed dramatically reduced acetylcholine esterase-positive fibres in the hippocampus and somatosensory cortex of those animals which survived to adulthood . A persoNTR immunostaining in the postnatal period, as well as a decrease in total ChAT expression in the basal forebrain across all ages investigated from P14 to P180. It is again unclear from these experiments whether this result represents a loss of the axons themselves or only down-regulation of ChAT and p75NTR expression. However the authors suggested that loss of NGF-TrkA signalling (demonstrated by reduced Akt phosphorylation) specifically decreased the level of ChAT (and p75NTR) synthesis within cells, and that this in turn was sufficient to affect cholinergic neurotransmission.However, contrasting findings were reported in a study published the same year which used a different conditional TrkA knockout mouse line, in which the gene was removed from E9.5 in dlx5/6-expressing telencephalon cells . These TNTR-deficient mice.It is likely that the explanation of these contrasting findings is, at least partially, due to methodological issues. In adult rats, down-regulation of TrkA in the magnocellular nuclei had no effect on cBF neuron survival but decreased the density of cortical cholinergic axons and reduced their capacity to release acetylcholine, impairing attention of aged animals . Similar−/+ mice show a reduced number and size of adult cBF neurons, as well as deficits in memory updating in a Morris water maze reversal learning task [exonIII−/− mice [−/− mice, have a 30% loss of cBF neurons, reduced ChAT expression in axons , and a reduced number of nerve terminals in the hippocampus and frontal cortex [The first study of complete NGF knockout mice indicated that the production and survival of cBF neurons was unaffected . Howeveing task , a phenoing task  and p75e−/− mice , but is l cortex . Similarl cortex . Experiml cortex ,152. Howl cortex .in vitro. However, at earlier or later time points, they are largely resistant to NGF deprivation. The addition of BDNF, NT3, NT4/5 or glial-derived neurotrophic factor to cBF neurons, or co-culture with cortical neurons that enable synaptic innervation, confers resistance to NGF deprivation during this critical period [NTR?) and survival during maturation, then primarily as a mediator of postnatal axonal growth, and subsequently to fine-tune cortical synaptic function.One explanation for this is that NGF signalling plays a different role in regulating synaptic activity in immature and mature cBF neurons. Cultured cBF neurons from E16/17 rats are dependent on NGF for survival between the second and fourth week l period . During l period . Therefo−/+ mice was not only able to reverse the learning deficit induced by chronic NGF reduction, but also to cause an increase in cBF cell size and ‘number’ in the adult, indicating that NGF-mediated signalling controls the adult cBF neuronal phenotype [Several studies have directly tested the role of NGF signalling in adult cBF neurons, revealing that NGF can control synaptic plasticity, circuit development and rewiring. Intraventricular infusion of NGF into adult NGFhenotype . Similarhenotype . NGF exphenotype . Stimulahenotype ,160. Morhenotype . NGF apphenotype . The timNGF mRNA [NGF mRNA-positive neurons are parvalbumin-positive, although ∼20% of parvalbumin-expressing interneurons do not express NGF. In contrast, only a subset of calbindin- and calretinin-positive interneurons  contain NGF mRNA [A particularly important detail is the target-derived source of NGF  that cBNGF mRNA . In the NGF mRNA . AlthougNGF mRNA .NGF mRNA [en passant cholinergic neurotransmission to that local area. Indeed there appears to be a link between cholinergic and non-cholinergic transmission in cortical microcircuits regulated by the basal forebrain. For example, we recently found that a specific change of p75NTR expression in cBF neurons has a feedforward effect on the cortical synaptic connectivity of non-cBF neurons as well as cBF neurons [It has been demonstrated that cholinergic-responsive layer 1 vasoactive intestinal peptide (VIP)- and/or somatostatin-expressing interneurons can disinhibit the layer 2/3 parvalbumin-positive interneurons, thereby regulating pyramidal neuron firing . HoweverNGF mRNA . One exp neurons . Whether−/+ mice display decreased ChAT activity [−/− cBF neurons to prefrontal cortical cells  is sign animals .In contrast with NGF, BDNF is expressed by pyramidal neurons and not interneurons and is therefore the best candidate for establishing pyramidal neuron cholinergic transmission via synaptic connections. Interestingly, topical application of BDNF has the opposite effect on the functional representation in the barrel cortex of a stimulated whisker to that of NGF application. BDNF results in a rapid and long-lasting decrease in whisker representation size and decreases the amplitude of the activity-dependent intrinsic signals . This fiThe effect of NGF, which mimics whisker training that causes the expansion of the receptive field for a specific whisker , may be In summary, additional investigations need to be undertaken to reveal the mechanism by which BDNF-TrkB signalling modulates cBF neurons and thus cortical neuronal activity. BDNF appears to be required for the specification of magnocellular cBF neurons during embryonic development, and subsequently regulates the developmental and/or adult cBF synaptic innervation of the cortex, likely mirroring the role of NGF, but in relation to a different cellular target.NT-3 mRNA include those in the CA2 region, in the most medial portion of the CA1 region, and in the dentate gyrus [Finally, almost nothing has been reported regarding the role of NT3-TrkC in cBF neurons. It is known that NT3 plays a role in thalamic innervation of the cortex , where ite gyrus . These cte gyrus . Surpriste gyrus . This coAlmost 40 years old, the first major evidence-based hypothesis regarding the aetiology of Alzheimer’s disease (AD) was the cholinergic hypothesis (reviewed in reference ). It proIn AD , it is now clear that atrophy of the basal forebrain (as measured by a reduced volume in MRI images) precedes and predicts clinical decline in cohorts with both mild cognitive impairment and idiopathic AD, as well as in individuals who have not yet been diagnosed with cognitive impairment . As in aNTR from cBF neurons  has shown similar or superior diagnostic accuracy to hippocampal atrophy in identifying AD subjects ,186, andsynapses ,125) impsynapses . These dNeurotrophin dysfunction has been strongly linked to cholinergic degeneration in AD, particularly decreased NGF/TrkA trophic signalling ,195. ThiNTR small molecule modulators are also being tested for the treatment of AD [NTR expression is induced in the cortex and hippocampus of animal AD models and human patients [NTR [NTR can bind to and sequester and/or endocytose Aβ [NTR prevents the degeneration of cBF neurons in animal models of familial AD [NTR may be a key player in mediating the ongoing selective degeneration of cBF neurons, which in turn could promote not only cognitive decline but further Aβ accumulation and cortical degeneration.p75nt of AD . Unlike patients . Furthernts [NTR . It has ytose Aβ . Conversilial AD ,206. ThiThe major question regarding neurotrophin-based therapeutic options is how best to prevent or restore the loss of cBF connectivity in order to preserve cognitive function. Is it sufficient to merely keep cBF neurons alive? The provision of NGF to the NBM appears to promote neuronal survival ,211, butcBF neurons are regulated by neurotrophins through their receptors from as early as cell specification and differentiation, mediating the extent of their cholinergic phenotype, and controlling axonal outgrowth and target innervation. In addition it is clear that the adult expression of neurotrophin receptors in cBF neurons regulates aspects of cell morphology, both growth and degeneration, which in turn can affect cortical function and thus behaviour. However, the precise role that each Trk receptor plays in these neurons is less clear, with redundancy being an unlikely explanation. Rather we propose that the neurotrophin receptors control specific aspects of cBF axonal innervation, and that the neurons remain in a developmental-like or plastic state throughout life, constantly rewiring in response to salient external stimuli to assist in encoding experiences required for future behaviourally relevant responses.NTR and each Trk receptor  during development, and depend on these receptors for their appropriate innervation of different targets based on the expression of target-derived neurotrophins. Furthermore, it has been proposed that neurons with overlapping axonal fields predominantly express TrkA receptors, whereas those neurons with restricted axonal fields predominantly express TrkB and/or TrkC receptors [en passant release sites. More akin to the peripheral nervous system, co-expression of multiple Trk receptors by a single neuron may ensure that all relevant cells receive innervation by cBF axons, to guarantee widespread coverage of the cortex. However, this too seems simplistic, given the apparent importance of the location and timing of cholinergic transmission for cortical function.The specificity of innervation using different Trk receptors is illustrated by the sensory neurons of the dorsal root ganglia and neurons within the spinal cord . These neceptors . ExtrapoIt has been proposed that the activity-dependent release of NGF could serve as a feedback signal between highly plastic cortical inhibitory microcircuits and cholinergic neurotransmission, whereby NGF acts not only to increase cholinergic activity but also to reorganise the circuits for