During neuronal development, the formation of neural circuits requires developing axons to traverse a diverse cellular and molecular environment to establish synaptic contacts with the appropriate postsynaptic partners. Essential to this process is the ability of developing axons to navigate guidance molecules presented by specialized populations of cells. These cells partition the distance traveled by growing axons into shorter intervals by serving as intermediate targets, orchestrating the arrival and departure of axons by providing attractive and repulsive guidance cues. The floor plate in the central nervous system (CNS) is a critical intermediate target during neuronal development, required for the extension of commissural axons across the ventral midline. In this review, we begin by giving a historical overview of the ventral commissure and the evolutionary purpose of decussation. We then review the axon guidance studies that have revealed a diverse assortment of midline guidance cues, as well as genetic and molecular regulatory mechanisms required for coordinating the commissural axon response to these cues. Finally, we examine the contribution of dysfunctional axon guidance to neurological diseases. However, despite the seemingly overwhelming challenge of orchestrating the proper wiring of the nervous system during development, neuroanatomical studies have demonstrated a striking regularity in the arrangement of neuronal projections, a consequence of their tendency to compartmentalize in the formation of discrete neuronal fascicles and their precise guidance to their proper target regions. Extensive study of the manner in which developing axons traverse the developing central nervous system (CNS) has presented strong evidence for a molecular logic underlying the organization and guidance of neuronal axons during nervous system development.The sensory and motor functions of the nervous system are central to the ability of an organism to sense and respond to the environment. These systems are inherently complex both due to the multiplicity of environmental stimuli and the extent to which an organism can sense and respond to them. The complexity of the nervous system is evident given neuronal population size and the degree of neuronal connectivity. The human nervous system is composed of over 10Ramón y Cajal first proposed the directed development of axonal projections based on his studies in embryonic chick spinal cord and the observation of a specialized structure located at the tip of the developing axon, the growth cone . In his The axonal target is not the lone source for guidance substances as proposed by Cajal . Rather In this review, we will begin by considering the general question as to why commissural projections are such a predominant neuroanatomical feature. We will then discuss the studies of axon guidance mechanisms that are relevant for commissural axon midline crossing, focusing particularly on the netrins, slits, and their growth cone receptors. While these mechanisms are widely applicable to commissural neurons in the developing CNS, we will also consider other commissural neuron populations that appear to use alternate mechanisms to cross the CNS midline, including commissural neuron populations in the forebrain and those that cross at the dorsal midline of the spinal cord. To provide a more complete picture of the role of the CNS midline in neuronal development, we will also briefly discuss ipsilaterally-projecting populations. Finally, we will explore how genetic dysfunction of genes implicated in axon guidance manifest in neurological diseases.Commissures are a common organizing principle found throughout the CNS, such that midline-crossing axons are a predominant neuroanatomical feature. Is there a functional or evolutionary advantage to this bilateral connectivity? At first glance, contralateral projections appear to be an obvious consequence of organism bilaterality and the need to coordinate sensory and motor function across the body. In the spinal cord, a prototypical example of bilateral motor control is the locomotor central pattern generator (CPG) that relies on commissural projections that cross at the ventral midline and contribute to the ventral commissure . The CPGen route to the spinal cord, where it ultimately activates spinal circuits for the initiation of voluntary movements. Proprioceptive and tactile information also projects to the contralateral CNS via secondary neurons in the caudal hindbrain that cross as internal arcuate fibers to form the medial lemniscus. This organization scheme results in the contralateral cortical processing of sensation and motor control, but it remains unclear why this neuroanatomical arrangement is present in the CNS and whether this arrangement was selected for according to functional advantage or evolutionary favorability.However, the purpose of midline crossing in other commissural neuron populations is less clear. The corticospinal tract (CST) is composed of cortical layer V pyramidal neurons. It crosses the CNS midline in the caudal hindbrain at the pyramidal decussation (a crossed tract of nerves) while Most theoretical discussions of midline crossing in the CNS begin with the observation that the first ‘decussation’ occurs outside of the CNS at the pupillary eye, where the visual representation of the external environment becomes optically transformed as in a pin-hole camera, resulting in an inverted image at the retina . ConsequAlthough Cajal’s theory remains one of the most compelling functional explanations for decussations at the optic chiasm and elsewhere in the CNS, some findings have challenged this model. Cajal hypothesized that decussation at the optic chiasm is needed for a continuous internal visual representation of the external environment. However, patients with non-decussating retinal-fugal fiber syndrome, where the optic chiasm does not form and all retinal projections are ipsilateral , show suAdditional theories of decussation have offered functional hypotheses, including the facilitation of escape behavior  and the Drosophila, these motor neurons are still able to properly respond to local, ipsilateral guidance cues to project to their mirrored target muscles on the ipsilateral side [In the formation of the decussation at the optic chiasm, both theories rely on the ability of developing RGC axons to sense ‘sidedness,’ requiring that RGC axons are capable of distinguishing between the ipsilateral and contralateral optic tectums. Several lines of evidence, however, demonstrate that commissural neurons do not necessarily exhibit an inherent preference for a contralateral target versus its mirrored, ipsilateral target. For example, despite disruptions in midline crossing of the RP3 and V motor neurons in ral side . Furtherral side –37. TogeCaenorhabditis (C.) elegans [C. elegans present an example in which decussation preceded the formation of complex visual organs, contrasting with the theory of decussation proposed by Cajal that stems from the organismal perception of the visual world.Studies of commissure formation have revealed remarkably conserved molecular mechanisms of axon guidance that coordinate midline crossing . Thus, r elegans . InteresLocusta migratoria, showed that early differentiating neurons in the peripheral sense organs and thoracic limb buds project highly stereotyped axonal trajectories toward their central targets [The formation of functional neural circuits during development requires that axons can properly sense and respond to axon guidance cues within the extracellular environment to navigate towards their appropriate postsynaptic partners. Initial evidence that axonal pathways are partitioned into a series of steps came from axon guidance studies in grasshopper embryos and observations of the trajectories of pioneering neurons. Extensions from these earliest differentiating neurons traverse the developing CNS and provide a scaffold for the construction of subsequent neuronal circuits . Observa targets , 42, sug targets . Ablatin targets , support targets , 44, the targets , 46. ThuSimilar to guidepost cells in the insect embryo, the columnar ependymal cells that comprise the FP at the ventral midline in the vertebrate embryo have been proposed to act as an intermediate target, guiding spinal commissural populations along their trajectory to the contralateral side of the CNS . StudiesThe first direct evidence of chemotropism in the CNS came from in vitro experiments using embryonic rat spinal cord, which suggested the presence of a FP-derived axon guidance cue capable of directing commissural axon outgrowth toward the ventral midline Fig. a 48, 4949. When netrin1 transcript was present in the FP, while netrin2 mRNA was present in the ventral half of the neural tube [unc6 gene product [C.elegans. Two homologs, netrinA and netrinB, were later also identified to play axon guidance roles in Drosophila [The molecular identity of the FP-derived chemoattractant was discovered by systematically screening tissues to find a factor that could mimic the outgrowth promoting activity of FP conditioned medium . Proteinral tube . The net product , 59, preosophila , 61.netrin1 mouse mutants, which included both a hypomorphic allele, identified using a β-galactosidase-encoding gene trap approach [netrin1 mutant spinal cords, Tag1+ commissural axons stall above the motor column, with the majority failing to cross the FP [+ commissural axons and neurofilament (NF)+ axons, defasciculate in the absence of netrin1, growing both dorsally and into the ventricular zone (VZ) [netrin1 mutant forebrain, including the corpus callosum, and hippocampal and anterior commissures [netrin1 mutants remain intact [+ commissural axons in the ventral spinal cord, again consistent with a long-range activity for FP-derived netrin1 [In vivo evidence for netrin1 acting as a long-range chemoattractant came from analyses of approach  and a nuapproach . In embrs the FP , 63–65. s the FP . Subsequone (VZ) , 17. Axomissures , howevern intact . More re netrin1 .netrin1 transcript in the dorsal-most spinal cord may in part permit sensory commissural axons to enter and cross to the contralateral spinal cord [Netrin1 has also been suggested to elicit repulsion for various neuronal populations during development, including trochlear motor axons  and sensnal cord , 69–72.netrin1 transcript is localized to the FP from early stages of development [netrin2 is expressed by neural progenitor cells (NPCs) in the VZ of the spinal cord [netrin2 is not expressed in the spinal cord; rather the expression pattern of netrin1 appears to be a composite of chicken netrin1 and netrin2. Thus, by the stage at which the commissural axons begin their trajectory to the midline, netrin1 transcript is present at high levels in both the FP cells and NPCs in the ventral two thirds of the spinal VZ [netrin1 transcript and netrin1 protein in mouse. The distribution of netrin1 on the pial surface has been proposed to stem from the ability of the netrin1+ NPCs to transport netrin1 protein along their radial process to their basal endfeet, where then it is deposited onto the pial surface , or from FP cells (netrin1ΔFP). In the absence of NPC-derived netrin1, commissural axons become locally defasciculated in the dorsal spinal cord and project dorsally towards the roof plate (RP), and medially into the VZ. The number of Tag1+ axons reaching the FP was profoundly reduced [Due to its complex expression in the spinal cord, the spatial requirement for netrin1 in commissural axon guidance has been assessed using conditional genetic approaches in mouse embryos –15, 17.  reduced . Similar reduced , 15. The+ axons project largely normally towards the ventral midline in the netrin1ΔFP mice. However, further analysis of the netrin1ΔFP mice revealed defasciculation of Robo3+ axons (discussed in the previous section), and more local perturbations as commissural axons reach and cross the FP [netrin1 mutant, arguing that FP-derived netrin is sufficient to guide commissural axons ventrally. While the interpretation of this study is complicated by the presence of dorsal NPC-derived netrin1 at early stages in the netrin1ΔVZ line, it seems likely that both NPC- and FP-derived netrin1 have key axon guidance activities for commissural axons. Ongoing research will resolve when and where netrin1 acts as a short-range vs long-range cue.The more recent conditional genetic studies suggested FP-derived netrin1 was dispensable for axon guidance , 14, becs the FP , 19. Recnetrin1+ domain [hedera) that is not itself penetrated by the ivy. Commissural axons always respect the edge of the NPC-netrin1+ domain, to grow around the VZ, and then adjacent to netrin1+ cells in the FP. When a small region of netrin1 expression was extinguished in the intermediate spinal cord, axons deviated from their normal trajectories to follow the new boundaries in netrin1 expression [netrin1 expression emerge adjacent to the dorsal root entry zone, which also serve as boundaries for spinal axon growth. Thus, netrin1 may supply both an adhesive substrate along which axons can grow in a fasciculated manner, while also providing a border to delineate axon tract formation. The mechanism that mediates the hederal boundary is not known, although it may require the deposition of netrin1 on commissural axons, since only netrin1− axons are observed to stray into the VZ [Studies in flies and vertebrates have also suggested that netrin1 has an additional guidance activity establishing boundaries , 76, 77.+ domain . This bopression . At lateo the VZ .C. elegans counterparts. Unc40 was proposed to be the receptor mediating ventral migration toward sources of Unc6 [Dcc is broadly expressed by spinal neurons, while Dcc protein decorates commissural axons as they grow around the VZ [Dcc null mutant embryos exhibit all of the axonal outgrowth defects observed in netrin1 mutants, including the complete defasciculation of NF+ and Robo3+ axons and their subsequent growth into the VZ [+ spinal commissural axons and severe reduction or absence of commissures in the forebrain [Dcc mutants, but it is not present on axons [Vertebrate netrin1 receptors were first identified based on homology with their  of Unc6 , 78. Clo of Unc6 , 80, and of Unc6 . By mousd the VZ  and towad the VZ . Dcc medd the VZ . Netrin1d the VZ . Most coo the VZ , the staorebrain . Intriguorebrain , 76. Neton axons .Neogenin transcript was initially thought to be absent from commissural neurons [The role of neogenin in commissural axon guidance has remained unresolved.  neurons . However neurons , 85 and  neurons . Neogeni neurons  suggest C. elegans, where the Unc5 protein is thought to mediate the repulsive activities of Unc6 [An additional family of netrin1 receptors was also identified by homology with  of Unc6 , 86. The of Unc6 , 88. Unc of Unc6 , 68. The of Unc6 , 89, 90,+commissural axons will reorient away from either RP explants or COS cell aggregates expressing Bmp7, which is present in the RP [Bmp7 mutant embryos and some axons are observed to cross the RP in Bmp7 mutant embryos in vivo [Commissural axons are never observed to cross the RP at the dorsal midline at spinal cord levels, which suggests additional mechanisms exist to orient spinal commissural axons. Commissural axons are directed initially ventrally in response to a RP-derived chemorepellent, mediated by the bone morphogenetic protein (Bmp) family . In vitrn the RP . This re in vivo . However in vivo , 94.+ commissural axons towards them [netrin1 mutants [An opposing reorientation activity is provided by the morphogen sonic hedgehog (Shh), present in the FP . In the rds them . This re mutants , but not mutants . Recent  mutants  exacerba mutants , further+ axons extends towards the midline in the dorsal spinal cord independently of netrin1 signaling [Finally, there is evidence that alternate mechanisms exist to mediate commissural axon midline attraction. Dcc and neogenin can also act in netrin1-independent manners, binding other families of ligands. Neogenin binds the family of repulsive guidance molecules (RGMs) . A Dcc iignaling .B lineage that contributes to the dorsal funiculus and dorsolateral fasciculus [In vivo studies have demonstrated that some axons approach the ventral midline only to turn abruptly away to follow an ipsilateral trajectory , suggestsciculus –105, as sciculus , 107.The mechanisms used to develop and maintain ipsilateral projections include the Robo/slit, Npn/Sema, and Eph/ephrin families of repellent guidance molecules , 108. Th+ spinal commissural axons appear to lose responsiveness to netrin1 and Shh after crossing the midline [Unlike ipsilaterally-projecting neurons, commissural neurons grow towards, cross, and exit the CNS midline  mutant: the midline glia do not form, and commissural axons accumulate at the CNS midline [slit as a key midline repellent.The  cuticle . Slit is midline , 124. Inrve cord  and ther midline , 124. A  midline , 127. Tocommissureless (comm) and robo. During development in comm mutant embryos, commissural axonal outgrowth is initially normally oriented toward the midline [robo mutant, ipsilaterally-projecting neurons acquire the ability to cross the midline, while commissural neurons now recross multiple times, resulting in thick commissures and minimal longitudinal connectives [comm and robo mutant embryos are able to appropriately reach their mirror image equivalent synaptic targets [A genetic screen identified two further genes required for commissural axon organization:  midline . However midline , therebynectives , 128. Im targets , suggestrobo mutants [comm; robo double mutant phenotype is strikingly similar to that in the robo mutant alone, Comm was proposed to function upstream of Robo, regulating its function to orchestrate midline crossing [comm functioned in relation to robo, remained elusive. Clues came from the expression patterns of Comm and Robo, which are tightly coupled with both respect to each other and the position of growth cone relative to the midline [comm hypomorphic alleles, Robo is present at higher levels on commissural axons suggesting that comm suppressed Robo levels on commissural axons [comm resembles the robo mutant phenotype: there are reduced levels of Robo protein in the commissural axons, which