Although the insect wing is a textbook example of morphological novelty, the origin of insect wings remains a mystery and is regarded as a chief conundrum in biology. Centuries of debates have culminated into two prominent hypotheses: the tergal origin hypothesis and the pleural origin hypothesis. However, between these two hypotheses, there is little consensus in regard to the origin tissue of the wing as well as the evolutionary route from the origin tissue to the functional flight device. Recent evolutionary developmental (evo-devo) studies have shed new light on the origin of insect wings. A key concept in these studies is “serial homology”. In this review, we discuss how the wing serial homologs identified in recent evo-devo studies have provided a new angle through which this century-old conundrum can be explored. We also review what we have learned so far from wing serial homologs and discuss what we can do to go beyond simply identifying wing serial homologs and delve further into the developmental and genetic mechanisms that have facilitated the evolution of insect wings. Recently, studies using molecular and evolutionary developmental (evo-devo) analyses have provided a new view through which this conundrum can be assessed. A summary of the history of the wing origin debate and an overall perspective on how the application of evo-devo approaches to this question can lead to new insights on the evolutionary origin of insect wings have previously been reviewed3. Here, we focus our discussion on how the identification of wing serial homologs through developmental approaches will help us to explore the history and origin of insect wings and on what we have learned so far. Then we discuss the challenges we face with evo-devo approaches and how we can overcome these challenges.The acquisition of wings is considered a major driving force for the success of insects, yet the evolutionary origin of this important novel structure remains one of the biggest conundrums in biology. Over a century of investigations into this question have resulted in two prominent hypotheses on the evolutionary origin of insect wings: the tergal hypothesis and the pleural hypothesis. The tergal origin hypothesis  proposes that wings originated from expansions of the dorsal body wall (terga), whereas the pleural origin hypothesis essentially proposes that wings evolved from pleural  tissues and their associated branches (exites) 5. In this body plan, these structures on different segments are considered to be serially homologous to each other, as the development of these structures was likely orchestrated by the same developmental system. Throughout arthropod evolution, these serially homologous structures have often undergone segment-specific modification or suppression, resulting in serially homologous structures with different morphologies and different functions 5  in evolutionary biology has been quite controversial . Since there are no obvious “wing-like” structures outside of the second and third thoracic segments (T2 and T3) in insects, only a handful of structures  have been proposed to be wing serial homologs10. But, as mentioned above, serially homologous structures can lose morphological similarity, even though they have historical common ancestry . Therefore, it is possible that wings are so drastically diverged from other serially homologous structures that we are not able to recognize them as wing serial homologs. The application of molecular and functional approaches (an evo-devo approach) may allow us to identify “hidden” wing serial homologs in wingless segments, which could provide us with critical information to unveil the origin of insect wings.The concept of gaining insights into evolutionary transitional states of wings through wing serial homologs is not new. In fact, this has been the main approach  used by insect wing origin studiesbona fide wings in segments other than the two thoracic segments in the history of hexapod evolution . Instead, we are suggesting that there are tissues in these wingless segments that have common ancestry with insect wings and that wings are “apomorphic”  among serially homologous tissues  often orchestrates the development of tissues that are serially homologous to each other; thus, this type of marker gene is useful for investigating the history of the tissue and for identifying serial homologs that are morphologically diverged. The wing and leg marker genes discussed in this review are in the second class of marker genes.Hox genes are the region selectors responsible for the individualization of the otherwise uniform segments and segmentally repetitive structures 17. Hox genes achieve this task via at least three distinct modes of action: (i) modification of pre-existing structures, (ii) suppression of pre-existing structures, and (iii) induction ofde novo structures  will often allow us to strip away the modifications applied to the serially homologous structures (via Hox action 1), revealing serial homologs that are otherwise morphologically distinct and difficult to recognize.Drosophila as an example to discuss how we can apply the above-mentioned evo-devo concept to the identification of serially homologous structures. Head and thoracic segments ofDrosophila possess a pair of appendages on their ventral side. In the head segments, these appendages form a series of mouthparts as well as antennae. On the other hand, each of the three thoracic segments possesses a pair of legs, with minor modifications making the three pairs of legs slightly different from each other.Distalless (Dll), which codes for a homeodomain transcription factor, is one of the important marker genes for leg development18.Dll is expressed in mouthparts, antennae, and legs18, and these ventral appendages can transform into one another upon various Hox mutations (including both gain-of-function and loss-of-function (LOF) mutations)5. These outcomes indicate that the head appendages and legs are serially homologous and that Hox genes modify these serially homologous structures in a segment-specific manner (Hox action 1). In contrast, there is no ventral appendage in the abdominal segments ofDrosophila, as Hox genes suppress the induction of ventral appendages19. This is also apparent from the fact thatDll is never induced in the abdominal segments18. Upon Hox LOF mutations, a new set ofDll-positive cells are induced in the abdominal segments, producing ectopic leg primordia19. Through this outcome, we can conclude that there are no leg serial homologs in theDrosophila abdominal segments (Hox action 2).Let us first use the legs and their derivatives  inDrosophila, later studies showed that it is conserved among other insect orders . Interestingly, ventral appendages in the abdominal segments have been regained in some insect lineages (such as the prolegs of lepidopteran larvae). The tissues that give rise to these ventral appendages are accompanied byDll expression21, further supporting the idea that the presence of serial homologs corresponds to the expression of marker genes. In addition, studies in horned beetles provided an example of the third Hox action in regard to the ventral appendage. Beetle horn hasDll expression22. However, this has been achieved via co-option22, and removing the responsible Hox gene will simply remove the horn23. Hence, we can conclude that horn formation has evolved via co-option of the leg gene network (includingDll) under the control of the Hox gene and thus that there are no horn serial homologs in other segments.Although this configuration was initially recognized inDrosophila, the situation of wings and their derivatives  somewhat parallels the situation of the ventral appendages. As mentioned, morphologically, wings are unique to the two thoracic segments (T2 and T3) in extant insects, includingDrosophila. Among the several wing genes identified fromDrosophila studies,vestigial (vg) is often considered one of the most critical wing marker genes because of its wing-specific function during the development of epidermal structures  and its ability to induce ectopic wings when overexpressed30. InDrosophila, dorsal appendage primordia  are induced during embryogenesis. These imaginal discs in T2 and T3, along with a pair of residual cell clusters in T1, are thevg-positive epidermal tissues inDrosophila31. In contrast, the segments outside of the thorax do not havevg-positive epidermal tissues that contribute to adult morphology27. Becausevg-expressing imaginal tissues are missing in the non-winged segments, it has been considered that the induction of wing-related structures  is suppressed in these segments, similar to the absence of the leg serial homologs in the abdominal segments ofDrosophila (Hox action 2).At least inDrosophila paradigm regarding the unique presence of wing-related tissues in T2 and T3, it came as a surprise when additionalvg-dependent epidermal tissues were found in non-winged segments in beetles. Ohdeet al. found that the formation of some of the dorsally originated tissues, such as hypomeron in T1 and gin-traps  in the abdominal segments, isvg-dependent in the mealworm beetle,Tenebrio molitor32. Clark-Hachtelet al. identified that there are two distinctvg-dependent tissues in T1 of the red flour beetle,Tribolium castaneum: (i) the carinated margin, a dorsally originated tissue that corresponds to the edge of the hypomeron, and (ii) two pleural plates (posterior trochantin and the epimeron), tissues that are considered to have originated from ancestral proximal leg segments 33. Furthermore, in both studies, thesevg-dependent tissues were shown to transform into wings upon Hox LOF mutation. These results strongly suggest that, although their shape and function are drastically different from those of wings, thevg-dependent tissues found in the non-winged segments of beetles are wing serial homologs.Because