cortical function . In otheNTR. However, in this model , or control of innervation that needs to be coordinated between brain areas such as the hippocampus and cortex. However, NT3 may simply regulate a third layer of innervation such as a specific interneuronal class.NTR acts to limit cBF neurotransmission, which could be crucial when the information that cBF connections encode is no longer salient or requires modification. It appears that p75NTR might act in a continuum, from inhibiting LTP and enhancing LTD at cholinergic synapses, to pruning cholinergic synapses and/or non-synaptic acetylcholine release sites, to pruning axons. It is possible that p75NTR also acts to temper cholinergic transmission presynaptically, being able to regulate G-protein coupled inwardly rectifying channels that reduce the membrane potential, acting to prevent excitotoxicity in these highly metabolically active neurons [NTR is activated by proneurotropins accumulating in the absence of sufficient circuit activity and cleavage to mature forms, or by other cell types that act to remove inactive synapses (e.g microglia), and/or in the absence of sufficient Trk receptor signalling, will require sophisticated in vivo experimentation to reveal. It is clear nonetheless that expression of p75NTR makes cBF neurons susceptible to degeneration in conditions such as AD, including in response to Aβ.Finally, p75 neurons ,217. WheGiven the importance of acetylcholine on cortical disinhibition, particularly dendritic gating, further elucidating the mechanisms which control the cBF axonal innervation that results in synaptic and volume neurotransmission, as well as deciphering which neurons within cortical columns are so innervated, and under what conditions, will significantly increase our understanding of the cortical computations underpinning associative learning. Moreover determining the time-frame of cBF axonal and synaptic change mediated by neurotrophin receptors in the adult is of key importance for further understanding the mechanisms by which this influential group of neurons affects higher order cortical function and experience-dependent learning. This knowledge is also crucial for designing efficacious therapies to prevent or restore cBF dysfunction and thus cognitive impairment in the context of neurological and degenerative conditions including AD.


Adipose tissue is a key nutrient-sensing depot that regulates excess energy storage and consumption. Adipocytes, the key components of the adipose tissue, have unique ability to store excess energy in the form of triglycerides, sense systemic energy demands, and secrete factors  to regulate other metabolic tissues. The presence of various types of adipocytes  characterized by the number/size of lipid droplets, mitochondrial density, and thermogenic capacity, further highlights how intricate is the communication of these cell-types with other metabolic tissues to sense nutrients. In obesity the inherent capacity of adipose tissue to store and sense nutrients is compromised, causing spillover of the intermediate lipid metabolites into circulation and resulting in their ectopic deposition in tissues not suitable for lipid storage, a phenomenon known as lipotoxicity. This results in a spectrum of cellular dysfunction, that underlies various metabolic diseases. Of the numerous lipid classes implicated in eliciting lipotoxicity, sphingolipid: ceramides are among the most deleterious as they modulate signaling pathways involved in regulating glucose metabolism, triglyceride synthesis, apoptosis, and fibrosis. Notably, recent experimental studies have strongly implicated ceramides in the development of numerous metabolic diseases such as insulin resistance, diabetes, cardiomyopathy, hepatic-steatosis, and atherosclerosis. Herein we discuss and summarizes recent findings that implicate ceramides as a key contributor to adipocyte dysfunction underlying metabolic diseases and how depletion of ceramides can be exploited to improve metabolic health. Metabolic diseases represent a significant health burden that impacts millions of households worldwide. According to the World Health Organization (WHO) global report, ~422 million adults were living with diabetes in 2014 –5. NotabIn this review, we intend to provide a perspective on ceramides and ceramide metabolites in the maintenance of adipose tissue homeostasis and how adipose tissue ceramides contribute to the development of metabolic diseases.CERS6 in adipose tissue positively correlates with Body Mass Index, body fat content, and hyperglycemia while negatively correlating with glucose infusion rate during euglycemic-hyperinsulinemic clamps , that activates β-adrenergic receptors in adipocytes, increases the thermogenic capacity of adipocytes by elevating the expression of thermogenic genes such as d Prdm16 . We recend CerS6 . We furtfunction .ex vivo, suggesting that endogenous ceramides could be autonomous regulators of adipocyte function  in adipose tissue and reduced sphingosine content, further highlighting the presence of ceramidase activity in AdipoRs that requires adiponectin  , induced profound changes in the adipose tissue. Importantly, this intervention significantly reduced adipocyte size, increased recruitment of M2 macrophages, and elevated numbers of brown/beige adipocytes in white adipose tissue, particularly in the subcutaneous depot . We furtpiration . Interesse (HSL)   . Based oMore recently, we found that inhibition of DES1 in adipose tissue improved glucose metabolism and resolved hepatic steatosis. Interestingly, these observed improvements were independent of browning/beiging of adipose tissue . Using aSptlc1 or 2, respectively, in adipose tissue impaired adipose differentiation and exhibited lipodystrophy  in beige adipocytes ex vivo  for ceramide actions, the numerous effects  elicited by ceramide seems unlikely to be fully explained solely by PP2A-Akt axis or PKCζ-Akt axis. Therefore, identifying additional molecular mechanisms will be crucial for understanding the roles of ceramides such that the therapeutic strategy could be developed accordingly.Second, what is the role of immune-cell ceramide content in maintaining adipose tissue homeostasis? How do ceramides regulate these cell populations and how do they interact with adipocytes to gauge nutrient content?Third, how do β-adrenergic's inhibit ceramide synthesis in adipose tissue? Delineation of the molecular targets downstream of the β-adrenergic receptor may offer additional interventional strategies to target ceramide synthesis.Fourth, are there additional regulators (nutrients/enzymes) that modulate intracellular ceramide content? The recent development of the ceramide flux assays coupled with secondary biochemical assays might favor the identification of more regulators and lead to additional strategies to safely target ceramide synthesis for its therapeutic use.Fifth, how various enzymes in sphingolipids synthesis and degradation pathways coordinate to maintain lipid homeostasis, particularly in response to various local and systemic stimuli.The future research elucidating these important queries holds great promise in not only understanding how ceramides modulate nutrient sensing that underlies metabolic disease processes but also potentially identifying new therapeutic targets to combat metabolic diseases epidemic.YL and BC concieved and wrote the manuscript with inputs from CT. All authors approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pediatric Acute Liver Failure (PALF) is a rapidly progressive clinical syndrome encountered in the pediatric ICU which may rapidly progress to multi-organ dysfunction, and on occasion to life threatening cerebral edema and hemorrhage. Pediatric Acute Liver Failure is defined as severe acute hepatic dysfunction accompanied by encephalopathy and liver-based coagulopathy defined as prolongation of International Normalized Ratio (INR) >1.5. However, coagulopathy in PALF is complex and warrants a deeper understanding of the hemostatic balance in acute liver failure. Although an INR value of >1.5 is accepted as the evidence of coagulopathy and has historically been viewed as a prognostic factor of PALF, it may not accurately reflect the bleeding risk in PALF since it only measures procoagulant factors. Paradoxically, despite the prolongation of INR, bleeding risk is lower than expected (around 5%). This is due to “rebalanced hemostasis” due to concurrent changes in procoagulant, anticoagulant and fibrinolytic systems. Since the liver is involved in both procoagulant  and anticoagulant  protein synthesis, PALF results in “rebalanced hemostasis” or even may shift toward a hypercoagulable state. In addition to rebalanced coagulation there is altered platelet production due to decreased thrombopoietin production by liver, increased von Willebrand factor from low grade endothelial cell activation, and hyperfibrinolysis and dysfibrinogenemia from altered synthetic liver dysfunction. All these alterations contribute to the multifactorial nature of coagulopathy in PALF. Over exuberant use of prophylactic blood products in patients with PALF may contribute to morbidities such as fluid overload, transfusion-associated lung injury, and increased thrombosis risk. It is essential to use caution when using INR values for plasma and factor administration. In this review we will summarize the complexity of coagulation in PALF, explore “rebalanced hemostasis,” and discuss the limitations of current coagulation tests. We will also review strategies to accurately diagnose the coagulopathy of PALF and targeted therapies. Pediatric acute liver failure (PALF) is a life-threatening illness, characterized by the sudden onset of coagulopathy, thrombocytopenia, and