Single trauma injuries or isolated fractures are often manageable and generally heal without complications. In contrast, high-energy trauma results in multi/poly-trauma injury patterns presenting imbalanced pro- and anti- inflammatory responses often leading to immune dysfunction. These injuries often exhibit delayed healing, leading to fibrosis of injury sites and delayed healing of fractures depending on the intensity of the compounding traumas. Immune dysfunction is accompanied by a temporal shift in the innate and adaptive immune cells distribution, triggered by the overwhelming release of an arsenal of inflammatory mediators such as complements, cytokines and damage associated molecular patterns (DAMPs) from necrotic cells. Recent studies have implicated this dysregulated inflammation in the poor prognosis of polytraumatic injuries, however, interventions focusing on immunomodulating inflammatory cellular composition and activation, if administered incorrectly, can result in immune suppression and unintended outcomes. Immunomodulation therapy is promising but should be conducted with consideration for the spatial and temporal distribution of the immune cells during impaired healing. This review describes the current state of knowledge in the spatiotemporal distribution patterns of immune cells at various stages during musculoskeletal wound healing, with a focus on recent advances in the field of Osteoimmunology, a study of the interface between the immune and skeletal systems, in long bone fractures. The goals of this review are to (1) discuss wound and fracture healing processes of normal and delayed healing in skeletal muscles and long bones; (2) provide a balanced perspective on temporal distributions of immune cells and skeletal cells during healing; and (3) highlight recent therapeutic interventions used to improve fracture healing. This review is intended to promote an understanding of the importance of inflammation during normal and delayed wound and fracture healing. Knowledge gained will be instrumental in developing novel immunomodulatory approaches for impaired healing. Inflammation is the first response in the process of wound and fracture healing, and appropriate activation of the immune system is integral for maintaining tissue integrity and facilitating a return to homeostasis. Innate immune cells respond rapidly to injuries by both releasing and interacting with pro- and anti- inflammatory mediators  in order to direct the inflammatory response through inflammation and toward resolution. In healthy individuals, an isolated musculoskeletal wound or a non-critical size fracture typically heals without the need for intensive care. However, in patients with severe or polytraumatic injuries, there are major challenges associated with healing of musculoskeletal wounds, particularly in conditions where the inflammatory response is skewed or dysregulated as a result of severe injuries –3. FurthOngoing research and clinical efforts have brought about many advancements in the development of wound care products over the past decades. Several bioengineered scaffolds, primarily skin equivalents, and one cellular scaffold product, GINTUIT, have received FDA approval and have been used in the clinic to aid in wound healing and regeneration , 8. FurtDespite advancements in the field of regenerative medicine, there is still an urgent requirement for better wound healing therapies to improve the quality of life post-injury. However, modulation of the immune response through the use of therapeutics must be carefully considered, as failure to follow the correct therapeutic regimen could result in enhanced immune suppression and unintended outcomes. It is our belief that, in-depth knowledge of the spatial and temporal patterns of immune and inflammatory cells and their responses, post-injury, is critical for the development of effective therapeutic interventions for musculoskeletal regeneration. With this review, the authors aim to provide an in-depth survey on the innate and adaptive immune cell responses that regulate the stages of healing, with particular emphasis on the critical findings that underscore the important link between inflammation and the musculoskeletal healing in both healthy and distressed conditions. It is well known that the immune cell populations and the time at which these cells are recruited differ significantly during the healing processes of muscle and bone , 10. As Muscle injuries are increasingly common place and are often caused by acute trauma, with traffic accidents and armed conflict accounting for a significant portion of injuries . Skeleta+ T helper cells, along with few CD8+ T cytotoxic cells, to the injury site is a hallmark of normal healing in traumatic muscle injuries (+ T helper cells (enriched with T regulatory cells/Tregs [CD4+CD25+FOXP3+ T cells)] are selectively recruited (+ infiltrating lymphocytes) and they decreased at 4 hpi (~4%) and soon disappeared from the injury site , differentiated (Pax7−MyoD+) and quiescent (Pax7+MyoD−) states of satellite cells and to estimate age of the muscle injury. Pax7 regulates satellite cell proliferation and prevents early myogenic differentiation and apoptosis. Pax7 expression is sharply downregulated before satellite cell differentiation  and growth factors like hepatocyte growth factor (HGF), nerve growth factor (NGF), heat shock protein 70 (Hsp70) and fibroblast growth factor (FGF) –24. Thesntiation . Whereasntiation . Both Pantiation , 30.Tregs and macrophages are also integral to the muscle regeneration process. Tregs migrate toward the injury site in response to interleukin 33 (IL33) released from recruited mast cells and secrete growth factors, such as amphiregulin, required for muscle regeneration . Tregs aMacrophages can also potentiate the activation of satellite cells from a quiescent state, inducing proliferation and differentiation . In inju+MyoD−) represented as resident muscle stem cells, ready to respond to the next episode of muscle injury  as a standard biological approach for bone regeneration was traditionally discussed as the triangle concept . Giannou+ T helper cells , mast cells, histocytes and eosinophils to form a tight clump of cells known as fracture hematoma . The hemNeutrophils are recruited to the fracture site from ~1 hpf through 5 dpf via sensing a chemical gradient released by chemo-attracting/pro-inflammatory mediators like growth factors, cytokines and chemokines  Figure . The neuMast cells are granulated cells found in circulation in their immature states. Apart from their roles in allergic responses and autoimmunity, they also have a positive role in regulating tissue remodeling, wound healing and fracture healing . Mast cein-vitro study reported suppression of NK cells when incubated with fluids from early fractured zone and plasma from human subjects  infiltrate the fracture site from 3 to 28 dpf , maintain immune surveillance, bone formation, functional hematopoietic stem cells niches, and erythropoiesis  (inflammatory) and T helper 2 (Th2) (immune modulatory) type cell responses. Additional CD4+ T cell subsets, such as Th17 cells and Tregs, also have immunomodulatory roles during fracture healing. Th17 cells via the secretion of IL17F promote the maturation and maintenance of osteoblasts (+CD25+Foxp3+) are pivotal in maintaining immune homeostasis , albeit they showed that once the other co-inflammatory mediators and cells entered the fracture microenvironment, the healing was slowed down and was not different from the WT mice. In these γδ T cell deficient mice, the early alteration on healing noted in bone and cartilage formation correlates with the reorganization of the immunologic milieu at the inflammatory stage. Therefore, at 5 dpf, the absence of γδ T cells exhibited a qualitative influence on the final bone regeneration outcome, a phenotype that did not last through 28 dpf  at this time , and only a few memory or immature B cells (B220+IgM+IgD+) are detected  to 35 weeks  depending on the severity, ~16% of all fractures, in human patients subjected to surrounding soft tissue damage and/or high energy trauma, results in non-union i.e., a failure to heal within 35 weeks of fracture . Moreove+T cells, i.e., CD8+TEMRA cells  in circulation and in the fracture hematoma from patients with delayed healing, ranging from 1 to 18 weeks post-fracture, compared to those with normal healing, indicating a negative impact of these cells on healing outcomes [+ TEMRA cells with CD57+/CD28− were predominantly seen in the fracture hematoma than in blood. In sterile conditions, the CD8+ TEMRA cells respond to stress signals released from endothelial cells at the injury site or by pro-inflammatory cytokines, and not via the classical professional antigen presentation. Modulation of various effector function of CD8+TEMRA cells greatly improved fracture healing, confirming the inhibitory effect of these callus infiltrating cells in bone repair. Importantly, the authors observed that the mice housed in specific pathogen free facilities had less CD8+TEMRA cell counts with little to no role in delayed fracture healing, indicating that these memory cells appear in higher counts in individuals with pre-clinical conditions. Thereby, the authors concluded that the increased population of CD8+TEMRA cells in delayed healing reflected the patient's individual immune response and not influenced by the fracture or surgery procedures  into the muscle injury