of this well-establishedTribolium results in the induction of almost complete wing tissues throughout the abdominal segments34. However, legs were never ectopically induced in the same condition. This indicates that there are tissues which can transform into wings upon Hox LOF mutations , while there are no leg serial homologs in the abdominal segments which can transform because those tissues are never induced (suppression via Hox action 2). Therefore, these results further support the idea that wing serial homologs are present in the non-winged segments and that the Hox action on the wing serial homologs is different from that on the leg serial homologs in the abdominal segments in beetles.Additional evidence for the presence of wing serial homologs in non-winged segments comes from the outcome of the abdominal Hox gene analysis in beetles. Knocking down the abdominal Hox genes during the larval stage innubbin (nub)35, another critical wing marker gene36. Although thevg dependency of this tissue has yet to be tested, given that the treehopper helmet is an expansion of the prothoracic tergum38, it is likely that the helmet is homologous to one of the twovg-dependent tissues identified in beetles and thus at least “partially” serially homologous to wings  are missing from adultDrosophila43. Also, the entire adult abdomen is formed from a set of unique tissues, called histoblasts43. Owing to this highly derived body plan,Drosophila appears to have lost most of thevg-dependent epidermal tissues that contribute to the adult morphology in the non-winged segments. This signifies the importance of investigating a wide variety of taxa to gain a comprehensive view of insect wing evolution. In summary, wing serial homologs appear to be widespread in various insect orders, and the evo-devo approach outlined above provides a new way of identifying them: namely, surveying forvg-positive epidermal tissues followed by Hox transformation studies to test whether the identified tissues transform into wings.Why, then, were these wing serial homologs not discovered in45. Later, through expression analyses in basal insects, Niwaet al. proposed a dual  origin of insect wings46. Studies on wing serial homologs in winged species (discussed above) have also resulted in somewhat varying conclusions on the origin of insect wings. The identification of dorsal wing serial homologs in the T1 (hypomeron) and the abdomen (gin-traps) ofTenebrio beetles provided evo-devo support for a tergal origin of insect wings32. Interestingly, two sets of wing serial homologs were identified in the T1 ofTribolium: one tergal and the other pleural in nature. The merger of these two wing serial homologs  appears to be essential for the formation of ectopic wings upon Hox LOF mutation, suggesting a dual origin of insect wings33. In more basal insects, Elias-Neto and Belles concluded that both tergal and pleural tissues contribute to the formation of an ectopic wing on T1 inBlattella40, and Medvedet al. provided transcriptional support for the contribution of both tergal and pleural tissues to the wings ofOncopeltus39. These results may provide further support for a dual wing origin.The seminal studies in non-insect arthropods performed by Averof and colleagues provided evidence for a pleural origin of insect wingsTwo important messages can be obtained from the wing  homolog studies: (i) as mentioned, wing serial homologs are widespread, and (ii) wing serial homologs can have drastically different morphologies from each other. For example, in regard to the wing serial homologs of tergal origin, they can be lateral expansions of dorsal body wall or an elaborated helmet in T1, gin-traps  in the abdomen, and wings in T2 and T3. Given that many of the dorsal wing serial homologs are modified body wall structures, the body wall character state appears to be more plesiomorphic  among the dorsal wing serial homologs , whereas insect wings may be an apomorphic version of this trait . A similvg expression sufficient to claim wing serial homology? How can we differentiate thede novo vg-dependent tissues from the true wing serial homologs? What if other wing marker genes, such asnub andapterous (ap)47, disagree withvg expression? (ii) Although several new wing serial homologs have been identified through evo-devo studies, it is far from being able to fully reconstruct a transition series for wing evolution. Below, we will discuss what we can do to go beyond simply identifying wing serial homologs and delve further into the developmental and genetic mechanisms that have facilitated the evolution of insect wings.Although the evo-devo-based approach is a promising method to provide new insights into the origin of insect wings, there are several weaknesses. (i) The first and foremost criticism to the evo-devo approach is its tendency to rely on a limited number of marker genes when identifying certain tissue lineages. Isvg expression sufficient to identify wing serial homologs? The answer is probably “no”. Althoughvg has been very useful in identifying wing serial homologs, it is risky to rely on just one marker because of the pleiotropic nature of the marker genes. Therefore, investigatingvg expression will be useful as the first step to identify potential wing serial homologs, but it is also important to analyze the expression of additional wing marker genes.nub andap are two popular wing marker genes; however, many more genes have been identified as being involved in wing development inDrosophila, some of which may be used as additional wing markers. Of particular interest are the genes involved in anterior/posterior wing patterning42 and the genes involved in wing vein patterning51. To our knowledge, these genes have never been used to investigate the development of wing serial homologs or to identify possible wing homologs in non-insect arthropods. In addition, given the pleiotropic nature of the transcription factors and signaling molecules important for wing development, it is crucial to investigate not only the gene expression repertoire but also the gene regulatory network to assess wing serial homology and to understand the evolutionary relationships between the identified wing serial homologs. Recent advancement in genetic techniques in insects (such as CRISPR/Cas953) may allow us to investigate the regulatory hierarchy among wing genes in various non-model arthropods.Isvg may be more reliable than other marker genes, asvg appears to be the only gene whose expression can induce ectopic wing structures inDrosophila30. However, this “master gene” aspect ofvg (and lack thereof in other genes) needs to be tested in other insects.What if, then, the wing marker genes disagree with each other? Analyzing the gene regulatory network in a wide taxonomy will help identify which part of the network is ancestral and which part is lineage-specific. Also, we may need to consider which genes in the gene regulatory network provide more valuable information when evaluating homology. For example,vg and of other wing marker genes could have emerged via co-option. It is a challenge to differentiate these tissues from true wing serial homologs. As discussed above, we believe that Hox analysis will be powerful to excludede novo vg-dependent tissues. Hox LOF mutations allow for transformations among serially homologous structures, while it is less likely that Hox LOF mutations can cause homeotic transformation between the original and thede novo structures evolved via co-option. For instance, the horn ofOnthophagus beetles requires the leg gene network for its formation, but it does not transform into the leg upon Hox LOF mutation23. Thus, it is critical to assess the ability of a tissue to transform into the wing upon Hox mutation before determining whether the tissue is a wing serial homolog.A tissue with the expression ofTribolium suggest that the primordia that give rise to wings and wing serial homologs are induced similarly in each segment during embryogenesis54. However, it is currently unclear how these possible primordial tissues contribute to the formation of wings and wing serial homologs and how these primordia differentiate into very different tissues over the course of development. By studying the molecular and developmental mechanisms that orchestrate the differentiation of wing serial homologs from wings (and vice versa), we will be able to identify the mechanisms that are operating uniquely in the winged segments.Identifying wing serial homologs has been quite helpful to gain new insights into the origin of insect wings; however, it is still largely elusive how insect wings have evolved. What can we do to go beyond identifying wing serial homologs and delve further into the molecular and developmental mechanisms that have facilitated the evolution of insect wings? One key direction is to study the development of wings and wing serial homologs in various species in detail. Previous expression analyses inOncopeltus, which led the authors to identify a set of genes unique to the true wing when compared with the ectopic T1 wing created by Hox knockdown39. This study has provided interesting insights into how ventral components have contributed to the evolution of insect wings39.Several approaches are useful for this direction. For example, tracing the development of wings and wing serial homologs will help us determine the developmental events that differentiate wings from other wing serial homologs and also will allow us to investigate the developmental origin of wings and wing serial homologs . Comparing gene regulatory networks between wings and wing serial homologs will also be useful to reveal the changes in gene regulation that have been crucial to the evolution of wings. Along the same line, transcriptome comparison between wings and wing serial homologs will allow us to comprehensively identify genes