systemic inflammation that may ultimately result in dreaded complications such as cerebral edema and hemorrhage. The liver is responsible for the synthesis of a majority of blood coagulation factors and plays a central role in the hemostatic system. As a result of decreased coagulation factor synthesis, acute liver failure patients were historically believed to be “autoanticoagulated” and protected from thrombosis. However, this belief has been challenged as the understanding of the coagulopathy in liver failure has evolved. In acute liver failure, there is a parallel reduction of anti-coagulant factors along with pro-coagulant factors and this leaves the patient in a rebalanced coagulation state that may sometimes favor thrombosis –7. ConseSince PALF is defined as severe acute hepatic dysfunction with prolongation of International Normalized Ratio (INR) >1.5 with encephalopathy, close monitoring of INR and correction of elevated levels occur frequently . AlthougThe hemostatic system consists of three major arms. Primary hemostasis involves formation of the primary platelet plug on the injured vascular endothelium. Secondary hemostasis involves the cascade of coagulation factors leading to formation of the fibrin clot. Fibrinolysis follows primary and secondary hemostasis; and involves a process that removes the fibrin clot and prevents uncontrolled thrombosis , 18.In acute liver failure, all three of these arms of hemostasis are affected. The delicate balance between prevention of bleeding and excessive clot formation is impacted by the concomitant changes in pro- and anti-hemostatic factors in PALF. These changes are a result of decreased hepatic synthesis of pro- and anti-coagulation factors, thrombocytopenia, elevated von Willebrand factor (vWF), reduced a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), reduced vitamin-K dependent carboxylation of pro- and anti-coagulant factors, and consumptive coagulopathy , 19.Platelets are responsible for the initial primary hemostatic response by adhering to the damaged blood vessels and promoting aggregation and clot formation with the aid of the endothelial-derived protein vWF. They are also responsible for facilitating the Tissue Factor (TF) and factor VII complex formation, which catalyzes the activation of factor X and formation of “priming thrombin”  Figure .Both platelet number and function are reduced in PALF, although the changes are less pronounced compared to chronic liver failure , 9. ThroThis thrombocytopenia is compensated, however, by elevated levels of adhesive protein vWF and decreased ADAMTS-13 . While vThrombocytopenia is also compensated by the presence of platelet-derived extracellular vesicles (EV); microparticles (0.1–1 μm) which are highly pro-coagulant and contribute to ongoing activation of coagulation . RecentlOverall, thrombocytopenia in ALF is compensated by elevated levels of vWF, reduced ADAMTS-13 levels and presence of platelet-derived extracellular vesicles that partially offset the impact of low platelet count.Secondary hemostasis is characterized by the formation of an insoluble fibrin clot by activated coagulation factors and thrombin. Fibrin in turn stabilizes the primary platelet plug to stop the hemorrhage.Reduction of coagulation factors is more extensive in acute liver failure compared to chronic liver failure, and factor levels can be as low as 1–10% of normal values , 21. DueFibrinogen, an essential coagulation factor produced by the liver, it is the precursor of fibrin and is necessary for clot formation. Pediatric acute liver failure is accompanied by hypofibrinoginemia, though the reduction in fibrinogen levels is modest compared to the other pro-coagulant factors . DysfibrVitamin K is essential for the synthesis of both pro-coagulant factors and anti-coagulant factors by the liver. Unlike in chronic liver failure, vitamin K deficiency is uncommon in PALF unless there is coexisting biliary tract disease or prolonged use of gut sterilizing broad-spectrum antibiotics. Deficiency has the potential to worsen the associated coagulopathy, and so differentiation between vitamin K-dependent and vitamin K non-dependent coagulopathy should be part of initial evaluation in PALF .Fibrinolysis is the process by which the body breaks down clots. Fibrinolysis limits the extent of thrombosis, begins clot degradation, and maintains vascular patency. It starts with conversion of plasminogen to plasmin by the pro-fibrinolytic drivers tissue Plasminogen Activator (t-PA), urokinase-type Plasminogen Activator (u-PA), and activated factor XII. At the same time these pro-fibrinolytic drivers are opposed by anti-fibrinolytic drivers such as Plasminogen Activator Inhibitor type 1 (PAI-1), Plasmin inhibitor, and Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)  which is an important index of fibrinogenolysis may help differentiate true DIC from liver associated coagulation dysfunction –32. DecrIn summary, contrary to the common belief that acute liver failure patients are “autoanticoagulated,” they may be more prone to thrombosis rather than bleeding. There is a combination of (a) thrombocytopenia compensated with elevated levels of vWF, factor VIII and reduced ADAMTS-13, (b) a hypofibrinolytic state due to elevated PAI-1 and low plasminogen, and (c) elevated levels of pro-coagulant microparticles that may all lead to an increased risk of thrombosis and worsen outcome , 24.in vivo inhibitors, fibrinolytic enzymes, and other cellular components. These tests do not provide information on actual fibrin formation or clot lysis. Lack of predictive power can be explained by the deficiency of anti-coagulant proteins which are reduced in parallel with pro-coagulant factors  have limitations for evaluating the coagulopathy of liver disease. Specifically, the PT/INR measures only procoagulant factors and omits the contribution of the platelets,  factors , 35.The international normalization ratio (INR) is commonly used as a prognostic and decision-making tool when calculating the Pediatric End Stage Liver Disease Model (PELD) score which includes INR, bilirubin, albumin, and growth velocity of the child. International Normalized Ratio is a valuable biomarker for prognostication and determination of organ allocation; however it is inadequate for the measurement of bleeding risk. In children undergoing liver biopsy, hemorrhagic complications can occur in 0.91–4.2% of cases and coagulopathy markers do not predict bleeding complications , 37.International Normalized Ratio calculation is derived from the Prothrombin Time (PT) and is standardized across laboratories among patients receiving vitamin K antagonists. Thrombomodulin is essential for PT measurement. Commercially available Prothrombin time tests do not contain a sufficient amount of thrombomodulin, and although the PT measures the function of pro-coagulants, it fails to measure the thrombin inhibited by the anticoagulants . ThrombiFurthermore, there is a significant laboratory to laboratory variation in the INR tests from patients with liver disease . One stuWhile standard assays of hemostasis are limited and misleading to evaluate hemostatic status in PALF, viscoelastic hemostatic assays (VHA) can accurately evaluate the current state of rebalanced hemostasis by measuring clot formation, ultimate clot strength, and the stability of the clot by integrating the contribution of platelets .Viscoelastic hemostatic assays (VHA) such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are whole blood tests that are a functional measure of clot formation and degradation in real time. Viscoelastic hemostatic assays measure the time to initial fibrin formation, the rate of clot formation, the strength of the clot, clot lysis and the contribution of sepsis-induced heparinoids to coagulation abnormalities. Since clot formation is the endpoint, VHAs have an obvious advantage over thrombin generation tests which have thrombin generation as the endpoint rather than formation of the fibrin clot , 14.Both TEG and ROTEM are commercially available point-of-care tests of whole blood coagulation. For TEG, blood is placed into an oscillating cup and treated with calcium and a kaolin-cephalin reagent. As the blood starts to clot, the viscoelasticity changes, and the blood starts to exert force on a pin that is suspended in the blood. The torque on the pin is converted into an electrical signal that results in a graphical representation of the clot formation and strength over time. As fibrinolysis begins and the clot starts to degrade, the force on the pin decreases and the resulting graph shows the clot dissolving. ROTEM is performed similarly, but with blood placed into a stationary cup, and an oscillating pin suspended into the blood sample. ROTEM also offers the possibility to use different activator reagents to assess different components of coagulation independently  TEG parameters are within normal limits for adult ALF patients . Data onHemostasis in children is an evolving process; there are age-dependent differences in the quantity and quality of hemostatic proteins that may further complicate the diagnosis of coagulopathy in children . A recenThe coagulopathy and bleeding risks in acute and chronic liver failure are significantly different. Since portal hypertension is not significant in PALF and the thrombocytopenia is mild, bleeding risk is usually less compared to chronic liver disease .There are no transfusion guidelines available for PALF and this leads to the substantial use of prophylactic blood products. There are no current pediatric cutoff values for the use of prophylactic transfusions of plasma, yet in adults an INR value of >7 with TEG confirming marked prolongation of clot