site at 3 dpf through 28 dpf, with a peak at 14 dpf, compared to isolated osteotomy , i.e., irrecoverable muscle injury occurring adjacent to the fracture site. VML is a debilitating condition and the associated hemorrhage that occurs with VML can be life threatening. Although ~250,000 civilians suffer per year from open fractures involving a component of VML injury, the occurrence of VML injuries is most prevalent and a major concern in wounded military soldiers accounting for 65% disability in soldiers in the orthopedic cohort . Importasteotomy  [Figure steotomy . Althougsteotomy  [Figure steotomy . M1 macrmy group . Additiomy group . Typicalmy group , 131.Some of the other pre-existing chronic inflammatory response mounting factors that influence delayed union include: (i) Diabetes; (ii) Geriatrics and (iii) Smoking.Diabetes mellitus (DM), in particular type 2 DM (T2D), is well documented to have a negative impact on fracture healing and diagnosis of T2D associated with increased risk of fracture injury, despite patients often having normal to high bone mineral density . TheoretThe negative effect of smoking on fracture healing has been well documented , 138–141Aging is associated with increased risk of fracture due to changes to bone mass and density. The innate and adaptive immune response is altered with age, often skewed toward a chronic pro-inflammatory state that has been referred to “inflammaging” . The chr+ Treg cells have provided promising results  is a key requirement for efficient and timely bone fracture healing. Temporal disruption of immune cells in the fracture callus can be detrimental to all the four stages of bone healing and modulating inflammatory response in sterile trauma-induced conditions through therapeutic interventions has been a challenge. Some of the past interventions extend from targeted inhibition of receptor molecules to enzymatic removal of naturally occurring endogenous molecules that trigger chronic inflammation , 171. A  results –175. Add results , 177. ThBone is a complex tissue that requires many nutrients, especially during fracture repair, and multi-nutrient therapy has been suggested to improve fracture healing , 185. DiAs mentioned earlier, a well-balanced inflammatory response is an integral component in the bone healing process. Inflammatory mediators are responsible for maintaining and keeping this balance. Cells at the site of trauma release large amounts of endogenous inflammatory prostaglandins and eicosanoid derivatives called pro-resolvin/lipid mediators, generated through activation of cyclooxygenase (COX) enzymes, COX-1 and COX-2. Prostaglandin-induced inflammation is an essential component of the fracture healing process, and COX-1 and COX-2 play important roles in fracture healing. Endogenous lipid mediators in lieu with M1 macrophages are a natural and integral part of osteogenesis because they maintain a tight regulation of the initial inflammatory immune response which is crucial to timely fracture healing . In caseNanoparticle modified composite scaffolds  and bio-in vitro in the presence of BMP (in vivo (FK506 is a FDA approved immunosuppressive compound with osteogenic effects –204 thate of BMP  and may (in vivo .Another recent advancement is the administration of minced grafts as potential therapeutics to restore VML, due to the ability of the minced grafts to promote fiber regeneration in wounded muscles . MoreoveMSCs function by orchestrating the host's cellular and molecular response to injury. The use of exogenous MSCs in fracture unions is one of the most widely studied therapeutic approach, with on-going pre-clinical trials investigating their ability to improve non-union fracture healing . The souThe therapeutic potential of exogenous MSCs has revolutionized the field of fracture care and many labs are still refining their application in the field. Much about MSCs and their beneficial role in fracture healing has been previously extensively reviewed at length .Developing highly effective interventions requires an in-depth knowledge about the immune response during the healing phase in injured muscles and bones, keeping in mind the different healing cascades and immune cells involvement. Recent advances in understanding of the molecular basis of healing and inflammation in complex wounds have provided crucial guidance for improving care for critically injured patients. In summary, we discussed the acute inflammatory response in normal/standard healing process of single trauma i.e., isolated wounds. Next, we discussed delayed healing impacted by dysregulated inflammation in muscle wounds and long bone fractures especially following severe isolated fractures, polytrauma and/or concomitant muscle loss as well as in the case of other comorbidities like diabetes, smoking and aging. We also highlighted recent advances in therapeutic measures for improving bone fracture healing.Normal wound and fracture healing processes require a well-balanced inflammatory response. A shift in the normal balance can be detrimental to the healing outcomes causing impaired/delayed healing. Immunomodulatory therapeutic interventions can prove to be beneficial, if applied in right proportions and in a timely manner during the healing process. There is an urgent requirement for developing targeted immunomodulatory therapeutic interventions especially for delayed fractures associated with polytrauma and concomitant muscle trauma. As we continue to extrapolate new information about the cellular and molecular players of osteo-immunogenic responses associated with delayed fracture healing, the better prepared we will be to develop new therapeutic strategies to treat these costly clinical problems. Moreover, the precise underlying cellular mechanisms that drive bone healing need further attention. Finally, the understanding of basic immune-biological functions of healing will allow wound care specialists greater insights about the importance of how their skills can improve musculoskeletal wound healing processes.PM refined the overall concept, wrote the first draft, and revised the manuscript. LM wrote sections of the manuscript and contributed to the revision. JW developed the broad focus and concept of the manuscript, and contributed to the revision. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Increased knowledge regarding the implications of gut microbiota in irritable bowel syndrome (IBS) suggests that a disturbed intestinal microenvironment (dysbiosis) might promote the development and maintenance of IBS symptoms and affects several pathways in the pathology of this multifactorial disease. Accordingly, manipulation of the gut microbiota in order to improve IBS symptoms has evolved as a novel treatment strategy in the last decade. Several different approaches have been investigated in order to improve the gut microbiota composition. Dietary modifications including supplementation with fibers, prebiotics, and probiotics are shown to improve symptoms and composition of gut microbiota in IBS; however, the exact probiotic mixture beneficial for each individual remains to be identified. The use of antibiotics still needs confirmation, although promising results have been reported with use of rifaximin. Fecal microbiota transplantation (FMT) has recently gained a lot of attention, and several placebo-controlled trials investigating FMT obtain promising results regarding symptom reduction and gut microbiota manipulation in IBS. However, more data regarding long-term effects are needed before FMT can be integrated as a customized treatment for IBS in the clinical routine.  Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder (FGID), affecting between 10% and 20% of the population globally [1]. The condition is characterized by a combination of symptoms including abdominal pain, bloating, distention, flatulence, and disturbed bowel habits seen as constipation (IBS-C), diarrhea (IBS-D), or a combination of both (IBS-mixed) [2]. Currently, the Rome IV Diagnostic Criteria, providing symptom-based criteria for diagnosis, is applied for the diagnosis of IBS and its subtypes [2]. Besides the gastrointestinal (GI) symptoms, most patients suffering from IBS also experience a broad spectrum of extraintestinal symptoms, such as fatigue, fibromyalgia, poor social functioning, and reduced emotional well-being. Thus, although not being a fatal and organic disease, IBS is shown to have severe impact on quality of life . The etiology of IBS is not fully understood, but it is evident that the condition involves a dysfunction in the gut microbiota, seen as a dysbiosis leading to altered composition and diversity of the microbes in the gut [5]. The increased risk of developing IBS that has been observed after an infectious enteritis and/or excessive use of antibiotics supports the assumption of a dysbiotic gut as a contributor to the onset of IBS symptoms . In addition to altered gut microbiota, alterations in one or more of the control systems that contributes to the regulation of bowel function, including the central nervous system, the enteric nervous system, the enteroendocrine system, and the enteric immune system is presumed to be involved in the generation of IBS . Suggested mechanisms includes alterations in the gut-brain axis, low-grade inflammation, visceral hypersensitivity, abnormalities in the GI endocrine cells, changes in the GI motility, postinfectious changes, bacterial overgrowth, malabsorption of carbohydrates, abnormalities in serotonin metabolism, gene interactions, and alterations in the gut microbiota composition . An overview of suggested mechanisms and factors involved in the pathophysiology of IBS is summarized in Figure 1. As the gut microbiota composition and activity is decisive for several pathways suggested to be involved in IBS, different therapeutic approaches aim to target and manipulate a potentially dysbiotic gut. The current review is an attempt to summarize the most recent developments in treatment options targeting the gut microbiota and point out future perspectives for novel treatment approaches in IBS related to disturbed gut microbiota.Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteri [12]. While the gut microbiota is the present community of microorganisms, the gut microbiome refers to the entire set of genomic elements decisive for the microbiota in the gut. A healthy composition of the gut microbiome is essential for a broad range of physiological functions, as the gut microbial genes are decisive for the bacterial richness in the gut and hence the function of the gut microbiota [13]. In healthy subjects, the composition of the gut microbiota is determined by both genetic and environmental factors, of which the genetic factors are suggested to explain only 5–10% of the bacterial variability among individuals [14]. The environmental factors decisive for the gut microbiota composition include diet, geographical location, surgery, smoking, depression, frequency of treatment with antibiotics and use of certain nonantibiotic drugs, among others [15]. Microbial richness, seen as bacterial diversity, is usually considered an indicator of good health. In contrast, reduced bacterial diversity and imbalance of the gut microbiota, referred to as dysbiosis, has been associated with impaired metabolism and a broad range of different diseases [16].The human gut is inhabited by huge amounts of microorganisms important for maintaining GI homeostasis, including bacteria, virus, fungi, archae, and eukaryotes. More than 2000 different bacterial species have been identified in the human gut, of whom the majority belongs to the four main phylaMethanobacterialles, have been reported to be more abundant in IBS-C and less abundant in IBS-D compared to healthy subjects, whereas butyrate-producing bacteria, such asErysipelotrichaceaeandRuminococcaceae, are found in lower concentration in IBS patients than in healthy subjects [22]. Although the findings are overall inconsistent regarding gut microbiota composition and diversity in IBS, data in general suggest that there is a relative abundance of proinflammatory bacterial species such asEnterobacteriaceae, and decreased presence ofLactobacilliandBifidobacteri [20]. In addition, an increase inFirmicutes toBacteroidetesratio, and increasedStreptococciandRuminococcusspecies are observed when IBS patients are compared to healthy subjects [23–26]. It is suggested in the literature that the gut microbiota profile of IBS patients differs from the gut microbiota profile of healthy controls [17–20]. In addition, several differences in gut microbiota composition among IBS subtypes have been detected [21]. Methane-producing bacteria, such asIBS patients are most often characterized by a reduction in bacterial diversity, referred to as dysbiosis, when compared to healthy subjects [19]. Of note, it is suggested that IBS patients who do not respond to dietary interventions suffer from a severe dysbiosis [15]. However, although it is well documented that IBS patients have alterations in the gut microbiota composition and can be characterized by a dysbiotic gut, the specific microbial signature characterizing these patients is still unknown [5].A multidisciplinary approach is the recommended treatment option in IBS [2]. A better understanding of the disease, combined with life-style interventions including diet, exercise, and breathing techniques, is proven to be effective for reducing symptom severity in IBS patients [3]. In addition, psychological and behavioral treatment and hypnotherapy has also been shown to reduce IBS symptoms and improve quality of life [27]. Nonpharmacological treatment options are preferably the first-choice in IBS, as available drugs are most likely to target only the predominant symptom. Due to the broad diversity of symptoms experienced by most IBS patients, drug treatment is often perceived as inadequate and do not lead to total control of symptoms [2]. Hence, the need of novel and more personalized treatment approaches in IBS is of great need. Diet composition and food intake have been shown to play an important role in the symptom generation in IBS patients, and most of them claim that their symptoms can be related to the consumption of different foods [28]. In recent years, the interaction between diet, gut microbiota composition, and severity of IBS symptoms has gained a lot of attention and emerged as an important pathophysiological basis for the treatment [29]. Lately, the “brain-gut” axis is referred to as the “brain-gut-microbiota” axis [30]. Diet contributes in the pathogenesis of IBS by modulating the gut microbiome and the normal gut microenvironment [31]. The food we eat and its breakdown products are capable of altering the gut microbiota composition and the colonic fermentation, in addition to interacting with the gut immune system and affecting the gut permeability, motility, and visceral sensation [32]. Several modifications in the diet are shown to enhance symptom severity in IBS. The first line dietary treatment for IBS patients is to follow the dietary guidelines from the modified National Institute for Health and Care Excellence (NICE) diet, as recommended by the British Dietetic Association [33]. The NICE-diet highlights the importance of regular meals and recommends reducing the intake of carbonated drinks, chewing gum, fatty and spicy foods, coffee, alcohol, onion, cabbage, and beans as well as supplementing the diet with fibers such as psyllium husk. Bifidobacterium, an oligosaccharide fermenting bacteria suggested to be beneficial for human health [40]. Dietary fibers can be divided into soluble and insoluble fibers, according to their chemical and physiological properties. Soluble fibers can further be divided into viscous (gel-forming) and nonviscous fibers [34]. The dietary fibers include nondigestible short-chain and long-chain carbohydrates and lignin, a complex polymer percent in plants [35]. Overall, including fibers in the diet has several beneficial health effects, such as improving stool consistency, lowering blood cholesterol levels, and improving glycemic control [36]. For a long time, it was believed that IBS was caused by a deficient intake of fibers; hence, it was widely recommended that IBS patients increased their intake [37]. However, the effects of different fibers have proven to be of importance, as soluble fibers are shown to improve IBS symptoms while insoluble fibers, on the other hand, are shown to exacerbate symptoms . Supplementation with soluble and moderately fermentable fibers such as psyllium and oats is a recommended treatment in IBS, shown to improve symptom severity and beneficially modulate stool consistency in all IBS-subtypes [36]. Several randomized controlled trials (RCTs) investigating the effect of fiber supplementation on microbiota composition in healthy subjects have suggested that supplementation with fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS), known for their prebiotic activity, increases the abundance ofA broad range of studies have investigated the effect of supplementation with different types of fibers in patients with IBS, and increasing amount of evidence indicates that the dietary fibers acting as prebiotics, such as inulin and oligosaccharides, are able to influence the composition of the gut microbiota [41–43]. Fermentation of dietary fibers by colonic bacteria leads to the production of short-chain fatty acids (SCFAs), and concentrations of fecal SCFAs have been investigated as a marker of fecal fermentation reflecting the gut microbiota activity in IBS patients [44]. Distinct alterations in fecal SCFA concentrations have been reported between IBS patients and healthy controls, and propionate and butyrate in particular have been suggested as possible biomarkers for the distinction between IBS and healthy individuals [45]. If basic dietary modifications do not improve symptoms, a diet with a restricted intake of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) is proven by several metaanalyses to significantly reduce symptom severity in IBS, especially abdominal pain and bloating . FODMAPs are short-chained carbohydrates that are poorly absorbed in the small intestines, and when fermented by colonic bacteria, production of gas and osmotic action causes symptoms such as bloating and diarrhea [47]. Notably, a diet with a restricted intake of FODMAPs is associated with distinct alterations in both function and composition of the gut microbiota, as well as modulations in fecal fermentation and production of SCFAs . Actinobacteria, Bifidobacterium, andFaecalibacterium prausnitzii), significantly decreased in response to the low-FODMAP diet. Ten days of supplementation with FOS increased the levels of these bacteria, whereas the levels of inflammatory markers and concentrations of SCFAs did not change [42]. Accordingly, the long-term health effect of adherence to the low-FODMAP diet as a dietary