abnormally cross and recross the midline [comm in ipsilateral neurons enables them to cross the midline [Robo was proposed to participate in a repulsive signal that prevents axons from crossing the midline, based on the observation that ipsilaterally-projecting axons make inappropriate contralateral extensions in  mutants . Since tcrossing . However midline . In cont midline . In commal axons . In cont midline . Similar midline .Subsequent in vivo studies have suggested that Comm acts as an intracellular sorting receptor for Robo, intercepting it before reaching the growth cone in vivo –132. OurC. elegans identified sax3 and slt1 as the respective homologs of Drosophila robo and slit [robo homologs, robo1 and robo2, and three slit homologs, slit1, slit2, and slit3 [A further advance in our understanding of commissural axon midline crossing came from the discovery that Robo is an evolutionarily conserved axon guidance receptor , 136 andand slit . Initialnd slit3 , 141. Surobo1/2 and slit1/2/3 transcripts, and Robo1/2 protein in the rodent embryonic spinal cord shows remarkable similarity to their counterparts in the Drosophila nerve cord. In rodent embryos, robo1 and robo2 transcripts are expressed in overlapping patterns in many populations of neurons, while the three slit transcripts are all present in FP [The distribution of the nt in FP , 135. Bont in FP , 139, cont in FP . Togetheslit1; slit2; slit3 triple mutants [Robo1, robo2, and robo1;robo2 mutants demonstrate axon stalling and recrossing defects at the ventral midline, as well as defects in axon sorting in the ventral and lateral funiculi [+ commissural axons from the ventral midline and thereby prevent them from re-entering the midline. The axon guidance phenotype in robo1/2 double mutants is less severe than that in slit1; slit2; slit3 triple mutants, suggesting that commissural neurons may possess an additional receptor for slit [robo1/2 double mutant pathfinding defect in spinal commissural neurons that normally cross at the dorsal midline [Mouse mutant studies also supported a role for slit/Robo signaling in midline crossing. Commissural axons stall or re-cross the ventral midline in  mutants . Robo1, funiculi , 11. Thufor slit , 11. Mor midline  may unmaDrosophila homologs, no vertebrate homolog of Comm has been identified. However there are multiple candidates for functional homologues, these candidates include 1) the WAGR syndrome PRRG4, which can re-localize Robo away from the cell surface in vitro [Retinoblastoma mutant embryos [Robo3 mutant mice similarly display a lack of commissures in the hindbrain, and at the ventral midline throughout the developing spinal cord [robo3 mutants, and follow an ipsilateral pathway [robo3 mutants [Drosophila Comm. Partial rescue of the robo3 phenotype is seen in robo1; robo3 and robo1; robo2; robo3 double and triple mutants [Drosophila Comm. While robo3 mutants display a striking loss of commissures at the ventral midline throughout the developing spinal cord and hindbrain [While the vertebrate slit and Robo family members demonstrate many functional similarities with their in vitro , 2) Rab in vitro  and 3) tin vitro . Alterna embryos . It was  embryos . Robo3 mnal cord , 147. Thnal cord . This ex pathway , suggest mutants , suggest mutants , 116, su mutants , making  mutants , possiblindbrain . Thus, oMultiple human neurological disorders result from developmental errors in axonal pathfinding –152. HerROBO3 gene, which results in the loss of midline crossing in the hindbrain [ROBO3 mutations result in the complete loss of ROBO3 function [robo3 mutant mice has shown a reduction in cochlear nucleus projections that normally cross the midline [HGPPS is a rare autosomal recessive disorder stemming from mutations in the indbrain . Human Rfunction , resultifunction . The faifunction . Imagingfunction . The audfunction , 156. Ax midline , suggestrobo3 was conditionally knocked out in the hindbrain supports this analysis by reporting a reduction in contralateral projections at the level of the abducens nucleus and marginal connectivity between the abducens and contralateral oculomotor nucleus [robo3 mutant mice [The deficits in horizontal eye movement in HGPPS patients suggest that contralateral extraocular motor pathways are also affected, including contralateral inputs onto the abducens nucleus from the paramedian pontine reticular formation and projections from the abducens nucleus that target the contralateral oculomotor nucleus via the medial longitudinal fasciculus . A HGPPS nucleus . The sevant mice , 157, asant mice .robo3 mutant mice have similarly reported the persistence of forebrain commissures [Despite defects in the formation of hindbrain commissures, a common feature in HGPPS patients is the persistence of commissures at other levels of the CNS, suggesting that ROBO3-independent mechanisms play a role in the formation of these commissures. For example, the major forebrain commissures appear to be intact in HGPPS patients, including the corpus callosum . There imissures  as well missures . Functiomissures , suggestmissures , raisingCMM are involuntary movements that simultaneously accompany voluntary movements on the contralateral side of the body. They often occur as part of a neurological syndrome, including the Klippel-Feil, Kallmann, and Joubert syndromes , 159. ThDCC gene in two unrelated families with CMM. These mutations are predicted to result in either a truncated form of the receptor that cannot bind netrin1 [DCC mutations produce mirror movements because of inappropriate ipsilateral CST projections from the hindbrain [Dcc disrupt the CST at the pyramidal decussation [Dcckanga/kanga mutant mice, which are viable to postnatal ages [DCC mutations.Because CMM are a symptom in a number of neurological syndromes that are likely to have mutations at multiple genetic loci, it has remained unclear which genetic mutations specifically result in CMM. However, defects in netrin1/Dcc signaling have recently been implicated as causal factors for CMM. First, genome-wide linkage analyses identified mutations in the  netrin1 , or a fo netrin1 , resulti netrin1 . These sindbrain , 166. Duindbrain  and mutaussation . Furthertal ages , 168, thNETRIN (NTN)1 gene had not been directly linked to an inherited neurological human disease. However, exome sequencing studies have now identified three variants of NTN1 in members of two unrelated families and an unaffiliated individual with CMM. The three variants, which include two missense mutations (Cys601Ser and Cys601Arg) and one in-frame deletion (Ile1518del), all localize to the netrin (NTR) domain found at the C-terminus of the protein. Through molecular modeling software Cys601 is predicted to be important for the formation of disulfide bridges, while Ile518 is part of a beta strand [NTN1 disruption and not a secondary consequence of a neurological syndrome. The patients do not have other observable neurological defects or mutations in any of the genes previously associated with CMM [NTN1 patients demonstrated that they have an increased proportion of ipsilateral CST projections compared to control subjects [NTN1 allele affects the localization and processing of netrin1 for secretion from the cell. HEK239 and Hela cell cultures were transfected with either the control or mutated allele of NTN1, cells were cultured, the supernatant was collected and the cells were lysed to collect the intracellular fraction [NTN1 exon 7 mutation reduces the level of netrin1 in the extracellular matrix, thereby leading to reduced or aberrant crossing of axons in the CST, resulting in CCM.Until recently, mutations in the a strand . While ta strand , the NTRa strand . In theswith CMM , 172. A subjects , suggestfraction . A higheAxon guidance studies have suggested a model in which developing axons traverse a sequence of intermediate targets during development. Navigating these intermediate targets requires that developing axons respond to extracellular attractive and repulsive guidance cues, including members of the netrin and slit families, which are provided by specialized populations of cells that reside along the axonal trajectory. Commissural neuron midline crossing has provided a valuable model for the study of axon traversal at the CNS midline intermediate target and has revealed evolutionarily conserved molecular mechanisms that underlie axon guidance. Interestingly, theories of decussation have suggested that midline crossing may have been evolutionarily selected for based on its property to minimize wiring errors during development, suggesting that axon guidance studies at the CNS midline may reveal some of the fundamental aspects of CNS development and organization. Of particular interest is the fundamental property of how commissural axons regulate their responsiveness to axon guidance cues so that developing axons appropriately extend from one intermediate target to the next without stalling or recrossing previous targets. Studies of these commissural populations will advance both our basic knowledge of axon guidance in the developing CNS as well as our understanding of how axon guidance defects lead to disease.