formation, FFP transfusion is advised to maintain an INR between 5 and 7 . In PALFViscoelastic hemostatic assay tests, in contrast, may be able to provide the clinician with a more accurate assessment of coagulopathy. TEG and ROTEM have been used to develop resuscitation guidelines in adult trauma patients –41 and t3 for liver biopsy, and INR > 1.5 and platelet count of >50,000 mm3 for intracranial pressure monitor placement  and recombinant activated factor VII (rFVIIa) . ProthrorFVIIa given at a dose of 20–40 mcg/kg, administered 30 min before the procedure has a rapid onset of action and a low volume of infusion making it another option for use prior to invasive procedures . HoweverTreatment goals in PALF should focus on acute bleeding management and prevention of infection, uremia, and GI bleeding, rather than prophylactic correction of laboratory values.Antifibrinolytic agents such as aminocaproic acid and tranexamic acid (TXA) inhibit plasmin and prevent fibrin clot degradation. Although it has been shown that TXA may reduce blood loss during liver trauma or liver transplantation, there is no proven benefit for acute bleeding in PALF . These aBacterial infections in ALF may potentiate the risk of bleeding, although the mechanism is speculative. Endotoxins and cytokines produced during infection may induce disseminated intravascular coagulation (DIC), inhibit platelet function, and enhance the effects of nitric oxide. Therefore, appropriate antibacterial prophylaxis and treatment of known infection is recommended .Renal failure is also a common complication of acute liver failure. Uremia may further hinder primary hemostasis due to platelet dysfunction and impaired platelet-vessel wall interaction . PreventClinically significant gastrointestinal bleeding is rare in PALF, however the American Association of Study of Liver Disease (AASLD) guidelines recommend histamine (H2) blockers or proton pump inhibitors for prophylaxis of gastric bleeding in ICU setting , 34, 48.There are no guidelines for the resuscitation of acute bleeding in acute liver failure and transfusion of blood products have been associated with increased morbidity . RestricA catastrophic bleeding in PALF is very rare, most bleeding complications are clinically insignificant. Spontaneous bleeding usually due to self-limited upper gastrointestinal bleeding or post-procedural bleeding due to ICP placement . In caseHemostasis in pediatric acute liver failure exists in a rebalanced state due to concomitant changes in pro- and anti-coagulant mechanisms. This rebalanced hemostasis and lack of additional risk factors for bleeding , leads to a low rate of spontaneous significant bleeding in PALF.Current screening coagulation tests, such as the INR and PT are not predictive of acute bleeding and have certain limitations. International Normalized Ratio is useful for assessing severity of disease, however its use to evaluate one's bleeding risk is limited. Prophylactic FFP transfusion has not been shown reduce the risk of spontaneous bleeding and is not recommended in PALF. Arbitrary FFP and blood product transfusions may harm the patient with increased risks for fluid overload, cerebral edema, and elevated intracranial pressure. In addition, they will obscure the INR trend as a prognostic marker. Coagulopathy should be carefully evaluated and treated prior to high-risk procedures. Utilization of viscoelastic tests in PALF patients may provide an accurate assessment of bleeding risk, prevent unnecessary administration of blood products, lessen complications, and improve outcome. Further studies are necessary to characterize the coagulopathy in children with acute liver failure and develop treatment guidelines.Understanding the pathophysiology of coagulopathy in PALF is important when evaluating pros and cons of transfusion and maintaining the delicate hemostatic balance. Etiology of PALF is different from adults, and there are age specific differences in pro-coagulant and anti-coagulant factors. It is important to maintain the fragile balance of hemostasis with restrictive transfusion strategies, and prevention of infection and uremia.YB prepared the initial draft. GR and AS revised and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sexual risk, substance use, and mental ill health constitute a syndemic of co-occurring, mutually reinforcing epidemics amongst men who have sex with men (MSM). Developed since 1995, e-health interventions offer accessible, anonymous support and can be effective in addressing these outcomes, suggesting the potential value of developing e-health interventions that address these simultaneously amongst MSM. We conducted a systematic review of e-health interventions addressing one or more of these outcomes amongst MSM and in this paper describe the theories of change underpinning relevant interventions, what these offer and how they might complement each other.We identified eligible reports via expert requests, reference-checking and database and Google searches. Results were screened for reports published in 1995 or later; focused on MSM; reporting on e-health interventions providing ongoing support to prevent HIV/STIs, sexual risk behaviour, substance use, anxiety or depression; and describing intervention theories of change. Reviewers assessed report quality, extracted intervention and theory of change data, and developed a novel method of synthesis using diagrammatic representations of theories of change.Thirty-three reports on 22 intervention theories of change were included, largely of low/medium-quality. Inductively grouping these theories according to their core constructs, we identified three distinct groupings of theorised pathways. In the largest, the ‘cognitive/skills’ grouping, interventions provide information and activities which are theorised to influence behaviour via motivation/intention and self-efficacy/perceived control. In the ‘self-monitoring’ grouping, interventions are theorised to trigger reflection, self-reward/critique and self-regulation. In the ‘cognitive therapy’ grouping, the theory of change is rooted in cognitive therapy techniques, aiming to reframe negative emotions to improve mental health.The synthesised theories of change provide a framework for developing e-health interventions that might holistically address syndemic health problems amongst MSM. Improving reporting on theories of change in primary studies of e-health interventions would enable a better understanding of how they are intended to work and the evidence supporting this. The novel diagrammatic method of theory of change synthesis used here could be used for future reviews where interventions are driven by existing well-defined behaviour and behaviour change theories.CRD42018110317PROSPERO The online version contains supplementary material available at 10.1186/s13643-020-01523-2. Despite major advances in treatments and pharmacological prevention, men who have sex with men (MSM) continue to experience increased risk of infection with HIV and other sexually transmitted infections (STIs)  and highSexual risk, substance use and mental ill health are increasingly considered to constitute a syndemic of simultaneous, mutually reinforcing epidemics. There is consistent evidence that these outcomes can inter-correlate strongly at the level of the individual and the sexual event –18. DrugStigma and discrimination pose barriers to appropriate services for MSM worldwide –26. Onlie-Health interventions, facilitated via mobile phones, internet or other electronic communication technology, can be delivered in a variety of formats such as text messaging, smartphone/mobile apps, games and other online content. e-Health interventions aim to promote healthy behaviours and mental health, for example, by setting and reviewing goals; increasing/maintaining motivation; providing feedback on behaviour and challenging thought patterns that obstruct change. There is good evidence from systematic reviews focused on general or mixed populations that e-health interventions can reduce alcohol use  and addrWe therefore conducted a systematic review of e-health interventions addressing sexual health, substance use and/or mental health amongst MSM to explore the approaches and theories of how the interventions were intended to work to improve targeted health outcomes (“theories of change”) employed; factors promoting or impeding delivery or receipt of such interventions; their effectiveness in improving health outcomes and whether such interventions are cost-effective. We reviewed relevant interventions that targeted our three health outcomes of interest separately or together to maximise learning about what interventions exist that might be harnessed to address this syndemic and what the evidence from interventions addressing some but not all of these outcomes suggest could be developed to target all three holistically.What theories of change inform e-health interventions addressing HIV/STIs, sexual risk behaviour, substance use or common mental illness symptoms amongst MSM?What overarching theories of change are suggested by the theories of change used for included interventions?In this paper, we report findings from our synthesis of intervention theories of change to understand how these interventions are intended to work. Intervention theories of change typically draw on existing scientific theories of behaviour (which consider factors that predict behaviours) and/or existing scientific theories of behaviour change . Intervention theories of change make explicit the hypothesised mechanisms through which intervention activities are intended to generate outcomes , helpingOur methods were guided by a protocol prospectively registered on PROSPERO in September 