intervention is not established. The health effects of the changes observed in the gut microbiota composition and fecal fermentation in response to a diet with low content of fermentable carbohydrates is currently unknown [49]; thus, the long-term use of a strict low-FODMAP diet can be questioned. In a recent publication, we reported on the effect of a low-FODMAP diet combined with supplementation with FOS or placebo, in 20 patients with IBS-D or IBS-M [42]. We found the low-FODMAP diet to overall consistently improve symptom severity, and significantly more patients reported symptom relief when supplemented with placebo  for 10 days, compared to the FOS supplementation (80% vs. 30%). The serum levels of proinflammatory interleukins (IL-6 and IL-8), total concentrations of SCFAs and n-butyric acid, as well as levels of bacteria in feces  investigating the effects of probiotics compared to placebo in IBS patients have concluded that probiotic supplements significantly improve overall IBS symptoms and abdominal pain [52–55]. Of note, the use of monostrain or multistrain probiotics varies between studies, and it is suggested that supplements containing a multistrain probiotic  might be more effective than monostrain supplements in improving symptoms. In a recent review article summarizing the most up-to-date research on the effects of probiotic supplementation on symptom severity in IBS patients, we reported that the effects of probiotic supplementation were more distinct with the use of a multistrain supplement [51]. In addition, we found the effect to be more distinct in the studies with an intervention period of 8 weeks or more, suggesting that probiotics supplemented over a period of time have the potential to improve IBS symptoms [51].Overall, much remains to be answered as to which strains facilitate the most beneficial effects in IBS. Although probiotic supplements seem to have beneficial effects in improving IBS symptoms, their function and mechanistic effects are relatively unknown. Current literature has suggested that probiotics act through improvement of gut barrier function, inhibition of pathogenic bacteria overgrowth, and prevention of pathogenic invasion of the host, as well as production of signal substances such as SCFAs and neurotransmitters [56]. The fact that the degree of dysbiosis and gut microbiota composition most likely varies greatly both between subtypes of IBS patients and between each subject leads to the question of whether it is possible to target these individual differences in future therapy. According to current literature, individual microbiota profiling may in the future enable personalized or customized probiotic therapy.Nonabsorbable antibiotics have been investigated in the treatment of IBS in several large-scale double-blinded and placebo controlled RCTs, and have been found to serve as an effective treatment option [5]. In 2015, rifaximin, a rifamycine derivate with a broad range of antibacterial effects against aerobic and anaerobic organisms in the GI tract, was approved by the US Food and Drug Administration for the treatment of adults with IBS-D [57]. Less than 0.5% of the oral dose of rifaximin is absorbed; hence, this antibiotic has low toxicity and no significant adverse effects and drug interactions [58]. Although the route of action of nonabsorbable antibiotics in IBS is unclear, relief of IBS symptoms is suggested to occur due to reduction in the total gut bacterial load and changes in the composition of the gut microbiota, as well as modulation of the intestinal permeability and the gut microbiome [20]. Three multicenter RCTs, named TARGET 1-3, have demonstrated a beneficial effect of rifaximin in improving IBS symptoms . TARGET 1 and 2 included IBS patients without constipation. The primary outcome was proportion of patients with relief of global IBS symptoms, with adequate relief defined as self-reported symptom relief for at least 2 of the first 4 weeks after the intervention. Both interventions revealed that rifaximin given in doses of 550 mg 3 times a day for 2 weeks significantly reduced overall symptoms and bloating when compared to placebo (40.8% vs. 31.2% in TARGET 1 and 40.6% vs. 32.2% in TARGET 2) [59]. The TARGET 3 study included adult patients with only IBS-D, and those patients responding to a 2-week treatment with rifaximin as provided in TARGET 1 and 2 but relapsed during an observation phase, were randomly assigned to an additional treatment with either rifaximin or placebo for another 2 weeks. The percentage of responders were significantly greater with rifaximin group with placebo (38.1% vs. 31.5%), and the rates of adverse events were low [60]. This study demonstrates that repeated treatments with rifaximin are well tolerated and can be considered effective in reducing symptom in patients suffering from diarrhea predominant IBS.Although rifaximin is not indicated in IBS-C, a small double-blind trial has suggested that rifaximin may improve symptoms also in patients with constipation [61]. The study showed that rifaximin given in combination with neomycin significantly reduced the degree of constipation and bloating, compared to neomycin given with placebo. The constipation severity scores were significantly lower in the neomycin and rifaximin group (28.6 ± 30.8) compared to neomycin alone (61.2 ± 24.1) [61].Bifidobacterium might be a positive predictor for successful FMT [62]. A systematic review by Halkjær et al. reported in 2017 that 28 out of 48 patients (58%) experienced a beneficial outcome of FMT treatment, and importantly, no adverse events were reported [63]. The review included only a restricted number of FMT studies, as the data from RCTs then were limited. In the last two years, several RCTs evaluating the effect of FMT in IBS have been published, reporting somehow conflicting results regarding alleviation of IBS symptoms. Fecal microbiota transplantation (FMT) has recently gained a lot of attention due to the strong evidence for the role of dysbiosis in the IBS pathogenesis. FMT is an approach involving application of a solution of fecal material from a healthy donor into the gut of a receiver, aiming to restore a dysfunctional microbial composition into a healthy homeostasis and hence improve the function of the gut microbiota [5]. A Japanese open-label study in 10 IBS patients reported in 2017 that FMT treatment improved both the psychological status and stool form. In addition, the authors suggest that a donor holding a high fecal concentration ofThree recent Norwegian studies, all using FMT administered through gastroscopy or colonoscopy, have reported beneficial effects. Johnsen et al. assessed the effect of FMT in a parallel-group RCT, with 60 patients assigned to FMT treatment and 30 assigned to placebo [64]. The trial included IBS patients with moderate-to-severe IBS-D or IBS-M, and the primary outcome was reduction in total IBS severity scoring system (IBS-SSS) of more than 75 points. Fifty-five patients assigned to FMT treatment and 28 assigned to placebo completed the trial. They reported that the FMT treatment significantly reduced symptoms compared to placebo, with a 65% response in the FMT group and a 43% response in the placebo group [64]. Ruminococcus gnavus, Actinobacteria, andBifidobacteria, in addition to significantly differences between several gut microbiota taxa and concentrations of SCFAs at baseline, which of note became nonsignificant 3 weeks following FMT [66]. At 20/28 weeks after FMT, the changes in the gut microbiota were similar between IBS patients and donors; thus, the patient’s microbiota profile became more or less similar to those of the donors. Those IBS patients responding to FMT had different gut microbiota profile and SCFA profile than the nonresponders, and significant correlations were reported between the gut microenvironment and IBS symptoms [66].Results from our study, including 13 IBS patients and 13 healthy donors receiving FMT, support the findings of Johnsen et al. . According to symptom scores, the symptoms were significantly improved following FMT [65]. When analyzing fecal samples, we found that the donors and the IBS patients had significantly different bacterial strains signals forEl-Salhy et al. recently performed a placebo-controlled RCT, evaluating the effect of two different doses of FMT [67]. This study, including 165 IBS patients allocated to either placebo 30 g or 60 g FMT, demonstrated that FMT was effective in reducing IBS symptoms and fatigue, as well as increasing the quality of life. The primary outcome was a positive response defined as a reduction of 50 points or more in the total IBS-SSS score. Response occurred in 23.6% of the patients in the placebo group, 76.9% in the 30 g FMT group, and 89.1% in the 60 g FMT group, suggesting that the response to FMT increases according to dose. In addition, a significant change in gut bacterial profile was observed in the groups receiving FMT [67]. Following, an open-label study including the 10 patients not responding to the 30 g FMT treatment was conducted [68]. The ten patients received a 60 g FMT transplant, of which 7 (70%) responded to treatment with significant improvements in IBS-symptoms, degree of fatigue, and quality of life. These results further highlight that increasing doses and/or repeated FMT increases the response [68]. Results conflicting with those of the Norwegian studies were presented by a Danish trial by Halkjær et al. [69]. This