expressed differentially between these tissues. Another intriguing approach is to induce a “wing serial homologs-to-wing” transformation series. Analyzing these transformed tissues  may help us reconstruct an evolutionary transition series from the origin tissue to the functional wing. Combining these approaches will be powerful to gain insights into the origin of insect wings. In fact, some of these approaches have been used in a recent study usingTribolium,Blattella, andOncopeltus). However, some classic studies hint that it may be worth applying at least some of these approaches to non-model insects. For example, it has been reported that wing tissue arises at a lateral  position and migrates dorsally to merge into the tergolateral margin during the nymphal stages in a dragonfly, which may support the pleural  origin of insect wings55. Similar dorsal migration of wing primordia from the lateral region has been described in the embryos of some hemimetabolous insects, such as the cockroach,Periplaneta americana56. Some of these situations may partially recapitulate phylogeny; thus, studying the development of wings and wing serial homologs in these species will be helpful to gain more information in regard to the evolutionary transitional state(s) from the origin tissue to the wing. However, caution must be taken when making this argument, as a developmental process unique to a certain lineage (apomorphic) can superficially mimic possible plesiomorphic conditions. For example, in the past, the wing disc formation from the leg disc inDrosophila was used to support a leg origin of insect wings2. However, this invaginated imaginal disc formation is a highly derived trait even in holometabola. In addition, the so-called “wing” and “leg” discs produce not only the appendages but also the entire adult epidermis . Therefore, theDrosophila situation likely does not reflect an ancestral state. In summary, detailed studies on the development of wings and wing serial homologs in various species will help us identify key developmental and genetic events that have facilitated the evolution of insect wings . Several leg branches  in the brine shrimp and the crayfish have been shown to express some wing marker genes44, but their functional dependency on wing marker genes, includingvg, still needs to be tested. In addition, it is yet to be determined whether other tissues in these crustaceans (such as terga) also share gene regulatory networks with the insect wing. Furthermore, given the vast diversity and the possible polyphyletic nature of the crustacean order58, it will be critical to analyze more crustacean species. Myriapoda (millipedes and centipedes) is another taxon that may provide interesting insights in regard to identifying tissues homologous to insect wings. The possible wing homologs in Myriapoda have not yet been investigated. However, some myriapods possess elaborated tergal expansions reminiscent of paranotal lobes. Therefore, it would be interesting to investigate whether these structures (and other tissues such as parts of the leg) have dependency on genes homologous to insect wing genes. The myriapod lineages are even more basal on the arthropod phylogeny than crustaceans58, and thus identification of potential wing homologs in the myriapod lineages can provide crucial information as to which tissues have given rise to insect wings. In summary, functional analyses for genes homologous to wing genes in a diverse array of arthropod taxa will lead us to a better understanding of what tissues are homologous to wings in these lineages, which will help us further evaluate the wing origin hypotheses from an evo-devo perspective.As mentioned, previous expression analyses for wing marker genes in non-insect arthropodsod taxa . For exaet al. reported very intriguing findings that provide support for a dual origin of insect wings from the paleoentomological point of view59. Collaboration among various fields, including paleontology and evo-devo, will be fruitful to tackle this century-old question regarding the evolutionary origin of insect wings.While we were revising this manuscript, ProkopDorsoventral insect anatomy: The insect thoracic and abdominal body wall can be subdivided into three distinct regions: dorsal (tergum), lateral (pleuron), and ventral (sternum).Tergum (pl. terga): A large plate that forms the dorsal body wall in the thoracic and abdominal segments. The thoracic tergum is callednotum.Pleuron (pl. pleura): The lateral portion of insect thoracic and abdominal segments that consists of several pleural plates. The pleural plates are thought to have stemmed from ancestral proximal leg segments that have fused into the body wall of extant insects.Sternum (pl. sterna): The large ventral plate of insect thoracic and abdominal segments.Tergal origin hypothesis : This hypothesis proposes that wings originated from expansions of the dorsal body wall (terga).Pleural origin hypothesis : This hypothesis proposes that wings evolved from pleural  tissues and their associated branches (exites).Dual origin hypothesis: This hypothesis proposes that both tergal and pleural components have contributed to the evolution of insect wings.Carinated margin: A lateral expansion of dorsal body wall (tergum) in the first thoracic segment in beetles. This structure and several pleural plates have been identified as wing serial homologs33.Gin-trap: A defensive structure formed at the lateral portion of the abdominal segments in some coleopteran pupae. This structure has been identified as a wing serial homolog32. The gin-trap structure is also observed in lepidopteran pupae; however, the evolutionary and developmental relationship between coleopteran and lepidopteran gin-traps is currently unknown.Helmet: An often highly decorated structure of the first thoracic segments in treehoppers. This structure has been proposed as a modified wing of the T1 segment in treehoppers35, which was later disputed from a morphological standpoint38. Clark-Hachtel and Tomoyasu have proposed that the treehopper helmet is serially homologous to the dorsal  wing serial homologs but not lateral  wing serial homologs; therefore, the helmet is “partially” serially homologous to wings33.Apomorphy: Within a group of organisms that have common ancestry, an apomorphy is a trait that is diverged or novel compared with the ancestral state.Plesiomorphy: Within a group of organisms that have common ancestry, a plesiomorphy is a trait that is maintained/conserved throughout the course of evolution from a common ancestor.


Successful development of high affinity radioligands for the translocator protein 18 KDa (TSPO) has contributed to a rapid rise in their use with positron emission tomography (PET) imaging to quantitatively detect the higher density of TSPO in neuropsychiatric conditions with putative microglial activation or reactive gliosis in vivo. [11C]PK11195 has been widely used to study TSPO in many neurological and psychiatric diseases, but the quality of quantified binding estimates using this first-generation radioligand is hampered by low signal to noise ratio, which also limits the sensitivity to detect group differences in binding. Second-generation radiotracers for TSPO such as [11C]DPA-713, [11C]PBR28, or [18F]FEPPA have superior specificity for the target and improved brain penetrance. However, in spite of these promising newer generation radioligands, their use with PET neuroimaging to study the immune response in psychotic diseases like schizophrenia has yielded inconsistent results of low, unchanged, or even tendency toward decreased binding to TSPO compared to data from controls.In this presentation, we will provide necessary biological and methodological perspective to help interpret better the recent results from imaging TSPO in psychosis. We will first review its expression by many cell types including activated microglia, the resident immune cells in the brain, and its diverse functional roles including TSPO as a biomarker of classic neuroinflammatory processes. We will then present optimized methods for estimating useful binding outcomes that go beyond correction for TSPO genotype to minimize effects of factors, including those related to the diverse roles of TSPO, which otherwise introduce limiting, inter-individual variability in binding.These methods include the reporting of relative binding in one tissue to another, where such global factors are cancelled out by their appearance in the numerator and denominator of the outcome ratio. Use of this ratio approach may decrease inter-individual variability in binding measures and improve the sensitivity and statistical power to detect differences between cohorts. In contrast, use of a relative outcome measure may limit the utility of TSPO imaging since a difference between cohorts or within a subject over time may reflect either abnormal TSPO density or a mere shift in possibly normal, relative distribution between the two tissue regions. The most useful pseudoreference region is therefore one in which the true regional density of TSPO is unchanged in the study population, and is yet unidentified in schizophrenia. Building on this biology and methodology, we discuss misconceptions about imaging TSPO in psychosis and cautiously remind the field that this technique should not be equated with ‘imaging microglial activation’ or ‘imaging neuroinflammation.’ Indeed, PET-based TSPO estimates have not correlated with increased peripheral or central pro-inflammatory cytokine levels in schizophrenia or other psychiatric diseases like major depressive disorder.Together, a less simplified approach to imaging TSPO may inform its utility in studying other biological processes captured by its use in psychosis, and may guide future, complimentary research in vitro and in vivo to enhance our understanding of altered neuroimmune processes in psychosis.