2018 (registration number CRD42018110317) .In this paper, we report what intervention theories of change were used in eligible studies that were retrieved as part of a broader systematic review. Reports eligible for the broader review were published in 1995 or later ; focused on gay, bisexual and other men (including cisgender and transgender men) who have sex with men; and reported on e-health interventions delivered via mobile phone, internet or other electronic communication technology that aimed to provide ongoing support to populations consisting entirely or principally of MSM to prevent HIV, STIs, sexual risk behaviour, alcohol and drug use or common causes of mental ill health (anxiety or depression). We excluded e-health interventions merely facilitating one-off as opposed to ongoing support, those addressing HIV self-testing, clinic attendance or STI partner notification only, and interventions delivered by human providers via electronic media. There were no limitations by language or geographical location of studies. Reports eligible for inclusion in the theory of change synthesis described intervention theories of change, logic models or theorised mechanisms of effect.ClinicalTrials.gov; the World Health Organisation International Clinical Trials Registry Platform; the EPPI-Centre Trials Register of Promoting Health Interventions); Google for non-governmental organisation and governmental publications (limited to the first 100 results); the OpenGrey database; and references of included reports. We contacted experts to request eligible reports. Citations were uploaded to EndNote and de-duplicated. Database and trials register searches were updated 22-27 April 2020.Our search strategy, refined after piloting, covered two core concepts: men who have sex with men; and e-health. Publication date was limited from 1995 to now and no language limits or limits by study methodology were added. We searched 19 health and social science bibliographic databases, 23 October-26 November 2018. These were ProQuest Applied Social Sciences Index & Abstracts (ASSIA) (1987-current); Campbell Library (complete database); CRD Databases (complete databases); EBSCO CINAHL Plus (complete database); Wiley Cochrane Library (complete database); EPPI-Centre Database of Health Promotion Research (Bibliomap) (complete database); ProQuest Dissertations & Theses Global (1951-current); OvidSP EconLit ; OvidSP Embase (1980 to 2018 week 43); OvidSP Global Health (1910 to 2018 week 41); OvidSP Health Management Information Consortium (HMIC) (1979 to July 2018); ProQuest IBSS (1951-current); Ovid MEDLINE(R) and Epub Ahead of Print, In-Process & Other Non-Indexed Citations and Daily ; OvidSP PsycInfo ; Web of Science Science Citation Index Expanded ; Elsevier Scopus (complete database); OvidSP Social Policy & Practice (201807); Web of Science Social Sciences Citation Index Expanded ; ProQuest Sociological Abstracts (1952-current as of 29 October 2018). Full details of the search strings for each are available at the London School of Hygiene & Tropical Medicine’s Data Repository  and fullTwo reviewers piloted an inclusion criteria worksheet by screening batches of the same 50 references, resolving disagreements by consensus. After piloting achieved an agreement rate of at least 95%, each reference was screened on title and abstract by one reviewer. A second round of screening with a comparable piloting process then focused on full study reports. Where reports provided insufficient information on the intervention to determine eligibility, we contacted study authors. Two reviewers independently extracted data from each eligible report on intervention theory of change constructs and mechanisms, the evidence presented in support of the theory of change and how the theory of change was developed. We extracted data on intervention theory of change as well as the existing scientific theories of behaviour and behaviour change that informed these.Two reviewers independently assessed the quality of each report included in the theory of change synthesis (i.e. each “theory report”) using a tool used in our previous systematic reviews , 52, modWe undertook synthesis of author narratives describing theories of how interventions were intended to generate outcomes. We aimed first to summarise theories of change for specific interventions and then to examine whether there were one or more overarching theories of change relevant across different interventions. Theory synthesis commonly uses a meta-ethnographic approach, originally developed to synthesise findings across multiple qualitative studies , and nowWe initially planned to undertake line-by-line coding of theory reports in order to identify narrative themes within, and common themes across, intervention theories of change as we have done in previous theory syntheses , 52. In Where more than one publication reported on the same intervention, reviewers drew on the theory of change descriptions from all relevant reports to inform the diagram. The two reviewers then met to compare the two diagrams for each intervention and reconcile discrepancies through discussion. Drawing on the strengths of each, we developed an overall diagram of each intervention’s theory of change, which included intervention components, mediators and moderators (where discussed by authors) and intended outcomes. Where author descriptions implied but did not explicitly state inter-relationships between components of the theory of change, reviewers made inferences and noted where the diagrams were in part based on such inferences.Finding that the theory of change approaches underpinning the interventions were not patterned by targeted outcomes, we took an inductive approach, grouping diagrams of theories of change that shared important constructs. Then, using reciprocal translation (to identify and describe similar concepts occurring across theories of change underpinning different interventions), refutational synthesis (to identify contradictory or opposing concepts occurring across theories of change) and line of argument (to synthesise distinct elements occurring across theories of change that form part of a broader whole) approaches from meta-ethnography , each reWe documented each stage of this process, noting where theory of change components or relationships between these components differed between individual diagrams within the grouping; the approach taken to synthesise these components ; and the resulting decision for the synthesised theory of change diagram. The two reviewers then met to compare their respective synthesised diagrams for each grouping, reconciling discrepancies and drawing on the strengths of each to develop a single synthesised theory of change diagram for each theory of change grouping. To demonstrate this process, Additional file In this application of meta-ethnographic methods to the synthesis of theories of change, our first-order constructs were the theory of change information described in theory reports and represented in data extraction forms; our second-order constructs  were the reviewers’ interpretations of these concepts, represented in the intervention-specific theory of change diagrams and our third-order constructs  were the higher-order interpretations, represented by the diagrams of the synthesised theories of change developed for each inductive grouping.Our search yielded 26,044 unique references, of which 37 were included in the overall review. Of these, 33 reported on intervention theories of change see Fig. .Fig. 1SCognitive Vaccine Approach, both reported by Davidovich et al.—share a single theory of change [The 33 reports included in the theory of change synthesis represent 28 unique studies and 23 interventions , 58–87. f change , resultiN = 1) to 5/5 (N = 15), with agreement of 4/5 or above for more than three quarters of reports . All disagreements on independent quality assessments were resolved by discussion. Table Agreement between independent reviewer assessments was good, ranging from agreement on none of the five quality criteria  meeting none or one of the assessed criteria , 86–88; N = 23; 70%) clearly defined constructs or concepts and about half described a pathway from intervention components to intended outcomes . Slightly more than a third clearly described how theoretical constructs were inter-related . Only four (12%) described underlying mechanisms and the same number described how mechanisms and outcomes might vary by context.Quality varied across the five criteria assessed. Nearly three quarters of theory reports  model and social cognitive theory were the most commonly cited, with the former informing seven interventions , 83, 87 nagement , and we nagement .Sexpulse intervention was informed by the sexual health model, which identifies ten components essential to healthy sexuality [Role-Playing Game intervention was informed by stress and coping theory and by the emotional learning framework [Other scientific theories of behaviour commonly used to inform health interventions informed between one and three interventions each: the health-belief model , 81, whiexuality , theorisexuality , p. 2). ramework . In regaramework , p. 6).MOTIVES intervention was rooted in behavioural economics [myDEx was informed by a ‘dual processing, cognitive-emotional decision making framework’ (p. 4), which recognises that affective states (emotions) and cognitive states (thinking) both influence decision-making, and that the former might be processed more quickly than the latter [SOLVE intervention was informed by learning from neuroscience highlighting the important role emotions play in decision-making [The conomics , which ee latter . The SOLn-making .Hot and Safe M4M intervention