randomized, double-blind, and placebo-controlled trial included 52 patients with moderate-to-severe IBS, randomized to capsules with FMT or placebo for 12 days, with 6 months of follow up. Here, a significant reduction of symptoms according to the IBS-SSS core was observed for placebo, but not for the FMT treatment. The patients receiving the FMT capsules had an increase in the fecal microbiota diversity that was not observed in the placebo group; however, the authors state that altered gut microbiota is not enough to obtain clinical improvement in IBS [69]. Accordingly, although several studies investigating FMT as a treatment option in IBS obtain promising results regarding symptom severity and gut microbiota manipulation, the results are still conflicting. The outcome of FMT seems to be dependent on the donor; hence, a so-called “superdonor” seems to be essential for FMT to be an effective treatment [14]. Currently, FMT is only investigated in a research setting, and the available data on potential effects are too limited to draw sufficient conclusions. More RCTs investigating the long-term effects of FMT and its route of administration need to be carried out in order to potentially be able to offer FMT as a standardized treatment approach for IBS in a clinical setting. As our understanding of the implications of the gut microbiota in IBS is constantly evolving, the potential of treatments manipulating both the gut microbiome and the gut microbiota aiming to improve GI symptoms and severity of IBS is widening. Hopefully, determining the exact microbiota profile and degree of dysbiosis in each IBS patient will soon be an available tool for personalized treatment options, such as customized probiotic or FMT treatments and/or targeted use of antibiotics. An overview of some suggested future therapeutic strategies in IBS is shown in Figure 2. There is strong growing evidence suggesting that dysbiosis is central in the pathophysiology of IBS. Manipulation of the gut microbiota in order to improve IBS symptoms has evolved in the last decade as a novel treatment strategy and several different approaches have been investigated in order to improve the gut microbiota composition and hence improve symptoms. Dietary modifications including supplementation with fibers and probiotics are shown to improve symptoms and gut microbiota in IBS, although the exact probiotic mixture beneficial for each individual remains to be identified. The use of antibiotics still needs confirmation, although promising results have been reported with the use of rifaximin. Several studies investigating FMT obtain promising results regarding symptoms and gut microbiota manipulation in IBS; however, more data including long-term effects of FMT are required before it can be used as a customized treatment in the clinic.Both authors have contributed to the paper. HFD wrote the paper, and GAL provided writing assistance and corrected the manuscript.


Eisenia fetida, which inserts large conductance pores in artificial and natural lipid membranes containing sphingomyelin. Its cytolytic and hemolytic activity is rather indicative of a pore-forming toxin; however, lysenin channels present intricate regulatory features manifested as a reduction in conductance upon exposure to multivalent ions. Lysenin pores also present a large unobstructed channel, which enables the translocation of analytes, such as short DNA and peptide molecules, driven by electrochemical gradients. These important features of lysenin channels provide opportunities for using them as sensors for a large variety of applications. In this respect, this literature review is focused on investigations aimed at the potential use of lysenin channels as analytical tools. The described explorations include interactions with multivalent inorganic and organic cations, analyses on the reversibility of such interactions, insights into the regulation mechanisms of lysenin channels, interactions with purines, stochastic sensing of peptides and DNA molecules, and evidence of molecular translocation. Lysenin channels present themselves as versatile sensing platforms that exploit either intrinsic regulatory features or the changes in ionic currents elicited when molecules thread the conducting pathway, which may be further developed into analytical tools of high specificity and sensitivity or exploited for other scientific biotechnological applications.Lysenin is a pore-forming protein extracted from the earthworm  The ability of pore-forming proteins and peptides to establish conducting pathways between two sides of a lipid membrane was exploited for decades for numerous analytical applications ,6,7,8,9.Adjustments of conductance in response to chemical stimuli are an essential biological function of canonical ion channels in living cells ,21,22, aE. coli ClyA toxin, lysenin, and motor proteins .,62.53,622 to the support electrolyte solutions strongly diminishes the ionic currents through lysenin channels . T. T2+ ion2+ ions show a different behavior that does not match the typical description of divalent ion effects in terms of inhibition efficiency and the shape of the inhibition curve (does not present an inflection point), hence resembling trivalent-like effects. To identify the origin of this behavior, single-channel experiments that employed Cu2+ ions as inhibitors were conducted similarly to the other divalent and trivalent metal ions. After insertion of only two lysenin channels in the bilayer membrane  completely cancels the individual conductance and reduces the ionic currents to zero in a stepwise manner [2+ induces transitions to sub-conducting states, as observed for other divalent metals, but the sub-conducting states are not stable and the channels may fully close by employing a second transition from the sub-conducting to fully closed states [Cumembrane a, Cu2+ ae manner . Howevere manner   is much smaller for trivalent metals (in the µM range) than for divalent metals (mM range). This naturally leads to the hypothesis that the charge of the cations is central for the ligand-gating mechanism manifested in the presence of multivalent metal ions. This may be easily seen in han Fe2+ . To bettermine4+ ,62. The Experimentations with multivalent inorganic and organic cations revealed conductance inhibitory effects dependent on both the charge and size of used ions. Conductance modulation is in most cases reversible, and inhibitor removal by chelation, precipitation, or buffer exchange restores the lysenin channel’s conducting properties. However, the inorganic and organic ions used for these investigations were still small compared to the channel’s opening and carried a relatively small charge. This led to questioning of the potential effects on the macroscopic conductance of lysenin channels presented by large and highly charged molecules, such as cationic polymers . In thisThis lack of reversibility is explained by considering a channel occlusion mechanism based on gating and trapping . Once th2+  are added to the support electrolyte . AdditiorG is the relative macroscopic conductance, Gmin is the minimum relative conductance measured at saturation , IC50 is the half-way inhibitory concentration, x is the purine concentration, and n is the Hill coefficient.IC50 and n vary with the chemical identity of the inhibitor; IC50 increases (lower binding affinity) and n decreases (less cooperativity) as the net charge of the anion increases. This suggests that the inhibitory mechanism relies on electrostatic binding of purines to specific sites present in the channel’s lumen [The results shown in ’s lumen , which aIC50 decreases as the ionic strength increases  and dwell time (Td) (D histogram (From ime (Td) , one mayime (Td) b and ID istogram a. This tistogram b. This mProtein channel gating in the presence of DNA may lead to “events” that resemble translocation and additional evidence is needed to demonstrate DNA passage. Irrespective of the origin of the differences between events, PCR provides irrefutable evidence of DNA translocation . In thisThe large interest in DNA translocation was fueled by the promise of fast and reliable sequencing ,66,87,88B> and dwell time tD performed with the Transalyzer software package [B> (Signal analysis in terms of the average current blockage <I package  provideskage [B> b and ovekage [B> c. The exAn important exploration of the same work provides evidence of translocation . Such st3+ ions, and EDTA [Lysenin channels are molecular tools anticipated to significantly contribute to the development of high-performance sensing devices. Such devices may be realized based on the intrinsic properties of lysenin channels to adjust their conductance in response to interactions with multivalent ions. While the response is non-specific, such a simple device may find applicability for fast screening purposes. Irreversible channel blockage by cationic polymers can be realized at concentrations in the nM range . Given tand EDTA , which mLysenin is a protein amenable to chemical and genetic modifications intended to adjust its sensitivity and specificity for analytes. A lysenin channel inserted into an artificial lipid membrane and endowed with a biorecognition element may lead to the development of single-molecule sensors for molecules and complexes too large to thread the pore; in this case, the binding event near the pore entrance may reduce the ionic flow and facilitate electrical detection from changes in ionic currents.