The authors interrogated tonotopic map dynamics in auditory cortex (ACtx) by employing a natural sound-learning paradigm, where mothers learn the importance of pup ultrasonic vocalizations (USVs), allowing Shepard et al. to probe the role of map area expansion for auditory learning. They demonstrate that auditory learning in this paradigm does not rely on map expansion but is facilitated by increased inhibition of neurons tuned to low-frequency sounds. Here, we discuss the findings in light of the emerging enthusiasm for cortical inhibitory interneurons for circuit function and hypothesize how a particular interneuron type might be causally involved for the intriguing results obtained by Shepard et al.This commentary centers on the novel findings by  The authors interrogated tonotopic map dynamics in auditory cortex (ACtx) by employing a natural sound-learning paradigm, where mothers learn the importance of pup ultrasonic vocalizations (USVs), allowing Shepard et al. to probe the role of map area expansion for auditory learning. They demonstrate that auditory learning in this paradigm does not rely on map expansion but is facilitated by increased inhibition of neurons tuned to low-frequency sounds. Here, we discuss the findings in light of the emerging enthusiasm for cortical inhibitory interneurons for circuit function and hypothesize how a particular interneuron type might be causally involved for the intriguing results obtained by Shepard et al.This commentary centers on the novel findings by Sounds guide most of our everyday behaviors, and the first cortical area that receives sound-evoked barrages is the auditory cortex (ACtx). A cardinal task for ACtx is to encode, process, and transmit species-specific communication sounds from the surrounding environment  is one of three major classes of interneurons found the cortex, and these make perisomatic synapses onto principal cells in multiple layers (+ interneurons position them favorable for potently influencing the spiking of principal cells (+ interneurons might be a key player enhancing the activity contrast between neurons tuned to high and low frequencies in response to ultrasound frequencies, as observed by + interneurons using optogenetics while recording the response to ultrasound frequencies in ACtx in maternal and naïve mice. If our hypothesis holds true, one might expect that inhibiting PV+ interneurons in maternal mice abolish, at least partially, the enhanced spiking suppression of neurons tuned to low frequencies. Opposite, activating PV+ interneurons in naïve mice might reproduce the effects observed in maternal mice. It is possible that the suggested effects mediated by PV+ interneurons are circuit and target specific, and thus, a broad increase or decrease in PV+ interneuron activity might not fully reproduce the effects occurring with auditory learning, but it would nevertheless provide insight into whether they are involved in auditory learning.In cortex, inhibitory barrages and overall inhibitory tone is regulated by local interneurons . The impe layers . The twoe layers . The peral cells , and we + interneurons could be the mechanism and site of plasticity resulting in the effects observed by + interneurons is the exclusive site of plasticity or, alternatively, whether changes in inhibition onto principal cells is merely a passive reflection of plasticity occurring earlier in the auditory pathway. Cortical PV+ interneurons receive feed-forward excitatory barrages from thalamic glutamatergic neurons (+ interneurons could be explanatory for the findings by de novo cortical plasticity and computations. Gaining experimental insight on this issue will likely not only be restricted to auditory processing but also extend well into the more canonical functional organization and universal motifs for how experience-dependent sensory plasticity occurs in the brain.So far, we have entertained the hypothesis that PV neurons , and act neurons . In mice neurons . Hence, + interneurons could be such a substrate but also that thalamic plasticity could be involved. The study by In summary, by employing careful experimental investigations,


Despite optimized therapeutic approaches, coronary heart disease with its secondary diseases, such as myocardial infarction and chronic heart failure, as well as embolic stroke are the main causes of morbidity and mortality. They result from inflammatory processes induced by lipoproteins such as oxidized low-density lipoprotein (LDL), VLDL and lipoprotein(a) (Lp(a)).Fifty years after the first publication by Kare Berg from Oslo, Lp(a) has meanwhile been accepted as an individual risk factor. However, in spite of the increasing scientific interest in Lp(a), biological function and biosynthetic and catabolic processes of this molecule are still widely unknown. Furthermore, the exact laboratory determination of Lp(a) plasma concentration and its different isoforms is still challenging.The individual Lp(a) serum concentration is genetically determined and remains relatively constant thoughout life. Some ethnic groups in Arabic and African countries show very high Lp(a) plasma concentrations.In the meantime, several registries have indicated that a high Lp(a) plasma concentration is associated with an increased incidence of coronary heart disease, aortic valve stenosis and heart failure. However, there is still no systematic analysis of the population in order to assess their cardiovascular risk depending on Lp(a).For the first time, the European Society of Cardiology has now included Lp(a) determination in the extended screening for the assessment of the cardiovascular risk.In Germany, there are still no effective drugs to lower Lp(a) plasma concentration. Neither are the recently approved PCSK9 inhibitors expected to significantly improve therapeutic options. Thus, lipid apheresis has to be regarded as the only effective therapeutic option to decrease cardiac events.Against this background, the contributions to the present supplement are intended to focus on the clinical aspects of Lp(a) and to emphasize the importance of lipid apheresis.


Multiple relationships exist between antimicrobial resistance and bacterial virulence, and the spread of clones combining multiple antibiotic resistance and a high virulence level is an increasing problem. These relationships have been previously reviewed, notably by Beceiro et al. , who desAlcade-Rico et al. established that multidrug efflux pumps are “at the cross-road between resistance and virulence of bacterial pathogens.” They describe how multidrug efflux pumps are involved, besides antibiotic resistance, in many physiological and virulence-related processes controlled by complex regulatory networks. A special focus is made on the role of these efflux pumps in bacterial cell-to-cell communication and in bacterial-host interactions.In an excellent review article, Staphylococci is given in a mini review by Qin et al. The authors summarized how the psm-mec locus present in some methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococci influences methicillin resistance and virulence-related phenotypes, including the ability to form biofilms.Another example of link between resistance and virulence in Wang et al. used an up-to-date iTRAQ protein labeling technology to identify proteins that are differencially expressed in Streptococcus suis treated with sub-MIC concentrations of tylosin. These concentrations were indeed capable of inhibition of biofilm formation by this zoonotic pathogen, which is of interest, since biofilms are known to display an increased tolerance to antibiotics.In another paper also dealing with biofilms, Xu et al. lifts a corner of the veil on this subject in Staphylococcus aureus by suggesting that heme biosynthesis, in addition to being necessary to a full expression of virulence, is also important in the formation of persisters in S. aureus.This antibiotic tolerance of bacteria present in biofilms is thought to be at least partially due to the presence of higher numbers of persisters than in planktonic bacterial populations. Persisters are phenotypic variants of bacteria which are able to survive antibiotic treatments in a dormancy state and to resume growth when the (antibiotic) stress is withdrawn. Like antibiotic-resistant bacteria, antibiotic-tolerant persisters are also often responsible for failures of antibiotic therapies. However, the mechanisms underlying their production are still not well-understood. An article by Chen et al. addressed the problem of antibiotic tolerance in extraintestinal pathogenic Escherichia coli (ExPEC). Authors studied the role of polyphosphate kinase (PPK), an enzyme already known for its implication in motility, quorum sensing or virulence, in antibiotic tolerance. Their results indicate that PPK is important for the antibiotic stress response and may therefore be a valuable antibacterial target, especially since it is absent in mammals.Another study by S. aureus and Pseudomonas aeruginosa but their existence was only recently reported in Listeria monocytogenes, a food pathogen producing recurrent infections despite being rarely resistant to antibiotics. In an original research article, Curtis et al. described a heme mutant displaying a SCV phenotype mutant with an increased tolerance toward most of the relevant antibiotics. They suggest that the SCV phenotype of L. monocytogenes should be screened in clinical laboratories, as it might be associated to complicated antibiotic treatments.Small Colony Variants (SCV) represent another state in which bacteria are more tolerant to antibiotics. The clinical importance of SCV has been well-documented in cases of chronic infections by Schaufler et al. who reported that the carriage of extended spectrum beta-lactamase (ESBL)-plasmids by strains of pandemic E. coli lineages did not lead to a fitness cost. Instead, these plasmids could enhance virulence or adaptation to specific habitats by influencing the expression of chromosomal genes.Genetic changes leading to antimicrobial resistance are expected to result in higher fitness costs in the absence of antibiotic selection pressure. On the other hand, increased resistance is sometimes associated with increased virulence or to a better adaptation to stress conditions. This topic is developed in a study by Agnello et al. presented results that possibly explain why highly virulent P. aeruginosa strains, which often possess the exoU toxin gene, are more frequently resistant to fluoroquinolones than the generally less virulent strains harboring the exoS toxin gene. Their results support the hypothesis that fitness cost imposed by fluoroquinolone resistance may be lower and “more easily” compensable by exoU strains than by exoS strains.In another article dealing with bacterial fitness, Hao et al. analyze a multidrug resistant and virulent Campylobacter jejuni strain isolated from a broiler chicken. By comparison to reference strains, they revealed that the phenotype of this isolate was associated with large differences in the genome structure and content. This study provides an example of how a stepwise accumulation of mutations in antibiotic target genes, acquisition of exogenous resistance genes and virulence associated genes can result in the emergence of isolates that are both multidrug-resistant and virulent.In a whole genome sequence-based study, P. aeruginosa, therapies based on quorum sensing interference (QSI) have been envisaged as possible alternatives to conventional antibiotic therapies against this pathogen. In a thoughtful perspective article, Garcia-Contreras, however, compiles many recent experimental evidence which actually indicate that early optimistic expectations about QSI should be put in perspective. Indeed, he estimates that a far better understanding of P. aeruginosa virulence and behavior during infection is needed before efficient QSI-based clinical applications can be designed.Since quorum sensing is a major player in the control of virulence factors in P. aeruginosa such as bacterial killing or the repression of multiple virulence factors. This antibiotic is therefore currently used in the treatment of cystic fibrosis patients, whose lungs are commonly colonized by this pathogen. However, P. aeruginosa responds to AZM by yet unknown mechanisms and is able to counteract its killing and virulence-inhibitory effects. An interesting work by Tan et al. revealed that PA3297, a P. aeruginosa gene encoding a DEAH-box helicase, is involved in this response. Indeed, deficiency of this gene renders P. aeruginosa more susceptible to the killing and to virulence suppression by AZM. Authors therefore suggested that targeting the regulatory pathway of PA3297 or its function might increase the beneficial effect of AZM in chronic P. aeruginosa infections such as those occuring in cystic fibrosis patients.In addition to some immunomodulating activities, azithromycin (AZM) has various inhibiting effects on Obtreska-Machaj et al. report the development of hydantoin derivatives that have increased activity as inhibitors of the AcrAB-TolC efflux pump of Enterobacter aerogenes. Close homologs of this multidrug pump are also present in other pathogenic enterobacteria and involved in their virulence. The authors defined important pharmacophoric groups whose modulation may allow to develop still more potent inhibitors capable of restoring antibiotic activity in AcrAB producing bacteria.At last, in a pharmacological perspective, It is likely that both resistant and virulent high risk clones will continue to emerge and spread in the next years. Several factors supports this hypothesis, among which the facts that highly pathogenic bacterial strains are more likely to be subjected to the selective pressure of antibiotics treatments, that virulence and resistance determinants are sometimes genetically associated and that fitness and virulence impairments that often accompany acquisition of resistance can be compensated in the long term by suppressor mutations (Beceiro et al., All authors have substantially contributed to this work and 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.