incorporated strategies from motivational interviewing by assessing, and delivering exercises based on, participants’ ‘readiness to change’ proaches  and the iver CBT .As evidence to support intervention components and theories of change, theory reports often cited evaluations of earlier iterations of the current intervention or of similar interventions, or previous research on the scientific theories in question. Where authors discussed how the intervention theory of change was developed, these were commonly informed by formative research, existing interventions, literature on the needs of the target group, components of the scientific theories underpinning the intervention or a combination of these.Internet-Based Safer Sex Intervention [All intervention theories of change identified intended intervention components, mediators and outcomes which could be incorporated into an intervention-specific theory of change diagram except for the theory underpinning the rvention , which drvention , 60, 74.Our grouping of theories of change based inductively on their key constructs resulted in three groups of intervention theories of change. The largest, the ‘cognitive/skills’ theory of change grouping , 87, 88,We drew on thirteen intervention theories of change, which varied in quality from low to high, to develop a diagram for the ‘cognitive/skills’ grouping Fig.  55, 56,, 87, 88.Interventions in this grouping provided information and exercises and incorporated modelling techniques. These activities were theorised to impact on mediators including knowledge, outcome expectancies, attitudes , peSex Positive! intervention characters modelled behaviours and this was theorised to increase self-regulatory skills [We inferred that exercises would develop behavioural skills, indicating this inference by use of a dotted line in Fig. y skills . Theory y skills , 73.Theory reports differed as to the activities intended to influence outcome expectancies, attitudes, perceived norms and perceived vulnerability/risk. We portray the general relationship between the intervention and these mediators in the diagram by use of a block arrow. Knowledge and behavioural skills were both described as impacting behaviour either directly or via self-efficacy, so both pathways are depicted in the synthesised diagram.WRAPP intervention [Sex Positive! intervention [HealthMinder highlighted outcome expectations [Outcome expectancies—positive and negative expectations of the results of a particular behaviour —were thervention  and thisrvention . The thectations  as a keyMotivation and self-efficacy both featured prominently in this grouping’s intervention theories of change. Whilst some reports suggested that motivation and self-efficacy influence each other in one direction or the other , 68, mosWhilst some intervention theories of change included either motivation , 65 or iWe interpreted the construct of perceived behavioural control , 70 to bTheorised moderators are shown at the top right of Fig. Finally, some interventions in this grouping , 77 and Smartphone Self-Monitoring [TXT-Auto intervention [We defined a grouping focused on theorised ‘self-monitoring’ Fig.  by synthnitoring , an intervention , a sexuaSmartphone Self-Monitoring suggested the need for users to first establish criteria for their desired behaviour, such as personal norms or standards, against which they can assess their actual behaviour [Both interventions in this grouping asked the user questions about their behaviour to prompt self-monitoring, and both were driven by self-monitoring as a key mechanism. Frequency of self-monitoring varied by study and outcome, ranging from four times per day  to weeklehaviour . HoweverTXT-Auto suggested the intervention worked via two non-intersecting pathways, one captured in the ‘social/skills’ synthesised theory of change and one featuring self-monitoring. Arguing that pathways from self-monitoring to behaviour change are underdeveloped, Swendeman et al.’s research drew on qualitative data to further explicate the theory of change for Smartphone Self-Monitoring [Whilst both theory reports included in this grouping suggest processes of change are more complex than depicted in this synthesised theory, neither specified further self-monitoring mechanisms prospectively. The theory of change underpinning nitoring .Online Mindfulness-Based Cognitive Therapy [Rainbow SPARX [We drew on theories of change underpinning  Therapy  and Rainow SPARX , a compuRainbow SPARX intervention) [Activities in the synthesised theory of change diagram include CBT approaches (forming the core of the vention)  and mindvention) . Whilst Rainbow SPARX [Online Mindfulness-Based Cognitive Therapy focusing on accepting and ‘letting go’ of them [Rainbow SPARX suggested this was done by providing information to promote young people spending more time with those who accepted them and reducing exposure to homophobic bullying [Online Mindfulness-Based Cognitive Therapy did not make clear how this would be achieved [Theory of change descriptions for both interventions referred to recognising, paying attention to or developing awareness of thoughts, feelings and situations, with ow SPARX  then cha of them . We use  of them , 75. Theachieved . DescripSexpulse intervention did not specify components or mechanisms and so could not be synthesised [MOTIVES [SOLVE intervention [Role-Playing Game aimed to improve mental health and reduce substance use via use of productive coping strategies with a focus on help-seeking [Internet-Based Safer Sex Intervention [MOTIVES intervention [Internet-Based Safer Sex Intervention [Whilst informed by the sexual health model, the theory of change underpinning the thesised , 86. Fouthesised , 74, 76.rvention  was driv-seeking . InformeFor this review, we synthesised the theories of change underpinning e-health interventions targeting HIV/STIs, sexual risk behaviour, substance use and mental ill health amongst MSM. We developed a novel approach to do so by using diagrams rather than narrative themes to summarise theories of change within and across interventions.Whilst we have previously conducted reviews synthesising intervention theories of change using line-by-line coding of descriptive text , 52, we Social cognitive theory and the IMB model featured most prominently in intervention theories of change and informed a small majority. Whilst reports cited a number of other existing scientific theories, each informed only a few interventions at most. In the ‘cognitive/skills’ synthesised grouping, based on theory of change descriptions assessed as ranging from low to high quality, information and exercises are theorised to influence behavioural skills and a range of distal cognitive mediators theorised to influence behaviour via motivation/intention and self-efficacy/perceived control. The synthesised theory of change suggests that particular strategies can boost intervention impact, whilst other factors relating to the participant or  their partner moderate impact.Though represented in fewer interventions, two other distinct theories of change groupings emerged. The ‘self-monitoring’ grouping, based on theory of change descriptions assessed as of low and high quality, focuses more narrowly on the role of self-monitoring in triggering reflection, self-reward/critique and behavioural self-regulation. Theory reports suggest this synthesised diagram represents the basic components of more complex cognitive pathways  and can Theories of change included in this review were informed by scientific theories that have been associated with greater impacts in reviews of e-health interventions specifically, including the transtheoretical model , 45 and As is common with e-health behavioural interventions , 48, theFurthermore, we found that included interventions drawing on multiple theoretical approaches , 75, 78 It was notable that some existing scientific theories that informed theories of change were theories of behaviour change  whilst others were theories of behaviour and its determinants . Authors generally did not report drawing on scientific theories oriented more explicitly towards selecting strategies for enacting behaviour change, such as the Behaviour Change Wheel  or the EOur synthesis aimed to develop overarching theories of change for different sub-types of e-health interventions to address HIV/STIs and sexual risk, substance use and mental ill health amongst MSM, which we hope will help inform future interventions. Intervention developers might also draw on existing scientific theories which aim to integrate existing scientific theories of behaviour and behaviour change, such as the PRIME and COM-B models , 104.Our synthesis is limited by the quality of the existing theory reports, which did not often describe clear pathways from activities to intended outcomes. In some cases, reviewers inferred relationships between theory of change constructs (denoting assumptions in the theory of change diagrams), and in others the relationships between specific activities, mediators and outcomes could not be determined. Whilst our approach to theory of change synthesis enabled us to systematically explore constructs and the relationships between them across intervention theories of change, synthesised diagrams do not capture aspects that theory reports suggest influenced theories of change where their role was not clear enough to be included in intervention-specific diagrams.We did not assess the parsimony of theories of change, because we have found in past reviews that this is very difficult to consistently operationalise as a criterion of quality assessment; however, this is an important feature of theories of change. We also did not aim to systematically