This series of nine articles  is presented by international leaders in biliary tract cancers (BTC). BTCs are a rare but heterogeneous group of malignancies with dismal overall prognosis and comprised of intrahepatic cholangiocarcinoma (CCA), extrahepatic CCA, and gallbladder cancer (GBC). Surgery is the only potentially curative treatment, but the majority (~65%) of patients present at unresectable stage at the time of diagnosis, with a 5-year overall survival (OS) rate of less than 5% .The high rate of unresectability has generated considerable interest in orthotopic liver transplantation (OLT). While OLT is currently not recommended outside of clinical trial for intrahepatic cholangiocarcinoma (iCCA) due to high tumor recurrence rates ,3,4, it While the surgical management of T3/T4 GBC has been controversial, primarily due to a high rate of lymph node positivity and occult metastatic disease, successful extensive radical resections can be achieved in selected patients . In thisFor most of the patients with BTC that are ineligible for potentially curative therapies, cytotoxic chemotherapy remains the cornerstone of management, despite dismal survival outcomes. To date, the combination of gemcitabine and cisplatin has been established as the preferred first-line regimen . The resWhile an anatomically diverse group, recent molecular profiling efforts of BTCs have revealed a wealth of genomic alterations with prognostic and therapeutic implications. These actionable alterations have been the subject of ongoing clinical investigations, with the most promising targets to date being fibroblast growth factor receptor (FGFR), isocitrate dehydrogenase (IDH) 1 and 2, BRAF, mismatch repair proteins and HER2-neu . Most reCCA exhibits a highly desmoplastic stroma that plays a key role in CCA tumorigenesis, invasiveness and therapeutic resistance through remodeling of the tumor extracellular matrix and cross-talk with proinflammatory immune subsets ,20. MeanIt is our hope that this thematic issue provides updated information on the evolving treatment paradigms and research progress in the field of BTCs. This series of unique articles highlights the rapidly growing precision medicine efforts and reflects on future directions which may lead to improved outcomes for patients with this lethal disease.


While the role of pHi on proliferation, migration, and tumorigenesis has been explored in cancer cells for some time, emerging evidence suggests an important role of the pHi in the intestinal stem cells (ISCs) proliferative rate under physiological conditions. The present review highlights the current state of knowledge about the potential regulatory role of pHi on intestinal proliferation and differentiation.During the maturation of intestinal epithelial cells along the crypt/surface axis, a multitude of acid/base transporters are differentially expressed in their apical and basolateral membranes, enabling processes of electrolyte, macromolecule, nutrient, acid/base and fluid secretion, and absorption. An intracellular pH (pH The activity of acid loaders is counteracted by the apical acid extruders, the Na+/H+ exchangers NHE2, NHE3, and NHE8 (SLC9A2/3/8); a process that results in salt and water absorption. Apical proton ATPases are also expressed in various cell types along the GI tract. The basolateral membranes also express both acid loaders, such as the Cl–/HCO3– exchanger AE2 (SLC4A2), and acid extruders, such as the NHE1 and the Na+/HCO3– cotransporters NBCn1 (SLC4A7) and NBCe1 (SLC4A4). Immunostaining and in situ hybridization techniques localized these transporters in the respective membranes, often with an expression gradient along cryptal or crypt/villus axis, and along the proximal to distal gut axis  and the Clgut axis . Figure i in the cellular physiology of the intestinal epithelium has not been addressed experimentally, partially because of experimental uncertainties in the calibration process that allows conversion of the fluorometric intensity into an actual pHi value , or of the colonic epithelium by short chain fatty acids , have been studied in detail. The epithelium counteracts this intracellular acidification by activation and/or rapid trafficking of a variety of ion transporters to the brush border and basolateral membrane, facilitating proton extrusion, and HCO3– import to re-establish the resting pHi  mouse duodenal mucosa. When techniques became available for intestinal stem cell maintenance and thus long term culture of native intestinal organoids, the same group used the technique to demonstrate a sustained alkalized resting pHi in the epithelial cells of small intestinal organoids grown from –/–Cftr crypts -labeled WNT transducer Disheveled (Dsv) and the cell membrane-targeted, two-color fluorescent Cre-reporter RosaTmT/mG enabled the group to study the proximity of Dsv to the membrane receptor Frizzled 7, which has been recognized as a key event in WNT signaling  is important for initiating DNA synthesis and proliferation [reviewed in i and/or proliferation was measured, but the molecular mechanism linking the two has yet to be explored.From the existing literature pool, only the study done by i compared to WT  is expressed predominantly in small intestinal villous  and coloed to WT . Due to ed to WT . The Cl–the ISCs . Loss ofithelium . Colonic/– mouse . Possibl/– mouse . Increasithelium .i on proliferation and differentiation? While many cell lines are viable in acidic medium, the pharmacological or genetic inhibition of acid extruders or base loaders generally curbs proliferation and has been repeatedly suggested as an antiproliferative treatment in tumors , BMP (bone morphogenetic protein), Notch, Hedgehog, and Eph–ephrin which mainly occur in gradients along the crypt/villus axis as depicted in Cftr–/– ISCs as described above. The molecular mechanism behind the increased Wnt activity involves the alkaline pHi-facilitated association of the Wnt transducer Disheveled (Dvl) to the plasma membrane and binding to the Frizzled-7 receptor (Fz) , which allows maintenance of alkaline pHi, is necessary for the binding and surface recruitment of Dvl by Fz  . Similarvl by Fz . AnotherPS cells . Intraceor model . However nucleus , while ltrusions . Recentltrusions  and KCNQtrusions , 2020. Agulation .i in A431 human epidermoid carcinoma cell line -ATPase (V-ATPase), a proton transporter involved in the acidification of endosomal compartments  has been observed in mouse embryonic stem cells during their differentiation in vitro (Drosophila, is also strongly influenced by pHi alterations.BMP, Notch, Hh, and Eph are more dominant in the upper sections of the crypt and involved in cell fate decision and terminal differentiation. Knowledge about the impact of the pHartments . Notch sartments , thus imartments  via finein vitro . Hh signi may trigger stimulation or inhibition of different signaling pathways active in proliferation and differentiation of the intestinal epithelium. However, the exact molecular mechanism correlating the pHi and signaling pathways gradient in the intestinal epithelium is yet to be determined.The reported studies are only examples, which, taken together, suggest that the pHi in intestinal epithelial homeostasis has been hampered by the absence of models that accurately assess the pHi in different epithelial compartments along the crypt-villus axis, and to induce long-term and selective pHi-alterations. The ability to generate intestinal organoids and monitor their growth over days in culture has enabled scientists to intensely study the process of intestinal cell renewal and differentiation. Some of the molecular events that link the elevated pHi secondary to loss of functional CFTR to intestinal epithelial hyperproliferation were elegantly explored by generating intestinal organoids from –/–Cftr and wt mice crossed onto a variety of transgenic reporter mouse lines (i-regulatory dysfunction and reduce cancer risk (i assessment with genetic, molecular biological and pharmacological tools, as already established for tumor cells (Addressing the role of the steady-state pHse lines . These scer risk . The potor cells , in inteMA, US, and KN designed and wrote the review. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cannabis sativa L.).Silicon is a non-essential element for plants and is available in biota as silicic acid. Its presence has been associated with a general improvement of plant vigour and response to exogenous stresses. Plants accumulate silicon in their tissues as amorphous silica and cell walls are preferential sites. While several papers have been published on the mitigatory effects that silicon has on plants under stress, there has been less research on imaging silicon in plant tissues. Imaging offers important complementary results to molecular data, since it provides spatial information. Herein, the focus is on histochemistry coupled to optical microscopy, fluorescence and scanning electron microscopy of microwave acid extracted plant silica, techniques based on particle-induced X-ray emission, X-ray fluorescence spectrometry and mass spectrometry imaging (NanoSIMS). Sample preparation procedures will not be discussed in detail, as several reviews have already treated this subject extensively. We focus instead on the information that each technique provides by offering, for each imaging approach, examples from both silicifiers (giant horsetail and rice) and non-accumulators ( The entry route from the soil follows water [Silicon (Si) is the second most abundant element on Earth after oxygen and it is considered non-essential for plant growth and development . Si is bws water ,7, amongws water ,9.2 × nH2O) and the process of biogenic silica precipitation is referred to as silicification. With respect to Si accumulation, plants are distinguished into excluders (Si <0.5% in dry weight), accumulators (Si > 1% in dry weight) and intermediate types (Si between 0.5 and 1% in dry weight) ,11,12: ain vitro (using an undersaturated solution of silicic acid) and in vivo, that silicification in Equisetum arvense follows callose deposition. Indeed, horsetail silica is found intimately associated with callose in different cell types [Arabidopsis thaliana [POWDERY MILDEW RESISTANT 4-PMR4 accumulated significantly less silica, as compared to wild-type and overexpressing plants [It is known that silica associates with the cell wall and that specific macromolecules can template its deposition . Celluloll types . The rolll types  and Arabthaliana , where pg plants . The celg plants . Yet, itin vivo detection in plants add information in real-time, which eliminate the bias caused by sample preparation.The study of metal(loid) uptake and accumulation in plants has received much interest in the last years ,24,25,26In this review, imaging techniques to study plant silicification are discussed with an emphasis on their advantages and drawbacks. Classical histochemistry with known stains/fluorophores is discussed together with techniques implying the extraction of plant silica and subsequent scanning electron microscopy (SEM), X-ray emission spectrometry  and mass spectrometry . Although other analytical techniques like Raman microscopy or micro computed tomography (MicroCT) have already shown their suitability to study the distribution of Si in plants, we have confined our investigations to histochemistry, SEM, X-ray emission spectroscopy and mass spectrometry imaging. More details about the use of Raman imaging applied to plant research can be found in . MicroCTA summary of the techniques surveyed is provided in Plant silica can be observed with light microscopy after staining with methyl red (MR), crystal violet lactone (CVL) and silver-amine chromate (SAC) . AmorphoCVL  staiMR and CVL give variability in staining; indeed, for specific cell types, like bulliform and silica cells, staining will not be visible, unless etching is done . It was suggested that the outer layer of these cells may be packed with silica and have tight surface pore sizes hindering the penetration of the dye . The rea3 with K2CrO4, then washing the precipitate in hot water at 50–60 °C and dissolving it in 3% v/v NH4OH, followed by filtering) gives a brown colour upon adsorption to amorphous silica due to the removal of ammonia and the unmasking of silver chromate [Pteris [2SO4 prior to SAC incubation enhances the intensity of the staining because of the partial digestion of neighbouring cells which facilitates penetration of the solution; however, excess acids may cause unspecific precipitation of SAC [SAC  of lignin and silica in different plant species and/or under different growth conditions. For example, silica and lignin are two compounds expected to play a mechanical role of support in plant tissues. Silica is however a cheaper alternative to lignin ,43, requin planta [Biosilicification can be visualised using a fluorescence microscope and the fluorophore 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole (PDMPO). PDMPO is a silica tracer used to monitor pH changes within acidic organelles . The flun planta .For example, we have used PDMPO staining of silica extracted after removal of organic material to study the fine cellular details of the silicified cell replicas in both Si accumulators and non-accumulators .Microwave acid digestion proved to be a suitable method to free inorganic silica deposits from organic plant material ,19,20,223 and H2SO4) within sealed containers. These containers are then subjected to a microwave digestion program (180 °C for 15 minutes and maintained at 180 °C for 30 minutes) which breaks down the sample matrix leaving behind its component elements in solution. It was demonstrated that, although this procedure successfully digests all organic plant tissue, the inorganic silica structures are left intact [During acid digestion, the plant sample is exposed to a cocktail of highly corrosive acids (HNOt intact ) and sevThe silica extracted from giant horsetail A,B showsLikewise, in rice, details of the silicified warts A, whiskePDMPO staining of extracted silica was also recently used in non-accumulators. Textile hemp does not accumulate the same levels of silica as horsetail or rice; nevertheless, when grown in the presence of sodium metasilicate, silicified trichomes and leaf epidermal cells are visible  Figure E,F. The Arabidopsis was indeed shown to accumulate Si when grown in the presence of the metalloid [PDMPO staining was also recently applied to the observation of fresh okra leaves by confocal microscopy . The fluetalloid  and a coHigh-resolution microscopy, such as SEM, can be used to image plant silica and estimate the extent of silicification by observing tissue and cell types at the nanometer scale. From the observation of silicified cell types, it is possible to infer the functional role of silica. The high resolution of SEM allows analysis of micromorphology at the cell level and of the surface microstructure in silicified tissues. Silicified ‘microsculptures’ from horsetail were already imaged with SEM almost three decades ago : it was By using microwave acid digestion followed by SEM observations , a staggering heterogeneity of silicified cell types can be observed in giant horsetail .The interdigitated lobes of the epidermal cells of the stem are perfectly replicated, together with recesses running all along the undulating margins A; in somin vivo needs further proof.The extracted silica shows that epidermal cells appear as silicified scales with thick margins E, while Microwave acid digestion of rice leaves followed by SEM provided a richness of structural details . Besides known silicified cells, namely bulliform and silica cells A–D, multThe difference in image quality between a benchtop SEM  and a conventional instrument is noteworthy , but expThe two different approaches to SEM imaging can complement each other: samples which have been imaged using a benchtop SEM are compatible with the conventional electron microscope. The easy to handle benchtop instrument can be used to quickly scan over multiple samples, identifying structures of interest which can then later be gold-plated and analysed using the higher resolution SEM. By combining the two instruments, the ease and simplicity of the benchtop SEM can be used to analyse large quantities of digested silica, whilst the higher resolution and better image quality of the conventional SEM can be used to image specific structures.The imaging of elements using X-ray-based techniques improves lateral resolution (down to the