We tested zebrafish shoals to examine whether groups exhibit collective spatial learning and whether this relates to the personality of group members. To do this we trained shoals to associate a collective spatial decision with a reward and tested whether shoals could reorient to the learned location from a new starting point. There were strong indications of collective learning and collective reorienting, most likely by memorising distal cues, but these processes were unrelated to personality differences within shoals. However, there was evidence that group decisions require agreement between differing personalities. Notably, shoals with more boldness variation were more likely to split during training trials and took longer to reach a collective decision. Thus cognitive tasks, such as learning and cue memorisation, may be exhibited collectively, but the ability to reach collective decisions is affected by the personality composition of the group. A likely outcome of the splitting of groups with very disparate personalities is the formation of groups with members more similar in their personality. Summary: Zebrafish groups were trained to make collective decisions and memorised locations by using cues. However, collective decisions were made slower by the splitting of groups comprising individuals of dissimilar personality. Organised groups are characterised by cooperative and synchronised behaviour, which allows for better resource acquisition and risk avoidance . HoweverMost studies on group navigation have focused on collective decision-making as a means of choosing between routes while maintaining group structure . Yet indDanio rerio. Fish were first tested as individuals to determine their levels of boldness  or the probability of splitting , suggesting consistency in collective behaviour and limited effects from differing individual learning during training. The mean boldness of shoal members did not significantly contribute to the probability of splitting . The only significant predictor was variance in shoal-member boldness, which strongly predicted both collective decision-times  and the probability of splitting . Groups with greater variance in boldness between their members were more likely to split and took longer to collectively reach an arm  in both the initial and probe trial, but some tended to split before reaching a decision . No significant differences were found between the initial trial (before training) and the probe trial (after training) for either decision times , but was not significantly predicted by the probability of splitting decision . Contrary to predicted speed–accuracy trade-offs .Decision accuracy (number of erroneous decisions during training) was only weakly predicted by the mean of shoal-member boldness .All shoals met the collective learning criterion of all fish being simultaneously in the rewarded location for eight/ten trials over three consecutive days . The rat2R<0.04; P>0.05). Indeed, the majority of shoals (eight/ten) showed preference for reaching the rewarded arm significantly more than predicted by chance , but the ability to reorient to the arm rewarded during training was unrelated to the variance and the mean of shoal-member boldness or the likelihood of splitting  were acquired from a local supplier, Grosvenor Tropicals, Lisburn, Northern Ireland. Given the supplier was not informed on strain variations in their stock, we used only males that show no strain preferences for shoaling  and used to calculate composite boldness scores. Greater latencies are linked to lower boldness , the boldness of each fish was assessed in their housing tank by measuring consistency in their approach latency towards novelty between two contexts often used to test differences in boldness [see review by boldness , thus thboldness .n=10) and housed together (tanks filled to 25 L) for a further week and then trained in a plus maze . During training internal landmarks were unavailable, but visual cues were available outside the maze, including white paper sheets on a distant wall, adjacent tank tops and the camera arm above the tank. To control for inter-shoal differences by differing information, these external cues and their locations were kept constant during trials and for all shoals. Shoal trials started in the bottom arm and the top arm was blocked during training. Trials commenced by removing an opaque divider that kept shoals constrained in the starting arm for 2 min. Shoals were then presented with the two remaining arms, left or right, with 5 min to choose between them. A collective decision was indicated by all individuals being in the same arm at the same time, training them to associate a collective decision towards one arm with a reward and towards the other arm with a mild punishment. The choice of direction, left or right arm, for the rewarded and mildly-punished arm was randomised across shoals. When reaching the arm randomly assigned to be food rewarded, shoals were blocked in until each fish received 1–2 chironomid larvae . However, in the unrewarded arm they were blocked in for 2 min and not fed [mild punishment; Following individual behavioural tests, fish were randomly sorted in shoals of five   tested whether each measure was predicted by the mean (5% trimmed to limit bias by minority fish with extreme phenotypes) or the mean absolute deviation of shoal-member boldness . Individuals with personality tendencies on the extreme ends of our distribution, mostly very shy individuals . The proportion of shoals reorienting at the probe trial was first tested against chance levels (0.5) by a binomial-proportion test. Speed–accuracy trade-offs were tested by rank correlations between time to decide and the number of erroneous trials during training (Spearman's rs) . Decisioividuals , can skeAll applicable animal-welfare guidelines were followed . Veterinary inspections by DHSSPS, Northern Ireland, deemed no need for licensing. Following the conclusion of the study, animals were kept for separate non-invasive tests.


Catheter-related central thrombosis is a rare complication of long-term central line. We describe the case of an asymptomatic boy who was diagnosed a calcified thrombus in right atrium eight years after the removal of a long-term central venous device. Although the most appropriate therapeutic approach for managing floating right heart thrombi remains to be determined, surgical removal is an effective and safe procedure for calcified long-standing thrombus and it is to be preferred in elective conditions especially in young asymptomatic patients without hemodynamic involvement, that are at low risk of surgery-related morbidity and mortality. Catheter-related central thrombosis is a rare complication of long-term central line , 2 especWe report the case of an asymptomatic boy who was diagnosed a thrombus in right atrium by echocardiogram eight years after the removal of a long-term central venous device used for chemotherapy administration.A 14-year-old boy was referred to our service to get a cardiologic evaluation with electrocardiogram (ECG) and cardiac ultrasonography as part of pre-partecipation screening to play rugby competitions. He was clinically fine, medical examination was unremarkable, and ECG was normal. Transthoracic echocardiography (TTE) evidenced a hyperechogenic polyglobulated calcified mass, suggesting it was an old formation, sizing 22 x 11 mm, consistent with an organised thrombus, with a thin pedicule of implantation in the floor of the right atrium, close to ostium of inferior cava vein, floating in atrium, without obstruction of systemic venous flow or tricuspidalic valve. He had never been symptomatic for embolic events and he had been performing sport activity (rugby) without any limitation for six months. The medical history included an ALL diagnosed when he was 6 years old. At that time a long-term indwelling central line (Broviac) via internal left jugular vein was placed without difficulty for chemotherapy administration. He was treated with Protocol AIEOP ALL 2000 that includes asparaginase. For standard evaluation of anthracycline toxicity at the beginning of chemotherapy he had a routine two-dimensional echocardiogram that did not show anomaly. During the treatment he had not bacterial nor mycotic sepsis and the Broviac was never used for parenteral nutrition. The catheter was removed uneventfully six months after completion of chemotherapy, thus eighteen months after implantation. To better define the characteristics of the mass and of interatrial septum, we performed a transesophageal echo (TEE) that confirmed the location and the extent of the lesion and showed a large patent foramen ovalis (PFO) through which a left to right shunt was detected after infusion of contrast media in basal conditions and right to left shunt after Valsalva manouver . TranscrMobile right atrial thrombi are a rare but potentially catastrophic phenomenon, because they can embolize at any moment requiring emergency treatment. Immobile thrombi can develop in situ favoured by blood stagnation and usually they have a good prognosis when treated with heparin. Right atrial thrombus is a known complication of long-term central venous catheter (CVC), and even if uncommon in children -12, it iWe emphasize the need for removing CVC as soon as possible after completion of treatment and for monitoring long-term indwelling central line especially in patients receiving chemotherapy and parenteral nutrition even in asymptomatic children in absence of signs of malfunctions, in order to promptly identify thrombus formation. Treatment for this condition is not standardized yet   and the The presence of intracardiac thrombi should be monitored in children with long-term central line for chemotherapy, and echocardiography is a quick and valid instrument to detect this life-threating but treatable condition. Although the most appropriate therapeutic approach for managing floating right-heart thrombi remains to be determined , we suggest that surgical removal is an effective and safe procedure for calcified thrombus and it is to be preferred in elective conditions especially in young asymptomatic patients without hemodynamic involvement, who are at low risk of surgery-related morbidity and mortality.