The management of patients with acute coronary syndromes (ACS) has improved markedly in the last decades through the development of new biomarkers such as the troponins, natriuretic peptides and risk stratification scoring systems. However, there has been a shift in the epidemiological characteristics of myocardial infarction (MI) with an increasing number of patients presenting with non-ST-segment elevation MI (NSTEMI) where the ability to predict short- and long-term outcomes is more limited ,2. ExploChemokines are small heparin-binding proteins that together with their chemokine receptors are critical mediators of cell migration during immune surveillance and homeostasis. The homeostatic chemokines, CCL19 and CCL21 and their common receptor CCR7, play a pivotal role in T cell and dendritic-cell trafficking into lymphoid tissue, and their function was originally thought to be restricted to immune cell homeostasis. However, findings during the last decade suggest that these chemokines may have pleiotropic effects and are involved in inflammatory responses in non-lymphoid tissue such as atherosclerotic lesions implying a complex role for CCR7 signaling in immune responses . We and Recently, we demonstrated in a large population of ACS patients that high serum CCL21 was associated with short- and long-term major cardiovascular events (MACE) after full multivariable adjustment including troponins, natriuretic peptides and GRACE risk score . The rolThe shift in presentation of ACS with more frequent NTSEMI coincides with a change in the character of atherosclerotic plaques with less lipids and inflammation and more fibrous tissue and interaction between neutrophils and endothelial cells that are vulnerable to superficial erosion rather than plaque rupture . Matrix Finally, CCL21 was strongly correlated with cardiac dysfunction and natriuretic peptide levels supporting a role for CCL21 in maladaptive remodeling . Both knWe suggest that the CCR7/CCl21/CCL19 interaction could be an interesting molecular axis in ACS, not only as a novel and promising biomarker in these patients and in particular in NSTEMI patients, but also as a novel therapeutic target attenuating not only plaque destabilization, but also development and progression of plaque erosion and maladaptive remodeling following MI.


Currently, research based on the technology and applications of 3D printing is being actively pursued. 3D printing technology, also called additive manufacturing, is widely and increasingly used in the medical field. This study produced custom casts for the treatment of mallet finger using plaster of Paris, which was traditionally used in clinical practice, and 3D printing technology, and evaluated their advantages and disadvantages for patients by conducting a wearability assessment. Mallet finger casts produced using plaster of Paris, when incorrectly made, can result in skin necrosis and other problems for patients. These problems can be mitigated, however, by creating casts using 3D printing technology. Additionally, plaster casts or ready-made alternatives can be inconvenient with respect to rapid treatment of patients. In contrast, 3D-printed casts appear to provide patients with appropriate treatment and increase their satisfaction because they are small in size, custom-made for each patient, and can be quickly made and immediately applied in clinical practice. Mallet finger refers to a deformity in which the finger cannot be extended due to severing of the extensor tendon at the distal phalanx or fracture of the bone to which it attaches. Mallet finger can be classified as tendinous, in which only the tendon is ruptured, or bony, in which it is accompanied by a broken phalanx. Surgical intervention is required in the case of bony mallet finger. However, tendinous mallet finger is treated conservatively, primarily by using a splint to extend the finger so that the tendon reattaches naturally. Traditional conservative treatments include fixation of plaster of Paris or metal casts, whereas surgical interventions include bone fragment fixation using steel wire, fixation with lag screws, tension band wire fixation, fixation using figure-eight tension band wire, intramedullary wire fixation, and dorsal suture.Three-dimensional 3D) printing technology is widely used in industry, biology, and medicine . Major aD printinMedical applications of 3D printing are rapidly expanding, and revolutions in medical services are expected . The preSubjects of the study were selected randomly. Casts were designed for subjects' index fingers using either the traditional plaster of Paris method or MediACE 3D software (based on 3D scanning to measure the finger's dimensions).Plaster of Paris casts, which are traditionally used in clinical practice to treat mallet finger, were created as shown in First, the index finger was scanned repeatedly while maintaining posture of the extensor tendon  using a A 3D model of the mallet finger cast was then created based on the STL file  using thThe MediACE 3D software is expected to create beneficial business value in the medical industry and to contribute to implementation, distribution, and diffusion of customised medical services using 3D printing, the foundation of the 4th Industrial Revolution. It has been verified through clinical application and evaluation.A mallet finger cast for 3D printing was designed using the mallet finger STL file and MediACE 3D and then converted into G-code. The cast, shown in Application of the plaster of Paris and 3D-printed casts is shown in 3D-printed lattice casts for treatment of mallet finger were produced based on STL files obtained through 3D scanning, the MediACE 3D software, and 3D printing technology. The advantages and disadvantages of the 3D-printed casts were then compared with those of plaster of Paris casts traditionally used in clinical practice.The 3D-printed lattice casts showed results similar to those of the traditional plaster of Paris casts in the QUEST evaluation . HoweverThe lattice design of the 3D-printed casts and their customised application can prevent necrosis or infection, offer a thickness and elasticity that guards against slipping off, and resolve other shortcomings of traditional plaster of Paris casts, including oedema, discoloration, hinderance of function and circulation, pain, pulselessness, dysesthesia, and burning from pressure. In future, 3D-printed lattice casts should be used instead of plaster casts to treat mallet finger, based on their many benefits, such as providing patients with the most appropriate, customised treatment.This study produced 3D-printed lattice casts for treatment of tendinous mallet finger based on STL models of the index fingers of randomly selected subjects, obtained using a 3D Systems Sense scanner. The casts were constructed from PLA resin using a 3D Edison Printer.The advantages of traditional plaster of Paris casts are the difficulty of taking it off by patients once it is worn and short application time. Disadvantages include possible pain, skin oedema, discoloration, burning, infection, and necrosis; the complications of splint treatments are mainly skin-related.Complications occurring in 123 cases of mallet finger treated both surgically and nonsurgically were reviewed. The 84 cases of splint treatment had a complication ratio of 45%; these complications were almost always temporary. Among the 45 cases of surgical treatment, 53% experienced complications and 76% of complications developed during the average monitoring period of 38 months. Six cases that involved surgical treatment after splinting (for a minimum of six weeks) were counted in both groups. Major complications experienced by surgically treated patients were deep infection (4%), total joint incongruity (18%), and nail deformity (18%). Seven patients (16%) required reoperation, and all had unsatisfactory results  except fThe 3D-printed lattice cases were compared with plaster of Paris casts in treatment of patients with mallet finger. The results of PPP evaluation indicated that 3D-printed lattice casts allow for accurate, rapid production of customised orthoses, improving upon existing production methods. The 3D-printed casts can additionally resolve problems associated with plaster of Paris casts, namely, hyperextension and skin problems, because production is customised to the patient using 3D scanning. The size is very appropriate for 3D printer output, and it uses less material than a plaster of Paris cast and requires little output time. The ability of 3D-printed casts to overcome the disadvantages of traditional plaster of Paris casts makes them beneficial for clinical application.Development of 3D-printed lattice casts is meaningful because they have the potential to help many patients if produced in clinical practice using 3D printers. Improvements in body scanning technology and product design software are expected to bring benefits to clinical practice. Future research should investigate the production of orthoses with various structures using the processes developed in this study. The investigators hope that the present study may serve as a reference for research on the production methods of finger orthoses based on various diseases and their treatment in clinical practice, such as the various types of 3D-printed finger casts in