assess the evidence base for each of the scientific theories underpinning the intervention theories of change, because this was outside the scope of feasibility for this review and would require assessing not only the evidence for the scientific theory but also the evidence for the application of that theory to the outcomes targeted in the theories of change it underpins.e-Health interventions are a promising approach for reaching MSM with targeted health interventions , and exiOur findings suggest that the quality of existing theory reports is low- to medium, with limited discussion of the inter-relationships between theoretical constructs and little attention to how mechanisms might vary by context. Improving the quality of theory reports would enable a better understanding of how interventions are intended to work and the evidence supporting this. It would also facilitate evaluations which can go beyond outcome assessment to identify which components are triggering which mechanisms of change, and to what effect . In partThe novel method of theory of change synthesis developed for this review worked well for the theories of change underpinning included interventions. These were largely driven by existing scientific theories and featured constructs commonly underpinning behaviour change interventions such as self-efficacy, motivation, behavioural intentions, attitudes and perceived norms. This approach can be used for similar reviews in which line-by-line coding does not effectively capture the relationships between theoretical constructs. Our forthcoming synthesis of outcome evaluations of e-health interventions addressing sexual health, substance use and/or mental health amongst MSM will aim to explore whether particular theoretical approaches are associated with greater impact.Additional file 1. Search terms and strategy for Medline database.Additional file 2. Individual and overarching theory of change logic models for the ‘Self-monitoring’ theory group.


Congenital heart disease (CHD) is a multifaceted cardiovascular anomaly that occurs when there are structural abnormalities in the heart before birth. Although various risk factors are known to influence the development of this disease, a full comprehension of the etiology and treatment for different patient populations remains elusive. For instance, racial minorities are disproportionally affected by this disease and typically have worse prognosis, possibly due to environmental and genetic disparities. Although research into CHD has highlighted a wide range of causal factors, the reasons for these differences seen in different patient populations are not fully known. Cardiovascular disease modeling using induced pluripotent stem cells (iPSCs) is a novel approach for investigating possible genetic variants in CHD that may be race specific, making it a valuable tool to help solve the mystery of higher incidence and mortality rates among minorities. Herein, we first review the prevalence, risk factors, and genetics of CHD and then discuss the use of iPSCs, omics, and machine learning technologies to investigate the etiology of CHD and its connection to racial disparities. We also explore the translational potential of iPSC-based disease modeling combined with genome editing and high throughput drug screening platforms. CHD is the most frequently occurring birth defect, and despite technological advances in healthcare, children afflicted with CHD continue to face significant morbidity and mortality , 2. MoreCardiac malformations present at birth make up a relevant component of pediatric cardiovascular disease that constitutes a significant percentage of clinically significant birth defects, occurring in about 4 to 50 per 1,000 live births . CHD is CHD can be classified either as simple, moderate, or complex based on survival, prognosis, and frequency of complications. Simple CHD typically does not require extensive surgery to repair. Common instances of simple CHD defects include isolated congenital valve disease , mild pulmonary stenosis, and minor atrial septal defect (ASD) or ventricular septal defect (VSD) , 11. ModThere are several risk factors contributing to CHD, including genetic/familial contributors as well as environmental/non-genetic factors promoting CHD development . PrenataStudies have shown that daily folic acid deficiency during the pre- and peri-conception periods can be a CHD risk factor . ModeratMaternal exposure to pesticides and some therapeutic agents such as anti-seizure medications, thalidomide, and indomethacin tocolysis have been known to contribute to CHD development , 37. AddMoreover, accumulating data suggest racial and ethnic disparities in multiple key outcome measures for patients with CHD . Multiplvia combined effects of multiple genes interacting with environmental and/or stochastic factors) may be further investigated using modern techniques . CNVs of DNA sequences are sub-chromosomal changes resulting in a large deletion or amplification of DNA segments due to inappropriate recombination that leads to alterations in genes . CNVs arThe term “race,” in its traditional genetic conceptualization, is often determined through patterns of human variation reflected from our evolutionary history to serve as a definitive measure . IndividWhile studies have suggested that racial and ethnic differences may influence the prevalence and outcomes of CHD, the precise genetic and/or environmental causation is not well-established , 59, 60.in vitro models of human physiology (A compelling new paradigm in cardiovascular disease modeling is the use of induced pluripotent stem cells (iPSCs) and their differentiated cardiovascular cells to establish ysiology . First iysiology , 65. Patysiology . A signiysiology .Another benefit of using iPSCs derived from adult somatic cells is that they can be tailored to the unique individual genetics of patients. This generates novel insights into the molecular mechanisms of heart disease that may allow clinicians to deliver patient-specific pharmacological, genetic and cellular therapies in the future . In 2020via SNP or CNV that are most prevalent among minorities . In 2016, Tomita-Mitchell et al. performed NGS on a multigenerational family with a high prevalence of CHD, identifying a rare variant in the α-myosin heavy chain (MYH6) gene  gene . AdditioH6) gene , 70. FutTraditionally, minorities are not as well-represented in clinical trials or drug development compared to their Caucasian counterparts, a gap that may be addressed at least partially by using iPSCs to help identify drug or therapeutic applications effective in these populations. Various studies have shown the value of iPSCs in elucidating the molecular and cellular mechanisms of cardiac arrhythmias in disease states while providing a robust platform for the development novel drugs for clinical therapy . HoweverUsing iPSCs to recapitulate a clinically relevant readout in a high throughput assay, researchers are now able to leverage chemical and functional genomics  to better understand and interpret disease mechanisms for identifying novel therapeutic targets . The appAdditionally, advances in genome-editing technologies have enabled biomedical researchers to precisely edit or introduce mutations in disease-causing genes to analyze the relative contribution of a single mutation on the severity of the disease phenotype . The utiin silico predictions. Further, machine learning is being used to identify and validate causes of CHD on a population level, in one instance elucidating the relationship between air pollutants and increased fetal CHD risk in pregnant women  datasets . scRNA-sIn summary, by utilizing advances in genomics and genetic technologies in combination with iPSCs, an innovative platform can be developed to address racial disparities in mortality rates among patients with CHD . By idenMM, J-WR, and JW contributed in drafting the manuscript. AZ, GL, and AR editing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drawing on the social cognitive theory of moral disengagement, this study examined the influence of the authoritarian-benevolent style of ambidextrous leadership on follower unethical pro-organizational behavior (UPB), mediated via moral disengagement. We tested the hypotheses using a sample of 175 participants at two time points. The results indicated that authoritarian-benevolent leadership affects moral disengagement. In addition, followers in congruent dyads with low authoritarian-benevolent leadership perceived higher levels of moral disengagement than those in congruent dyads with high authoritarian-benevolent leadership. Furthermore, high authoritarian-benevolent leadership had an indirect effect on follower UPB via moral disengagement. Theoretical and practical implications and future research directions are suggested. Leaders may pay close attention to either ensuring that duties are performed or the well-being of personnel and need to choose between self-centric or follower-centric approaches to dealing with the different demands of followers in organizations. Previous studies showed that a single leadership style can no longer satisfy the diverse needs of organizations and their members. The effectiveness of ambidextrous leadership can drive organizations or leaders to manage multiple conflicts in organizations . AmbidexWe, therefore, consider that the leader should understand holistic thought that encompasses paradoxes  in the organization. Indeed, a leader in the organization tends to contemplate management issues from the perspective of paradox integration ; thus, tIn recent years, instances of UPB have taken place in domestic and foreign enterprises, which has become a key issue of concern to all