Necrotizing fasciitis is a devastating infectious disease process that is characterized by extensive soft tissue necrosis along deep fascial planes, systemic toxicity, and high mortality. Ultrasound imaging is a rapid and non-invasive tool that can be used to help make the diagnosis of necrotizing fasciitis by identifying several distinctive sonographic findings. The purpose of this study is to describe the construction of a realistic diagnostic training model for necrotizing fasciitis using fresh frozen cadavers and common, affordable materials.Presently, fresh non-embalmed cadavers have been used at medical institutions for various educational sessions including cadaver-based ultrasound training sessions. Details for the preparation and construction of a necrotizing fasciitis cadaver model are presented here. This paper shows that the images obtained from the cadaver model closely imitate the ultrasound appearance of fluid and gas seen in actual clinical cases of necrotizing fasciitis.Therefore, it can be concluded that this cadaver-based model produces high-quality sonographic images that simulate those found in true cases of necrotizing fasciitis and is ideal for demonstrating the sonographic findings of necrotizing fasciitis. Ultrasound is a rapid and non-invasive tool ideal for the imaging of soft tissue infections, and it has been associated with a change in clinician management in 50% of emergency department soft tissue infections . NecrotiAlthough various diagnostic imaging modalities such as computed tomography (CT) and magnetic resonance imaging (MRI) are often used in the diagnosis of necrotizing fasciitis (NF); the diagnostic gold standard for NF relies on surgical exploration and tissue cultures  . Due to Fresh non-embalmed cadavers are used at medical institutions, including ours, for various educational sessions including cadaver-based ultrasound training sessions -10.The following materials are needed to prepare this training model: A fresh cadaver, ultrasound gel, normal saline, and an 18 guage needle with a 20 cc (or larger) syringe.To create the NF model, the following steps are taken:1) Ultrasound the desired musculoskeletal region of interest Figure  and idenThe surface quality of a fresh cadaver is certainly realistic; as such, the model mimicked the tactile feel of a true clinical scenario quite well. Furthermore, the unique anatomy of each cadaver was similar to the variable anatomy found in true clinical encounters. The cadaver skin lacked warmth and erythema; however, this was determined to be beneficial given that many cases of NF do not present with these symptoms. The cadaver model closely mimicked the ultrasound appearance of fluid and gas as seen in actual clinical cases of NF Figure -1F. The Necrotizing fasciitis is a devastating soft tissue infection characterized by the rapid spread of inflammation and necrosis that progressively involves the deep fascia, subcutaneous fat, overlying skin, and even skeletal muscle -3. BecauThe diagnostic gold standard for NF is surgical exploration with tissue cultures, and its treatment centers around source control through aggressive necrotic tissue debridement, fluid maintenance, and appropriate antibiotic therapy -3. WhilePoint of care ultrasound has proven to be useful in rapid clinical decision-making scenarios and has gained popularity because it is fast, non-radiating, and readily available . StudiesSince ultrasound imaging can be highly operator dependent, suitable teaching models should be available to properly train clinicians on how to best diagnose and recognize sonographic findings of NF. A mannequin-based model for NF (in addition to plain radiographs and CT scans demonstrating NF) has been shown to improve diagnostic ability of learners; however, this study did not include the use of ultrasound . To our The major limitation of this model is the cost and availability of cadavers since we are dependent upon the Willed Body Program and the monumental and generous gifts of our donors. As with most cadaver-based educational sessions, it is prudent to maximize the number of training simulations. We did not evaluate the effectiveness of the model for teaching and retaining the use of ultrasound as a diagnostic tool for NF, although these issues will be topics for further investigation using our cadaver model.Necrotizing fasciitis is a disease with significant risk for morbidity and mortality. With the ubiquitous use of ultrasound in emergency departments, it is pivotal that emergency clinicians be aware of the sonographic appearance of NF on ultrasound. Our model successfully replicates the clinical and ultrasound appearance of necrotizing fasciitis as compared to real clinical cases. Our model is well suited to simulate how point of care ultrasound can be used to aid in the diagnostic workup of necrotizing fasciitis while minimizing time to surgical consultation and intervention.


Tubulin proteins, as components of the microtubule cytoskeleton perform critical cellular functions throughout all phases of the cell cycle. Altered tubulin isotype composition of microtubules is emerging as a feature of aggressive and treatment refractory cancers. Emerging evidence highlighting a role for tubulin isotypes in differentially influencing microtubule behaviour and broader functional networks within cells is illuminating a complex role for tubulin isotypes regulating cancer biology and chemotherapy resistance. This review focuses on the role of different tubulin isotypes in microtubule dynamics as well as in oncogenic changes that provide a survival or proliferative advantage to cancer cells within the tumour microenvironment and during metastatic processes. Consideration of the role of tubulin isotypes beyond their structural function will be essential to improving the current clinical use of tubulin-targeted chemotherapy agents and informing the development of more effective cancer therapies. The microtubule cytoskeleton is an extensive network of filaments that span the cell interior. Microtubules are composed of αβ-tubulin heterodimers that associate to form protofilaments, which then laterally associate to form microtubules . These hDuring interphase, the majority of microtubules are nucleated at the centrosome and radiate towards the cell periphery . One of vinca alkaloids and epothilones, directly target β-tubulins to suppress the dynamics of spindle microtubules, thereby disrupting chromosomal segregation to induce cell cycle arrest and cell death . TubuliMitochondrial dynamics can significantly impact tumour progression ,166 and Tubulins are known to regulate endoplasmic reticulum (ER) homeostasis, with ER dynamics tightly linked to microtubule dynamics ,185,186.The microtubule network is involved in the initiation and execution of macroautophagy (reviewed in ), which TMPRSS2:ERG rearrangement, ERG expression and PTEN deletions, three key oncogenetic features in metastatic prostate cancer [A growing body of evidence has pointed to a role for the tubulin proteins in the metastatic progression of cancer. Research conducted over the past decade has indicated several tubulin isotypes as potential prognostic markers whose expression levels in primary tumours are associated with aggressive disease with greater metastatic potential and the propensity of patients to suffer metastatic relapse, although the major focus has centred on βIII-tubulin expression. In particular, clinical data has identified that high βIII-tubulin protein expression strongly correlates with aggressive clinical behaviour and poor patient outcome in many forms of cancer, including breast, ovarian, gastric, glioblastoma, prostate, colorectal, pancreatic and NSCLC ,212,213.e cancer .In melanoma, βIIb-tubulin mRNA transcript levels were significantly elevated in melanoma multinucleated giant cells which are frequently observed in metastatic sites and correlate with poor prognosis, indicating that this tubulin isotype may play an important role in melanoma metastasis . IncreasTumour metastasis requires completion of a multistep process, with dissemination of cancer cells into distant organ sites and their subsequent adaptation to foreign tissue microenvironments. Altered tubulin isotype composition may increase the metastatic potential of cancer cells by altering their capacity for migration and invasion. Emerging evidence indicates that activation of the epithelial-to-mesenchymal transition (EMT) program is critical in regulating invasion and metastasis  and thatFunctional links between βIII-tubulin and metastasis have also been reported using lung and pancreatic cancer mouse models ,66,123. The aberrant expression of β-tubulin isotypes in a range of cancers is correlated with aggressive, drug refractory disease with poor clinical outcome. While the effect of isotype composition on the interaction of tubulin binding agents with their targets, and on microtubule dynamics may partially explain drug resistance to these classes of common chemotherapeutics, the current evidence suggests that tubulin isotypes are involved more broadly in biological processes that modulate tumour progression and metastasis. Through interactions with oncogenic signalling and cell survival programs that facilitate adaptation to the harsh tumour microenvironment, altered tubulin isotype composition in cancer may contribute to drug resistance and more aggressive disease . DefininIn order to develop a more comprehensive understanding of the tubulin code in cancer, the context in which tubulin isotypes impact clinical outcome in this disease will need to be more robustly defined both within and between different types of cancers. Critical to this will be a firm understanding of the mechanisms that drive the individual and collective expression of tubulin isotypes in health and disease. The advent of gene-editing technologies provides great potential in the selective manipulation of isotype expression while retaining the endogenous regulatory framework. Defining the larger intracellular network in which the microtubule system operates will no doubt reveal the complex integration of this component of the cytoskeletal network with fundamental cellular processes and reveal novel therapeutic strategies for targeting altered tubulin isotype expression in cancer.