Nuclear receptor coactivator 4 (NCOA4) is a selective cargo receptor that mediates the autophagic degradation of ferritin (“ferritinophagy”), the cytosolic iron storage complex. NCOA4-mediated ferritinophagy maintains intracellular iron homeostasis by facilitating ferritin iron storage or release according to demand. Ferritinophagy is involved in iron-dependent physiological processes such as erythropoiesis, where NCOA4 mediates ferritin iron release for mitochondrial heme synthesis. Recently, ferritinophagy has been shown to regulate ferroptosis, a newly described form of iron-dependent cell death mediated by excess lipid peroxidation. Dysregulation of iron metabolism and ferroptosis have been described in neurodegeneration, cancer, and infection, but little is known about the role of ferritinophagy in the pathogenesis of these diseases. Here, we will review the biochemical regulation of NCOA4, its contribution to physiological processes and its role in disease. Finally, we will discuss the potential of activating or inhibiting ferritinophagy and ferroptosis for therapeutic purposes. Iron is required in multiple biological processes  includinCellular iron is mainly stored in ferritin, a protein complex composed of 24 subunits of ferritin light and heavy chains . Ferritin can chelate up to 4500 iron atoms in its ferric-Fe(III) form . Iron is−/−), Asano et al. showed that in iron-depleted cells, autophagy is required for ferritin degradation via the lysosome. Based on their work they hypothesized a selective ferritin autophagic process dependent on an as yet undetermined cargo receptor ..10]. UndThe mechanism by which NCOA4-FTH1 reaches the lysosome is not entirely clear. Despite NCOA4 association with multiple ATG8 proteins in in vitro binding assays, no canonical LC3-interaction region (LIR) motif has been identified in NCOA4, although the presence of other non-canonical ATG8-binding motifs may suggest the use of this alternative motif for interaction . While cWhile the role of NCOA4 in mediating ferritin degradation and cellular iron homeostasis appears to be a conserved function of NCOA4 across cell types, earlier reports suggested NCOA4 acts as a nuclear receptor coactivator. NCOA4 was originally identified in a yeast-two hybrid screen as a coactivator of the androgen receptor (AR), enhancing its transcriptional activity in prostate cancer . SubsequGiven the importance of NCOA4 in intracellular iron homeostasis, several studies have addressed the role of NCOA4 in physiological cellular processes that depend upon iron availability, such as erythropoiesis. Erythropoiesis consists of several differentiation steps between the hematopoietic stem cell progenitor stage and the mature enucleated red blood cell (RBC) that are characterized by gradual degradation of cellular content  and prodInitial connections between NCOA4 and erythropoiesis on an organismal level were made in zebrafish developmental studies where NCOA4 mRNA expression was found to be upregulated at sites of erythropoiesis . ConsistNCOA4 KO mice had higher ferritin in serum , suggestTo date, multiple studies have evaluated the role of NCOA4 in erythropoiesis in vivo but all of them have evaluated it in a systemic KO setting. Despite in vitro evidence in K562 and G1E-ER4 cells suggesting NCOA4 has a cell-autonomous role in erythropoiesis, it remains to be elucidated whether there are non-autonomous contributions of NCOA4 to erythropoiesis. Since NCOA4 knockout is non-fatal in these models, compensatory mechanisms may develop in erythroid cells or other cell types to overcome NCOA4 loss to support hemoglobin synthesis. Identifying these mechanisms will be of interest in the future. Finally, these data support study of NCOA4 in the context of multiple pathological conditions with imbalances in iron homeostasis like hemochromatosis or iron deficiency anemia .Ferroptosis is an iron-dependent form of regulated cell death characterized by lipid peroxidation ,55. GiveAs iron is necessary for the generation of lipid peroxides and thereby initiation of ferroptosis, ferroptosis sensitivity is impacted by compounds that decrease iron availability (iron chelation: deferoxamine (DFO)) and alterations in proteins involved in iron homeostasis that ultimately affect the cellular labile iron pool . For insInterestingly, Bellelli et al. showed the in vivo relevance of altering ferritinophagy to the ferroptosis pathway. They showed that NCOA4 depletion in mice fed with an iron-rich diet increased expression of GPx and SOD, likely due to overloaded FTH1 leading to iron leakage in tissues followed by upregulation of GPx and SOD as a compensatory mechanism to cope with oxidative stress . AdditioFerroptosis has been linked to several pathological conditions including neurodegenerative disease, ischemia/reperfusion injury and cancer. As such, there is significant interest in identifying pharmacologic means of blocking  or triggering (cancer) ferroptosis for therapeutic purposes. Given NCOA4 can modulate ferroptosis sensitivity, understanding whether NCOA4 has a role in the pathophysiology of these various diseases is critical.Neurodegenerative diseases (ND) have historically been associated with inappropriate iron accumulation and oxidative stress. More recently, ferroptosis has been shown to play a part in neurodegeneration pathophysiology . In ParkWDR45, leading to decreased autophagy, has been identified in a number of patients with Neurodegeneration with Brain Iron Accumulation (NBIA) [To date there is no direct evidence linking ferritinophagy, ferroptosis, and ND. While elevated ferritinophagy sensitizes cells to ferroptosis, the data connecting autophagy and ND instead suggest that decreases in global autophagy activity are correlated with development of ND. Indeed, defective autophagy can cause ND in genetic mouse models of autophagy loss ,74. Mutan (NBIA) . In genen (NBIA) , iron len (NBIA) . As defen (NBIA) . This coMany cancers accumulate large amounts of iron to support increased proliferation. This is accomplished in a cell-autonomous manner by increasing iron import or decreasing export ,80,81,82While robust evidence for the role of ferritinophagy in tumorigenesis is lacking, there is a significant evidence for the importance of autophagy in cancer with recent links between ferroptosis sensitivity and tumor autophagy dependence. The role of autophagy in cancer is exceedingly complex with autophagy acting in a tumor suppressive or tumor promoting manner depending on the tumor context (reviewed in detail ). AutophEscherichia coli survive by forming reservoirs within urothelial cell autophagosomes. These bacteria traffic with ferritin-bound iron to the autophagosome to support their proliferation. The authors show that ferritin trafficking is NCOA4-dependent and that NCOA4 depletion reduces bacterial load [Ferritinophagy is also involved in modulating susceptibility to infectious diseases. Uropathogenic ial load . Autophaial load . USP24 dial load . These sNCOA4-mediated ferritinophagy is integral to iron homeostasis in normal and pathological conditions. Significant progress has been made identifying the molecular mechanisms that regulate NCOA4 activity. However, a number of outstanding questions regarding the biochemical regulation of NCOA4 remain. First, how NCOA4 is regulated on a transcriptional and post-transcriptional level has yet to be determined. Importantly, NCOA4 mRNA does not appear to have a 5′ or 3′ Iron Regulatory Element (IRE) that would engage the iron responsive IREB1/2 system of post-transcriptional regulation. NCOA4-FTH1 and NCOA4-HERC2 binding appears to be regulated by iron levels; however, the mode of NCOA4 iron binding as well as the structural requirements for FTH1 and HERC2 binding are unclear. Further, where and when NCOA4 first interacts with FTH1 or HERC2 in the cell is unclear. The precise mechanism of NCOA4-FTH1 delivery to lysosomes is unclear and there may be multiple routes including a canonical autophagic route and/or an ESCRT complex dependent endosomal-lysosomal transport pathway ,34. NCOAThe role of NCOA4-mediated ferritinophagy in physiological processes has so far been assessed mainly in the context of erythropoiesis in zebrafish and murine models of systemic NCOA4 depletion. However, these studies have been unable to identify if the impact on erythropoiesis is due to cell-autonomous effects on iron metabolism in erythrocytes or due to impairment of whole body iron availability. Further studies are required to address this question using conditional knockout mouse models that interrogate NCOA4 in red blood cells or in other cell types that contribute to erythropoiesis. Furthermore, results from Gao et al. suggest there might be a temporal variation in NCOA4 dependency , with a The role of ferritinophagy in disease is an underexplored area. Based on the importance of NCOA4 in erythropoiesis and systemic iron homeostasis, future studies of NCOA4 function in the context of anemia and hemochromatosis and whether inhibition or upregulation of ferritinophagy can impact these and other disease processes where iron homeostasis is dysregulated will be informative. The recent discovery that ferritinophagy can modulate sensitivity to ferroptosis, an iron-dependent form of cell death linked to neurodegeneration, cancer and ischemia/reperfusion injury, suggests ferritinophagy may be similarly important in these diseases. Understanding how ferritinophagy contributes to ferroptosis sensitivity in each pathological setting in vivo will be key to designing therapeutic interventions that could trigger or block ferroptosis. The recent discovery of NCOA4 has opened a new area of biology with connections to a wide range of physiologic and pathophysiological processes. Future studies will clarify the above described unanswered biochemical and in vivo functional questions fundamental to our understanding of NCOA4 biology.