stakeholders . UPB refmoral neutralization) emerged in earlier theoretical models of Nonetheless, a few pieces of research have examined the interpersonal process, which is the top-down effect of authoritarian-benevolent leadership on followers’ organizational outcomes . ScholarOverall, this study makes several contributions to the current literature. First, we add to the leadership literature by examining the authoritarian-benevolent style of ambidextrous leadership. As previous research has focused on transformational leadership and transactional leadership to test ambidextrous leadership , we examAuthoritarian-benevolent leadership is an ambidextrous leadership style in organizations. In the workplace, authoritarian leadership concentrates on followers’ performance, and the leader asks his/her followers to obey orders; benevolent leadership shows holistic concern for followers’ well-being and cares for them within the workplace . TherefrAuthoritarian leadership can be conceptualized as leaders’ behaviors that exert control over followers, exercise absolute authority, and demand unconditional obedience . When leBenevolent leadership refers to a behavior that involves long-term concerns for followers’ performance in the workplace and personal well-being in life . It is aTo advance this line of research, we take a follower-centered perspective to explore the psychological process that links authoritarian-benevolent leadership to follower behavior. From this perspective, we can better understand how leaders shape follower behavior through the self-construction and experiences of the follower. Social cognitive theory proposes that personal cognition is determined by the interaction between situational and personal factors . AccordiThe first set of moral disengagement mechanisms concerns the cognitive construal of conduct, which includes moral justification, euphemistic labeling, and advantageous comparison , and thrDrawing on the social cognitive theory of moral disengagement, we propose that perceived authoritarian-benevolent leadership will cause followers’ moral disengagement for three reasons. First, previous studies have dealt with the positive effects of authoritarian leadership and benevolent leadership on follower deviant behaviors such as unethical decision making , turnoveLast, to a certain extent, moral disengagement depends on how the actor views the recipients as the target of the behaviors. Authoritarian leadership has been found to be powerful and controlling , but wheHypothesis 1: The more aligned the levels of authoritarian leadership and benevolent leadership are , the greater the moral disengagement.As indicated earlier, ambidextrous leadership integrates two different complementary leadership behaviors: authoritarian leadership and benevolent leadership . We, theLeaders may show different degrees of authority and benevolence, which lead to changes in followers’ psychological perceptions involving moral disengagement. Specifically, when authoritarian-benevolent leadership is at a high level, on the one hand, the leader seizes all the power to enable followers to actually execute the work instructions . Also, tWhen authoritarian-benevolent leadership is at a low level, followers observe and endure an unfriendly leadership style, which is low in benevolence, and they sense that they can do whatever they want. That is, leaders do not pay attention to their followers inside and outside the workplace. The low level of leader authority prompts followers to slacken their vigilance and become negligent in the workplace . Thus, iHypothesis 2: Moral disengagement is higher when leadership is low in both authoritarianism and benevolence  rather than when benevolent leadership is aligned with a high level of authoritarian leadership (high authoritarian-benevolent leadership).According to the social cognitive theory of moral disengagement, moral disengagement deactivates normative action and moral self-regulation processes , so thatHypothesis 3: Moral disengagement mediates the relationship between authoritarian-benevolent leadership and follower UPB.Data were collected from multiple companies that provided IT services, located in Shandong province, China. We followed the suggestion of Most measurement scales were originally written in English; thus, we translated them into Mandarin Chinese using We adopted Moral disengagement was measured with We used As prior research has shown that demographic characteristics may influence the extent to which individuals engage in unethical behavior , we contWe used SPSS 22.0 to analyze the correlations and regressions and conducted a set of confirmatory factor analyses (CFAs) using AMOS 18.0. To test hypotheses 1 and 2, we used the polynomial regression and response surface methodology . CompareZ represents moral disengagement, X is benevolent leadership, and Y is authoritarian leadership. Hypotheses 1 and 2 suggest that authoritarian-benevolent leadership influences follower moral disengagement, so we used the estimated coefficients as well as the slopes and curvatures along the (in)congruence line for the polynomial regressions in predicting moral disengagement. To test the indirect effects of authoritarian-benevolent leadership on follower UPB via moral disengagement (hypothesis 3), we chose the block variable approach  had a superior model fit over a one-factor model , as shown in Before testing our hypotheses, we conducted CFAs to examine the discriminant validities . ResultsWe conducted Harman’s one-factor test by using exploratory factor analysis for all the independent variables (except demographics) to check common method bias . The firr = 0.28, p < 0.01); benevolent leadership was positively related to UPB ; authoritarian-benevolent leadership was positively related to UPB ; authoritarian-benevolent leadership was positively related to moral disengagement ; moral disengagement was positively related to UPB .We present the means, standard deviations, and correlations among all the variables in p < 0.05). X = −Y line shows that moral disengagement was higher when authoritarian leadership was aligned with benevolent leadership, and any deviation from the congruence line increased moral disengagement; hypothesis 1 was supported. Second, to test hypothesis 2 , the results show that the slope of consistency (X = Y) was significant , illustrating that when authoritarian-benevolent leadership was low, followers had more moral disengagement. Thus, hypothesis 2 was verified.To test hypothesis 1, Finally, to test the indirect effect of authoritarian-benevolent leadership with UPB via moral disengagement (hypothesis 3), we referred to the suggestion of In the present study, based on the social cognitive theory of moral disengagement , we propFirst, our research contributes to the leadership literature field. Current research in this field has predominantly focused on linking outcomes of leadership with positive paradigms , but theSecond, we contributed to the study of leadership by adopting a follower-centered perspective and a psychological perspective to discuss the link between authoritarian-benevolent leadership and moral disengagement. Previous research has investigated the mechanism and effects of authoritarian leadership or benevolent leadership on followers’ destructive behaviors cf. . AdoptinThird, we uncovered a unique and important psychological mechanism to explain the effects of authoritarian-benevolent leadership on followers’ UPB by adopting a social cognitive perspective. Although research has utilized social identity theory to explain that leaders’ UPB or leadership style might cause another party’s UPB , social This research provides several valuable managerial implications for organizations. First, leaders must give thought to analyzing and discussing ethical problems in organizations. Thus, authoritarian-benevolent leadership is one style of ambidextrous leadership. Leaders can provide effective ambidextrous leadership in different situations to integrate problems and manage the organization well. Second, leaders should not simply show benevolent leadership or authoritarian leadership but must attach great importance to and cultivate authoritarian-benevolent leadership. In particular, authoritarian-benevolent leadership influences follower performance , as leadOur research has several limitations and indicates several suggestions for future research. First, the data were collected from a single source who may have answered the sensitive self-report in a socially desirable way , which could introduce common method variance, despite the fact that we collected the data at two different time points. Therefore, future research could collect variables from different sources or conduct a longitudinal study and also follow the methodology of The datasets generated for this study are available on request to the corresponding author.An ethics approval was not required per the author’s institutions’ guidelines and national regulations. For the participants, we sent online questionnaires, which directly link to individual members. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The cover page of the questionnaire explained the study objectives, the voluntary nature of the survey, and an assurance of confidentiality to participants. We were exempt from further ethics board approval since this research did not involve human clinical trials or animal experiments.K-HS was responsible for idea generation, manuscript writing for the theoretical part, and data collection. NT has been involved in idea generation and data analysis. H-YL was responsible for the initial method part writing. All authors reviewed and approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.