The vasomotor axon reflex can be evoked in peripheral epidermal nociceptive C-fibers to induce local vasodilation. This neurogenic flare response is a measure of C-fiber functional integrity and therefore shows impairment in patients with small fiber neuropathy. Laser Doppler flowmetry (LDF) and laser Doppler imaging (LDI) are both techniques to analyze vasomotor small fiber function by quantifying the integrity of the vasomotor-mediated axon reflex. While LDF assesses the flare response following acetylcholine iontophoresis with temporal resolution at a single defined skin point, LDI records flare responses with spatial and temporal resolution, generating a two-dimensional map of superficial blood flow. LDF is characterized by a high intra- and interindividual measurement variability, which is smaller in LDI due to its spatial resolution. Nevertheless, LDI still lacks standardized methods for image analysis. Consequently, use of the technique currently remains on an experimental level. Here, we sought to review the current literature on laser Doppler assessment of vasomotor function and discuss potential future applications of established techniques as well as those that are still experimental. In small fiber neuropathy, small somatic fibers and/or autonomic fibers are affected. Small fibers are unmyelinated C-fibers or thinly myelinated A-delta fibers that mediate thermal sensation, pain, and autonomic function, such as blood vessel and sweat gland control. A selective damage to these peripheral nerve fibers can be caused by a number of conditions and therefore comprises a variety of pathophysiologically heterogenous disorders, among them diabetes, neurodegenerative diseases, or paraneoplastic syndromes. Small fiber neuropathy is associated with increased cardiovascular morbidity and mortality, therefore early diagnosis and continued assessment of disease progression is important , 2. AsseBased on the assessment of the Doppler shift of backscattered laser light by moving red blood cells , change Laser Doppler flowmetry investigates vasomotor axon reflex response with temporal resolution in fixed points of the skin. It is widely used in research but its clinical use is limited due to a high intra- and interindividual variability. LDI assesses the vasodilatory response after acetylcholine iontophoresis using spatial and temporal resolution, thereby reducing variability. However, it has not attained widespread use, and only few clinical studies have used this technique to assess neurovascular dysfunction Table .This review provides a summary of the current literature on laser Doppler assessment of vasomotor function and critically discusses the utility and drawbacks of available and experimental techniques in research and clinical practice as well as potential future implications.Specific examination of quality and integrity of peripheral autonomic nerve fibers can be performed with vasomotor  and sudomotor (sweat gland function) tests that are usually only performed in specialized academic centers. In the beginning of the twentieth century, the term “axon reflex” has been described in vasomotor fibers that were studied and investigated afterward in different clinical settings , 7. The via a drug-delivering capsule is a common technique for studying pharmacological aspects of the microvascular function , coherent laser light source on a fixed area of the skin. Processing the frequency distribution of the backscattered light allows an estimation of the microvascular blood perfusion . LDF quaLaser Doppler imaging constitutes a newer axon reflex-based measurement method, which records the vasomotor axon reflex-mediated flare response with spatial and temporal resolution and generates a two-dimensional map of the cutaneous blood flow . The imaLaser Doppler imaging measures the microvascular blood flow sequentially over a defined skin area, representing skin blood flow two-dimensionally over time . Thus, LThere are several techniques available to analyze the axon reflex-induced vasodilatory response following acetylcholine iontophoresis. All analyzing techniques have in common that a baseline perfusion threshold is predetermined initially and used as reference for subsequent perfusion measurements.The flare area method sets the threshold by averaging the maximum perfusion reaction of all cells. Thereafter, the axon reflex flare is determined by the number of cells above the set threshold after acetylcholine iontophoresis subtracted from the number of cells above the threshold before the iontophoresis :[axon−rThe baseline perfusion method defines the blood flow threshold as two standard deviations of the baseline perfusion. Thereafter, the total axon reflex-mediated flare area is calculated based on the total number of cells exceeding the defined threshold .After acetylcholine iontophoresis the axon reflex-mediated flare response is represented by the measurement of the total number of cells exceeding the previous identified blood flow threshold , 14. ComBoth LDF and LDI should be applied under constant temperature, humidity, and light conditions to ensure reliable measurements. The distance to the tested skin area should be constant as well in all measurements as it influences the outcome of the imaging processes of both techniques. A calibration of each device before starting the measurement is recommended. Therefore, the use of both methods in bedside testing is challenging from the clinical point of view. Moreover, the LDI as well as the LDF technique require a thorough preparation of the studied skin area. Hairs should be removed from the skin, and skin scales should be removed carefully to avoid any disturbances in the imaging processes. The use of both methods on irritated skin is not recommended. In addition, for LDI, movement of the scanned area has to be avoided since this will introduce movement artifacts in the recording, limiting quality of the data obtained.Laser Doppler flowmetry assessment is characterized by a high intra- and interindividual variability and is limited by technically demanding settings. Since most investigators agree that it lacks sensitivity to detect vasomotor neuropathy in individuals during clinical routine diagnosis, LDF is being limited to specialized clinical centers with specific demands and research studies , 33–35. Using the LDI technique in clinical or research setting requires training. It is recommended to set up the LDI equipment before acetylcholine iontophoresis. The LDI needs to be placed at an appropriate distance over the iontophoresis capsule parallel to the skin before starting the imaging process. The presence of different analysis techniques to measure the extent of LDI axon reflex-induced flare area limits its consistent application and generalization of results. Most importantly, to improve utility standardization of image analyses presets is necessary. Therefore, studies to define parameters relevant to diagnostic discrimination such as sensitivity and specificity are needed. Especially, a set of normative data gained in studies in large populations of healthy subjects compared with patients with neuropathy should form the basis for future application. Furthermore, technical independence from environmental factors such as light, temperature, and humidity would improve its application. Until now, application of LDI axon reflex flare area test remains on an experimental level in clinical diagnostics and is limited to specialized centers , 31, 32.Even though clinical studies assessing these tests are still rare, some studies have been published proving their utility. It has been suggested that LDI is a simple method to detect early neuropathy in subjects with type 2 diabetes and may have some value as an endpoint to assess therapeutic interventions aiming to prevent or reverse C-fiber dysfunction . FurtherLaser Doppler imaging has shown a high sensitivity for diabetic mixed fiber neuropathy detection and could be performed with immediate results . Its repIn neurodegenerative disorders, reduced laser Doppler blood flow in patients with Huntington’s disease supports the hypothesis that autonomic nervous system dysfunction is part of the pathogenesis of Huntington’s disease . In patiAssessment of microvascular function in peripheral small fiber neuropathy is useful to diagnose vasomotor dysfunction and supplements other well-established autonomic function tests, such as tilt-table testing and the measurement of heart rate variability. Hence, vasomotor axon reflex-mediated flare measurement techniques have been gaining importance in recent years not only in research studies but also in specialized academic and clinical centers.Laser Doppler imaging presents increased accuracy and reproducibility when compared to the single-point LDF technique by using temporal and spatial resolution. However, standardized blood flow image analysis warrants further validation in larger study groups before the technique can be established in clinical practice.MK: conception and drafting the manuscript. AK, JP, SB, and KB: reviewing the manuscript for intellectual content. BI: creating figures and reviewing the manuscript for intellectual content. TS: conception, creating figures, and reviewing the manuscript for intellectual content.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.