Despite numerous strategies introduced to promote the safe use of insulin, insulin-related medication errors persist. Our aim was to examine the knowledge and self-reported confidence of a range of healthcare professionals regarding insulin use in a large teaching hospital in the North of England. A 16-item electronic questionnaire was prepared in light of locally reported insulin-related incidents and distributed electronically to all healthcare professionals at the hospital over a 4-week study period. A range of healthcare professionals, including nurses, pharmacists, pharmacy technicians, junior doctors and consultants, completed the questionnaires (n = 109). Pharmacists achieved the greatest percentage of mean correct answers overall (49%), followed by consultant doctors (38%) and pharmacy technicians (37%), junior doctors (34%) and nurses (32%). Healthcare professionals were mainly “slightly confident” in their knowledge and use of insulin. Confidence level positively correlated to performance, but number of years’ experience did not result in higher confidence or performance. This small-scale study allowed for a broad assessment of insulin-related topics that have been identified both nationally and locally as particularly problematic. Identifying knowledge gaps may help tailor strategies to help improve insulin knowledge and patient safety. Insulin is a high-risk medication used in the treatment of both type 1 and type 2 diabetes mellitus . The neeInsulin remains one of the top medicines implicated in adverse drug events, as well as being involved in medication errors worldwide . In the Insufficient knowledge regarding the safe use of insulin among healthcare professionals may contribute to medication errors and patient harm ,11. The Previous studies have reported low confidence and gaps in nurses’ knowledge with respect to insulin use, with only around 50% of participants correctly answering questions on aspects of insulin administration ,17,18. DIt has been suggested that a lack of knowledge on insulin prescribing, dispensing, dosing and administration can contribute to medication errors , and thaTo identify specific knowledge gaps amongst healthcare professionals regarding insulin products and dosing regimens.To determine correlations between insulin knowledge, self-reported confidence level, professional group and years of experience of healthcare professionals.To describe healthcare professionals’ experiences of previous involvement in insulin-related medication errors, and their recommendations for improving insulin safety.The aim was to measure and examine staff knowledge and self-reported confidence of a range of healthcare professionals regarding insulin products and administration in hospitals. The objectives of the study included:The study was conducted at a large teaching hospital in the North of England between December 2016 and January 2017. The hospital is part of one of the largest and busiest NHS hospital trusts in England, providing services to 2.3 million patients across five hospitals and 40 community sites. An electronic questionnaire was prepared by two pharmacists and a pharmacy student in light of previously reported insulin-related incidents at both a national and local level . AnswersValidity and content of the questionnaire were examined by piloting the questionnaire with a convenience sample of 10 academic and clinical pharmacists. Several iterations addressed any reported ambiguities and a refined version was agreed by consensus of the study team. A specialist panel from the hospital’s clinical effectiveness unit also reviewed and approved the questionnaire and study procedure. Ethical approval was not required as per the hospital’s criteria for research studies.The questionnaire consisted of 16 items, including both multiple-choice and open questions, in order to allow achievement of the study objectives. All qualified and registered healthcare professionals practising at the hospital who were involved in the care of patients were invited to complete the questionnaire, irrespective of profession or number of years of experience (n = approx. 6000). Due to the range of healthcare professionals involved in the prescribing and administration of medicines , broad inclusion criteria was thought to better reflect current practice.The hospital communications team distributed an email to all staff employed at the hospital in January 2017, inviting those involved in patient care to complete the online questionnaire. The email included a link to allow staff to directly access and self-complete the questionnaire via their web browser, and was resent after 2 weeks to increase visibility and participation. The online questionnaire remained live for 4 weeks, after which it was closed, and the results analysed. Informed voluntary consent was implied by completion of the online anonymous questionnaire. Participants were requested not to refer to any information resources and to provide answers on the basis of their knowledge. There was no set time limit for completion of the questionnaire; participants were advised that it should take around 5 min to complete. Participants were asked to disclose their professional group, number of years in practice, their clinical area, and confidence level regarding insulin products and their use. Due to the size of the organisation , such information was not thought to compromise the anonymity of the data obtained.Data was generated and inputted directly and electronically via the online questionnaire platform and translated into a Microsoft Excel 2016 document for descriptive analysis. Any questions that were answered incorrectly, as “I don’t know” or left unanswered, were considered incorrect. Questions that required selection of more than one option to comprise a correct answer were only considered correct if all correct options were chosen. Answers to open-ended questions were inductively thematically analysed by a single researcher. Findings were confirmed by an independent pharmacist to increase rigour.A total of 113 questionnaires were completed. The total number of clinical staff who were available to access and answer the questionnaire during the study period was unknown due to the nature of central electronic distribution. Out of those who responded, 4 participants were either pre-qualification or not actively involved in direct patient care  and were therefore excluded from the final analysis. Respondents included in the final analysis (n = 109) comprised of 36 nurses (33%), 33 hospital pharmacists of all grades (30%), 20 doctors (19%)—of whom 16 (15%) were junior doctors (foundation doctors and registrars) and 4 (4%) were consultants—, 18 pharmacy technicians (17%), 1 dietician (1%) and 1 operational department practitioner (1%). All 109 participants included in the final analysis completed the questionnaire, allowing analysis of all participant answers. A range of clinical specialities were represented in the study, including surgery (24%), cardiology (18%), diabetes and endocrine, acute medicine, haematology and care of the elderly, (8% each), respiratory, critical care, renal (6% each) and psychiatry and head and neck (4% each).Most professionals reported being “slightly confident” in their knowledge of insulin products and regimens; only 21% of respondents stated that they were confident, or very confident in their knowledge of insulin. The mean score overall for all professional groups answering all questions was 38%. All respondents answered the questions involving insulin products better than the questions involving insulin regimens . Mean percentages of correct answers returned for each professional group are illustrated in Pharmacists achieved the greatest percentage of mean correct answers overall (49%), followed by consultant doctors (38%), pharmacy technicians (37%), junior doctors (34%), nurses (32%) and others (13%). Pharmacists returned the most correct answers with respect to insulin products (75%), followed by pharmacy technicians (60%), with doctors and nurses scoring similarly for these questions (42–48%). Consultants returned the most correct answers with respect to insulin regimens (31%), followed by pharmacists, junior doctors, and nurses, who scored similarly for these questions (19–23%). The responses for each individual question are shown in Questions concerning long-acting insulin (Q9 and 10) were answered very well by most groups. Questions regarding duration of action (Q11 and 12) were answered poorly by all groups; the question returning the least amount of answers (Q6) concerned administration of insulin before meals. The other mealtime administration question (Q7) was answered more successfully by all participants.Doctors and nurses showed greater awareness of the duration of rapid acting insulin (Q12) than their pharmacy and nursing colleagues, whereas pharmacy staff demonstrated greater knowledge regarding the duration of action of mixed insulin (Q11). Pharmacy staff and consultants were more aware of products that contained non-standard concentrations of insulin (Q13) than their junior doctor and nurse colleagues. Junior doctors showed the greatest variety in performance between questions amongst the professional groups.Overall, the number of years’ experience had only a very slight impact on performance. Interestingly, those with less than 5 years’ experience had the greatest mean score (40%) compared to those with between 5 and 10 years of experience (38%) and more than 10 years’ experience (36%).Twenty-nine (27%) professionals reported being involved in, or identifying, a previous insulin prescription, administration or management error, or near-miss. Most responses (45%) described incidents involving the wrong form of insulin being prescribed , others described insulin being prescribed at the wrong time(s) of day, inappropriate omission of insulin (prescription or administration) and erroneous use of intravenous insulin and concomitant fluids.Seventy-two (66%) participants suggested measures to improve insulin safety in inpatients, most of which (58%) stated a need for more education on this topic, explaining that more regular training and education sessions, study days and regular e-learning modules could help increase their knowledge and help them to deal with patients with diabetes better. Seventeen people (24%) suggested increasing the number and use of available resources to improve insulin safety, for example, quick-reference guides and increased specialist input. Seven people (10%) suggested tighter regulation with respect to insulin . Other suggestions included allowing patients to self-administer where capable, prescribers having their mistakes highlighted so they may reflect and improve their practice, and better communication between care providers.This study allowed for a broad assessment of insulin-related topics that have been identified both nationally and locally as particularly problematic. It is the first study of its kind to include the range of professional groups that are involved in insulin prescribing and administration processes in hospital, for example pharmacy technicians, who routinely gather and present important insulin information to their medical, pharmacy and nursing colleagues.We report a general lack of insulin-related knowledge and confidence across all professions, despite interventions to improve insulin safety in recent years. This echoes the findings of Derr et al., who report that only a minority of the nurses and doctors included in their study felt very comfortable managing diabetes . We founThis study found that pharmacists achieved the greatest mean score amongst the professional groups, although certain topics seemed to be understood by different professions. For example, more nurses returned correct answers regarding insulin regimens than their junior medical colleagues. These results support those of Lee et al.  and may We also found that apart from pharmacists, pharmacy technicians demonstrated a greater level of knowledge regarding insulin products compared with other professions, which is a previously unreported result. Our findings would, therefore, also support pharmacy technician involvement in relevant insulin-related medication gathering and technical screening processes in hospital.Both nurses and junior doctors did not score highly regarding non-standard insulin concentrations in this study. This supports Segal’s findings , which iThe number of years worked did not have a significant impact on performance in this study, and supports the findings of Derr et al.  who specStaff reported previous incidents involving inappropriate omission of insulin, which was previously highlighted by the National Patient Safety Agency  and a prAlthough the questionnaire was assured for content validity, further benefit may have been achieved with multi-professional input in the initial stages. Convenience sampling, although respectful of busy front-line staff pressures, may have introduced bias, and the response rate was limited; however, the study was conducted in a typical large teaching hospital in England. The setting would not be predicted to be very different from most other hospital settings, and therefore the study findings could be applied in the broader context.This study demonstrates significant knowledge gaps around insulin products and regimens across a greater variety of healthcare professions than has been reported previously. Combined with a low level of self-reported confidence with respect to insulin knowledge, this highlights the need for further interventions to tackle insulin-related medication errors in hospitals. Healthcare professionals’ opinions regarding improving insulin safety are reported, and support further educational and resource-based interventions. Results highlight the relative strength of pharmacy professionals’ knowledge in this area, demonstrating their value in supporting the safe use of insulin in hospitals.


Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney  tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults.While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian  Iridoviridae) are significantly contributing to the worldwide amphibian declines [Escalating infections of amphibians by ranaviruses such as Frog Virus 3  cytokines are very important to the antiviral immune responses of all vertebrates . More reIFNs , of whicIFNs . While tIFNs . By contIII IFNs . AmphibiIII IFNs . MoreoveNs [IFNs ,13. IndeFN genes ,13; thesFN genes ,15,16. Ipe I IFN ,18,19. IFNs in the X. laevis tadpole and adult frog responses to FV3 challenge, as well as to a range of pathogen pattern recognition receptor agonists such as lipoprotein, lipopolysaccharide, polyinosinic:polycytidylic acid, CpG DNA, imiquimod, single stranded RNA and several viral DNAs.Here, we expand on our previous work by examining the roles of a panel of intronless and intron containing type I and type III Xenopus laevis were purchased from the Xenopus 1 facility, and housed and handled under strict laboratory and IACUC regulations .Outbred tadpole and adult The cell culture media and conditions have been previously described .2 for 5 days. The FV3-containing supernatants were collected over 30% sucrose by ultracetrifugation, re-suspended in saline, and the viral titers were determined by plaque assay over BHK-21 cells.The FV3 production has been described previously . In brie6 PFU/mL of FV3 for 1 h in 100 mL of water, were washed in FV3-free water and housed for an additional 6 h. After 6 h, the animals were sacrificed by tricaine mesylate (TMS) overdose and the tissues were collected for RNA and DNA isolation. Mock-infections were carried out in parallel using cohort animals by exposing tadpoles and adults to equal volumes of FV3-free water for the respective amounts of time as above. Tadpoles and the adult frogs were infected by water-bath containing 1 × 102 in the presence of respective stimuli.The tadpole and adult frog skin and kidney tissue were excised and incubated in 7.5 μg/mL of liberase  for 30 min at 27 °C. After this incubation, the samples were homogenized by passage through the progressively larger gage needles, washed with saline and the cells collected by centrifugation. The cells were then resuspended in a medium and enumerated. The cells were cultured in an amphibian medium Iscove’s Modified Dulbecco’s Medium containing Penicillin and Streptomycin (200 U/mL), gentamycin (15 μg/mL), non-essential amino acids, primatone, and 10% fetal bovine serum, and were adjusted to amphibian osmolality. The cells were incubated at 27 °C with 5% COX. laevis tadpole (N = 5) and adult frog (N = 5) skin and kidney cells were seeded at densities of 105 cells/well into 96 well plates and were stimulated with the following PAMPs. There was a high molecular weight of Poly I:C ; FSL-1 lipoprotein ; LPS from E. coli K12 ; Imiquimod ; ssRNA 40 ; and CpG ODN 2006 . The cells were also stimulated with 10 μg/mL of the following cytosolic DNA sensor agonists: HSV-60 , VACV-70 , and IFN-stimulatory DNA . The Poly I:C, HSV-60, VACV-70, and ISD were added to the cells in conjunction with the LyoVec transfection reagent. All of the treatments and stimulation conditions were performed in accordance to the manufacturer’s instructions . The tadpole and adult frog skin and kidney cells were incubated with the respective PAMPs for 6 h before they were harvested and processed for RNA isolation and cDNA synthesis.Isolated X. laevis tadpole (N = 5) and adult frog (N = 5) skin and kidney cells were seeded at densities of 105 cells/well into 96 well plates and mock-infected  or infected at a multiplicity of infection (MOI) of 0.5 plaque forming units (PFU) of FV3 per cell for 6 h. Subsequently, the cells were washed in saline, collected by centrifugation and processed for RNA and DNA isolation.The isolated For all experiments, tadpole and adult tissues and cells were placed in Trizol reagent , flash frozen on dry ice and stored at −80 °C until RNA and DNA isolation. The tissues and cells were homogenized by passage through progressively higher gage needles, and RNA isolation was performed using the Trizol (Invitrogen), according to manufacturers’ directions. Following the RNA extraction, the remaining Trizol layer was mixed with a back extraction buffer  and centrifuged to isolate the DNA containing aqueous phase. The DNA was precipitated overnight with isopropanol, pelleted by centrifugation, washed with 70% ethanol, and resuspended in TE  buffer. The DNA was then purified by phenol-chloroform extraction, resuspended in molecular grade water and quantified.IFNs, pattern recognition receptors (PRRs) and FV3 immediate early (82R), delayed-early (95R), and late (93L) genes was performed using the delta^delta CT method, with all of the gene expressions examined relative to the GAPDH endogenous control. The expression of the individual IFN genes was normalized against the respective control expression.The total RNA and DNA were extracted from the frog tissues and cells using the Trizol reagent and following the manufacturer’s directions (Invitrogen), and all of the cDNA syntheses were performed using iScript cDNA synthesis kits, in accordance to manufacturers’ directions , using 500 ng of the total RNA. The gene expression analyses of type I and type III 1–108 vDNA Pol fragment-containing plasmid copies. These were used as the standard curve in the subsequent absolute qPCR assays of the FV3 genomic DNA copies, using primers against the FV3 DNA polymerase. All of the absolute qPCR analyses were performed using 50 ng of total isolated DNA.The FV3 viral loads were determined by the absolute qPCR against the DNA , using a serially diluted standard curve. Briefly, the pGEM-T vector  bearing the FV3 vDNA Pol (ORF 60R) fragment was serially diluted to yield 10All of the experiments were performed using the CFX96 Real-Time System and iTaq Universal SYBR Green Supermix. The BioRad CFX Manager software (SDS) was used for all of the expression analyses. All of the primers were validated prior to use.t-test, using Vassar Stat (http://faculty.vassar.edu/lowry//anova1u.html) and Graph Pad  statistical programs, respectively. A probability level of p < 0.05 was considered significant.The statistical analysis was performed using a one-way analysis of variance (ANOVA) and post hoc X. laevis tadpoles and adult frogs undergo distinct type I and type III IFN responses at their skin following water exposure to FV3 [IFN responses are mounted following the recognition of viral (and other types of pathogen) infections by pathogen pattern recognition receptors (PRRs) [PRR genes, including those responsible for the recognition of viral as well other types of pathogens  and frog-specific  TLRs as well as the retinoic acid-inducible gene I  of the four examined cytosolic DNA sensor genes  ,22,23,24athogens . With th I RIGI, A. Moreovor genes B.PRR genes . Considy: [PRRs A,B, we nc clades  in the ic clades . We chosIFN genes  and examined their type I and III IFN gene expression , delayed-early (95R), and late (93L) genes as well as their FV3 DNA loads C,D. AkinIFN responses by those tadpole and adult skin and kidney cells that would be present within the respective tissues at a steady state. However, the above expression studies cannot account for the potential additional IFN-expressing cell population that may be recruited into these respective tadpole and adult tissues upon the FV3 challenge. To reconcile this possibility, we infected tadpoles and adult frogs with FV3 by water bath and 6 h later examined the gene expression of type I and type III IFNs in their skin and kidney tissues  skin cells and skin tissues, it was quiet striking that all of the other examined IFNs possessed very different expression patterns between the cells and tissues  kidney cells, the FV3-infected adults exhibited greater gene expression of  kidneys B. Intrigulations B.IFN responses. Considering that the FV3 replication time is around 12–24 h, we predict that the detected viral loads represent FV3 that has disseminated to the animal kidney from the skin, gills, and/or gastrointestinal tissues; all of which would be in contact with, and potential entry points for FV3.Consistent with our past studies  and in cXenopudinae frogs possess the most diverse and expanded repertoires of type I and type III IFN cytokines [IFN repertoires during amphibian tadpole and adult frog antiviral responses are imperative. Indeed, the results presented here indicate that the X. laevis tadpoles and adults mount vastly different IFN responses to FV3, as well as to distinct viral PAMPs; possibly accounting for the susceptibility differences between these developmental stages.It is interesting to consider that of all of the vertebrates examined to date (many of which are now known to be infected by ranaviruses), the ytokines ,13. It iytokines ,3, whileytokines ,5,6,7,8.IFNs at their skin, whereas the adult frogs mounted more robust intronless type I and type III IFN responses. Similar results were derived when the adult skin cells were stimulated with the cytosolic DNA sensor agonists (FV3 is a DNA virus), suggesting that the adult frogs utilize these particular IFNs when dealing with DNA viruses. Conversely, the FV3-infected tadpole kidneys possessed significantly more upregulated levels of an intronless type I IFNs, as well as an intronless and an intron-containing type III IFNs, whereas the FV3-infected adult frogs mounted an exclusively intron-containing type I IFN response within their kidneys. Both of the amphibian skin and kidney tissues undergo drastic changes during metamorphosis, including changes in their cellular composition [IFN responses and may mark differences in the ability of the two developmental stages to control the FV3. For example, after 6 h of infection, the tadpoles possessed lower kidney FV3 loads than the adult frogs, which may be accounted for by their distinct IFN responses at this site. Indeed, the tadpole kidney cells responded more robustly to the viral PAMPs with their IFN gene expression than the kidney cells from adult frogs, possibly indicating that the X. laevis tadpoles are more effective at recognizing and containing viral pathogens at this site. It is notable that the adult frog kidney cells possessed significantly greater IFN responses to CpG, suggesting that the adult kidney may be more equipped at dealing with bacterial infections. Many of the X. laevis IFNs are thought to participate in antibacterial responses [IFNs during bacterial infections.It is notable that following FV3 challenge, the tadpoles responded predominantly with intron-containing type III position ,26. Presesponses  and it wIFN responses, it may indicate the presence of distinct cellular populations. Notably, less PRR expression differences were observed between the tadpole and adult kidney tissues. This in turn suggests that the distinct tadpole and adult IFN responses may be not only a function of the cellular composition of the respective tissues, but may reflect the distinct immune strategies of the two development stages. Unfortunately, at present, the cellular compositions of the tadpole and adult frog skin and kidney tissues remain poorly defined. Thus, further research into this aspect of amphibian physiology will help define the facets of pathogen susceptibility and may explain the differences in the IFN responses reported here.Our expression analyses indicate that at the skin, the adult frogs express greater levels of most of the examined PRRs. While this does not explain the drastic differences in the tadpole and adult PRRs that recognize these agonists , whereas the tadpole and adult kidney cells responded to these respective stimuli. Interestingly, the stimulation of tadpole and adult skin cells with another TLR7 agonist, ssRNA also resulted in relatively modest IFN responses, with fewer IFN genes being upregulated than observed with the other PRR agonists. Possibly, these results reflect the transfection efficiencies of these compounds into the tadpole and adult frog skin (but not kidney) cells. Alternatively, as all of these agonists mimic the RNA viruses/viral products, together our results may suggest that the X. laevis are less sensitive or less effective at recognizing viral RNAs. While RIGI and TLR3 (PRRs that recognize dsRNA/Poly I:C) were expressed in animal skin tissues, the putative imiquimod/ssRNA receptor, TLR7 was very poorly expressed in both the tadpole and adult skin tissues. This would be a convenient explanation for the observed non-responsiveness to imiquimod, except that the animal kidneys, despite possessing a meager TLR7 expression, exhibited robust IFN responses to imiquimod and ssRNA. Considering that fresh water environments such as those inhabited by amphibians contain vast amounts of biologically diverse RNA viruses [X. laevis skin and kidney responses. It would be most interesting to see whether the fish skin cells are likewise desensitized to the RNA viruses.The tadpole and adult frog skin cells did not respond to Poly I:C or imiquimod stimulation, despite expressing the putative  viruses , the nonIFN responses to LPS. Presumably, since the tadpole skin is a much weaker barrier than the skin of the adult frogs combined with the fact that these animals reside in bacteria-rich environments may account for why tadpoles may be desensitized against immune recognition/activation in response to LPS.The notion of sensitization to environmental stimuli may also be extended to tadpole and adult skin responsiveness to LPS. Aquatic vertebrates are notorious for their resistance to LPS stimulation and much larger concentrations of this gram-negative bacterial cell wall component are required to activate immune cells of aquatic species . InteresIFN gene expression in the isolated tadpole and adult frog cells and the infected animal tissues indicate that while the respective resident cells (the identity of which remains awaits the development of new X. laevis-specific reagents) are capable of recognizing FV3 and other pathogen threats and mounting specific IFN responses, both tadpoles and adults rely on the recruitment of additional cell populations to coordinate their IFN responses. Indeed, while the responses of some IFNs  appear to be mediated by the cells that are present within the skin and kidney tissues of healthy animals, the drastic differences reported here between the FV3-challeneged cells and the tissues isolated from infected animals suggest that the additional IFN-expressing populations are rapidly (within 6 h) recruited to the infection sites to mediate the IFN responses. It will be most invaluable to learn which tadpole and adult frog immune (and possibly non-immune) cell types are recruited to the sites of FV3 infection and what the roles of the respective IFNs produced by the resident and recruited cell types during anti-ranavirus immune responses are.The comparison of the As we move forward to gain greater insights into the infection, replication, and immune evasion strategies of FV3 and other ranaviruses, it is imperative that we continually re-evaluate our understanding of these pathogens and acknowledge the fact that there is much that we do not know about how these pathogens replicate in the different cells and tissues of their numerous cold-blooded vertebrate hosts. In so doing, it also important to not build false dogmas upon previous literature, some of which may have employed much less-sensitive approaches (than presently available) in the context of cell lines that may not accurately reflect the natural cell tropisms of these pathogens, or representing one alternative cellular host to these viruses. Indeed, it has been more recently demonstrated that the modes of FV3 replication and the ensuing cell cytotoxicity (including apoptosis) and immune responses greatly vary with the infected cell type . LikewisIFNs. While the evolutionary reason for this stark divergence from other vertebrates is difficult to speculate upon, clearly some aspect of their physiology and immune pressures has been selected for the expansion and maintenance of these diverse gene families. When considering that the tadpoles and adult frogs have drastically different physiologies and pathogen susceptibilities, it is intuitive that they would take advantage of the distinct repertoires of these IFN cytokine in response to unique immune challenges. In turn, gaining a greater insight into the functional significance of those IFNs that are respectively utilized by tadpoles and adult frogs will yield new insights into the tadpole and adult immune efficacies and to the evolution of these cytokine families.From an evolutionary standpoint, it is intriguing to consider that amphibians have diverged to encode so many distinct type I and type III