In 2014, the all-cause age-adjusted death rate in the United States reached a historic low of 724.6 per 100,000 population . In mostPotentially excess deaths  are defined as deaths among persons aged <80 years in excess of the number that would be expected if the death rates for each cause were equivalent across all states to those that occurred among the three states with the lowest rates . During 2014, the number of potentially excess deaths from the five leading causes in rural areas was higher than those in urban areas  organize systems that coordinate acute stroke care across both urban and rural hospitals . These programs build coalitions that develop and implement strategic plans for cancer prevention and reduce morbidity and mortality for persons affected by cancer.To address the rural-urban gap in cancer-related potentially excess deaths, comprehensive approaches that encompass the cancer continuum  are needed at the local and state level to reduce risks associated with potentially excess deaths from cancer in rural areas (CDC also supports cancer screening programs that address health disparities among adults who are uninsured or underinsured (As differences in cancer-related death rates are addressed at the local and state level, opportunities are available to address them at the federal level. The Cancer Moonshot is focused on accelerating the understanding of cancer and its prevention, early detection, treatment, and cure, including improving access and care (During 2008–2010, the annual age-adjusted death rates for unintentional injury were highest in rural counties . SeveralInterventions to address the disproportionate unintentional injury death rates in rural areas include increased adherence to guidelines for triaging ambulatory transport destinations (In the United States, there is a rural-urban gap in age-adjusted death rates and potentially excess deaths from the five leading causes of death. Rural communities experience higher age-adjusted death rates and a higher number of potentially excess deaths from the five leading causes compared with urban areas. Higher death rates and potentially excess deaths are often associated with various interconnected societal, geographic, behavioral, and structural factors. Historic trends indicate that focusing on access to health care in rural areas of the United States alone is not sufficient to adequately address complex health outcomes, including mortality among rural populations (


The contribution of adaptive immune responses driven by T-helper cells and B cells will be reviewed as well. Each form of immune activation will be discussed in the context of its relationship to environmental and genetic factors, disease outcomes, and potential therapies. We conclude with discussion of unanswered questions and opportunities for future study in this area.The contribution of the immune system to idiopathic pulmonary fibrosis (IPF) remains poorly understood. While most sources agree that IPF does not result from a primary immunopathogenic mechanism, evidence gleaned from animal modeling and human studies suggests that innate and adaptive immune processes can orchestrate existing fibrotic responses. This review will synthesize the available data regarding the complex role of professional immune cells in IPF. The role of innate immune populations such as monocytes, macrophages, myeloid suppressor cells, and innate lymphoid cells will be discussed, as will the activation of these cells  Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic disease of unknown etiology characterized by the radiographic and histopathologic pattern of usual interstitial pneumonia (UIP) , 2. It iThe pathogenic cascade of lung fibrosis is thought to be initiated by perpetuated microinjuries to the alveolar epithelium that engenders a dysregulated wound healing response . ThroughIn order to understand the controversy surrounding the role of the immune system in IPF a brief overview of the disease state is required. IPF is defined as the presence of UIP in the absence of an identifiable underlying cause. Examination of lung tissue from patients with IPF reveals a paucity, but not absence, of inflammatory cells, when compared to pathologies, such as non-specific interstitial pneumonia, acute respiratory distress syndrome, organizing pneumonia (OP), and granulomatous processes such as hypersensitivity pneumonitis (HP) . SimilarThe immune response is stratified into innate processes, which respond immediately to chemical or physical patterns of the stimulus, and adaptive immunity, which involves a highly specific antigen-driven response. Both arms of the immune system appear to be activated in IPF. The data supporting this concept are presented below.The innate immune system forms the first line of defense against pathogens. Its recognition of antigens is mainly dependent on pattern recognition by innate immune receptors. These cell populations are central to both host defense and tissue homeostasis. Macrophages and neutrophils are among the best studied innate immune cells in regard to IPF, though a contribution of monocyte-derived cells, such as fibrocytes and myeloid-derived suppressor cells (MDSCs), and of innate lymphoid cells (ILCs), has also been proposed. It should also be noted that parenchymal cells, such as epithelial cells and fibroblasts, also show abnormalities in innate immune activation . HoweverMacrophages are innate immune cells that not only act as antimicrobial phagocytes in the lungs but also play a key role in the pathogenesis of fibrotic lung disease . Of the via a paracrine mechanism involving the production of soluble mediators typically associated with alternative activation . Ligands for PRRs fall into two classes. Those derived from invading microorganisms are called “PAMPs” and those derived from injured cells and tissues are called “DAMPs” . While bvia upregulating TGFβ1 expression and inducing mesenchymal properties in lung epithelial cells  is enriched in bronchoalveolar lavage (BAL) fluid and lung biopsy tissue of IPF patients when compared with healthy controls , 53, andal cells . In addial cells . A stronal cells . Consideal cells  and a smal cells  althoughThe potential contribution of bacteria to IPF pathogenesis is also an area of active investigation. Specifically, a relationship between total bacterial load and poor prognosis was observed in a study in which enriched detection of organisms, such as Haemophilus, Streptococcus, Neisseria and Veillonella, was found in BAL fluid of IPF patients . In addiImmune responses are also initiated in the absence of pathogen recognition. Here, damage to previously intact cells and tissue results in the accumulation of substances with the potential to function as “DAMPs.” In uninjured tissues, DAMP-mediated inflammatory processes contribute to homeostasis by allowing the regulated removal of debris, thereby facilitating the resolution of injury and the achievement of repair . Abnormavia the exocytosis of membrane bound vesicles or endosomes. Still another class of DAMPs is generated by the transformation of inert proteins such as collagens into signaling molecules such as collagen fragments. These entities are recognized by innate immune receptors that for the most part have the ability to be activated via pathogens as well  as detection of this PRR  inflammasome activation leads to IL-1β production and fibrosis in bleomycin treated animals , 81. Whithis PRR  and its this PRR . In factthis PRR  or synththis PRR . Howeverthis PRR , likely Neutrophils are innate immune cells that possess several functions through which they might participate in fibrosis. Neutrophilia in BAL fluid has been associated with early mortality in IPF  and concvia their regulation of ECM turnover. Neutrophil elastase (NE), the main proteolytic product of alveolar neutrophils, is increased in BAL fluid of IPF patients  axis  axis , but proilicosis . In summB cells represent another arm of the adaptive immune system. Functioning primarily as producers of antibodies, increased detection of CD20+ B cells has been reported for IPF lungs . A varieAs can be seen, the cells of the immune system show a rich and multifaceted contribution to IPF. When viewed in contrast with the epithelial cells that are believed to be the primary site of injury, and with fibroblasts, which are viewed as the driver of matrix deposition and remodeling, the more heterogeneous contribution of the innate and adaptive immune systems shown in Figure Box 1To what extent do data obtained from mouse models reflect the situation in the fibrotic human lung? Can mimetics be developed that more accurately simulate the IPF disease state?Do events in the peripheral blood truly reflect events occurring in the diseased lung?Do the innate immune abnormalities seen in IPF represent a unique form of immunosenescence?Can therapies targeting macrophage activation stabilize or restore lung function in patients with IPF?Does the altered microbiome cause pathogen-associated molecular pattern-driven innate immune activation in IPF and are antimicrobial therapies efficacious in IPF?Does perpetuated microinjury cause danger-associated molecular pattern (DAMP)-driven innate immune activation in IPF and are therapies targeting DAMPs and their receptors efficacious in IPF?Are neutrophil extracellular traps an important part of IPF pathogenesis?What is the role of fibrocytes and myeloid-derived suppressor cells in IPF?Do innate lymphoid cells participate in IPF?How does the relative balance of T-helper cells participate in IPF and can this contribution be targeted in a safe and efficacious manner?Are B cells involved in the development of IPF?Can immune events detected in the circulation be used to guide personalized therapies in IPF?OD wrote manuscript, JW made figures and wrote manuscript, MM wrote manuscript, and EH wrote manuscript. All authors approved final 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.