sub-micron under vacuum) and sensitivity . Micro-PThlaspi praecox, a known Cd and Zn accumulator, a study analysed the distribution of Cd, Zn and Pb in leaves [Micro-PIXE has been used extensively to map element distribution in plant tissues, especially hyperaccumulators that can accumulate high concentrations of metal(loid)s . For exan leaves ,57 and sn leaves .Lagenaria sphaerica (a member of Cucurbitaceae), where it probably has a role in containing the hypersensitive lesions [PIXE was previously used to detect Si in plant tissues challenged by pathogens: the metalloid accumulated at sites of necrotic lesions in  lesions .Micro-PIXE can be successfully used to detect Si distribution in the leaves of accumulators such as rice, after cryofixation, cryosectioning and freeze-drying . A faintin vivo [XRF has a higher resolution and better sensitivity as compared to PIXE and also allows analysis in vivo , which iPhragmites australis, Phalaris arundinacea, Molinia caerulea, Deschampsia cespitosa and in the sedge Carex elata, in combination with the analysis of the leaf optical properties [Sorghum root tissues using LEXRF, thereby indicating a role for Si in Al detoxification [Si distribution was studied via XRF in the grasses operties . Grassesoperties . It shouoperties . The posoperties . Evidencfication .−1 was found above the vascular bundle. The location and dumbbell appearance suggest that the element observed is a silica cell . The secondary ions are analysed by a mass spectrometer. The measurement is carried out under ultra-high vacuum conditions. During sputtering, the acquisition of successive images also allows to obtain a subsequent reconstruction of the volume sputtered (3D imaging).SIMS nano-analysis is a chemical imaging technique characterised by a high spatial resolution  and a hSIMS already proved to be a good complementary imaging technique for determining the distribution of elements in plant tissue ,68. Samplsi2 mutants: the metalloid was found to accumulate at higher levels in the proximal side of the root endodermis, in a region of the cell wall coinciding with the presence of a dark layer as imaged by transmission electron microscope (TEM). SIMS was also used to map the distribution of Si and germanium (Ge) in the roots and leaves of the grasses Poa annua and Dactylis glomerata [SIMS was used on rice to map Si distribution  in wild-lomerata : the sigSIMS nano-analysis was recently used to map Si in the accumulator horsetail . The pre12C14N signal indicates the gelatinous organic layer which overlaps with the 28Si linescan (High-resolution SIMS imaging was also used recently to map Si distribution in the stem tissues of textile hemp . A signalinescan F. This sSorghum [Our understanding of plant silicification is still partial and many open questions remain, especially in relation to which macromolecules can template silica deposition. The majority of the studies available in the recent literature have focused on the identification and functional characterisation of the membrane channels mediating the entry of silicic acid. Only a handful of studies, some of which date back to the early 80s, have analysed the spectacular silicified structures of plants. Molecular approaches can be used together with fluorescence microscopy after PDMPO staining, elemental imaging or electron microscopy following microwave acid digestion to infer the role of candidate genes in plant silicification: databases providing a collection of mutants of known silicifiers, such as rice  or SorghSorghum , will be


The focus of this review is the emerging role of PET and potential PET radiotracers for studying tDCS-induced functional changes in the human brain.Transcranial direct current stimulation (tDCS) is a form of non-invasive neuromodulation that is increasingly being utilized to examine and modify several cognitive and motor functions. Although tDCS holds great potential, it is difficult to determine optimal treatment procedures to accommodate configurations, the complex shapes, and dramatic conductivity differences among various tissues. Furthermore, recent demonstrations showed that up to 75% of the tDCS current applied to rodents and human cadavers was shunted by the scalp, subcutaneous tissue, and muscle, bringing the effects of tDCS on the cortex into question. Consequently, it is essential to combine tDCS with human neuroimaging to complement animal and cadaver studies and clarify if and how tDCS can affect neural function. One viable approach is positron emission tomography (PET) imaging. PET has unique potential for examining the effects of tDCS within the central nervous system  Transcranial direct current stimulation (tDCS) is a form of non-invasive neuromodulation that is increasingly being applied to examine and modify several cognitive and motor functions, ranging from motor learning and working memory to performance fatigability and muscle strength ,2,3,4. tAlthough tDCS has great potential, it is difficult to determine optimal treatment procedures to accommodate configurations, the complex shapes, and dramatic conductivity differences among various tissues , gray matter, etc.). Usually, placing the stimulating electrodes directly over the targeted brain areas cannot guarantee the proposed modulation (excitation or inhibition) of those areas; although this is typically assumed when designing stimulation protocols. Computer simulations and tDCS modelling studies have helped identify the behavior of induced electrical currents over recent years . While tin vivo, including imaging cerebral metabolism , neuroreceptor occupancy, and neurotransmitter activity/binding. Additionally, because PET imaging can provide a comprehensive  image, it is ideally suited to not only investigate the effects of tDCS in areas directly under the stimulation electrodes but also in remote or functionally connected brain areas. Consequently, it is essential to combine tDCS with human neuroimaging to complement animal and cadaver studies, and further clarify if and how tDCS can affect neural function. The physiological changes associated with tDCS in humans can be assessed with a variety of neuroimaging techniques. However, to-date the field has been led by non-invasive neuroelectrical techniques  and structural/functional magnetic resonance imaging (sMRI/fMRI). One viable alternative approach is positron emission tomography (PET) imaging. PET has unique potential for examining the effects of tDCS within the central nervous system 18F]fluoro-deoxyglucose (FDG), a glucose analogue. Since the brain almost exclusively uses glucose as a fuel source, mapping FDG uptake provides insight into the distribution of brain activity. In addition, since the brain does not have nutrient stores, the delivery of glucose and oxygen, and the removal of waste products via cerebral blood flow (CBF), is tightly coupled to cerebral metabolism. The brain is extremely metabolically active and “activation” or “deactivation” of a particular brain region necessitates respective increases or decreases in metabolism of that area, accompanied by corresponding changes in CBF. FDG provides a means of mapping glucose metabolism, whereas, [15O]water, a freely-diffusible PET radiotracer, provides a means of assessing changes in CBF. The “metabolic trapping” properties of FDG and diffusivity of [15O]water PET tracers allows for the examination of the neuronal mechanisms underlying various tDCS applications in unconstrained conditions . Furthermore, the interaction of neurotransmitters and receptors can also be measured using PET with various [11C]-labeled  or [18F]-labeled ligands. However, as revealed in this review, PET-tDCS studies in humans are surprisingly sparse. Despite some inherent disadvantages of PET, in particular radiation exposure and high costs that limit large-scale application, many human tDCS research questions can be addressed exclusively with PET. PET is a functional imaging modality that characterizes a given functional process ) contingent on the radiopharmaceutical employed. The most commonly used PET radiotracer is water concentration is linearly proportional to BF to the tissue at lower flows and non-linear at higher flows (>65 mL/min/100mL); thus, [15O]water concentration at higher flows will underestimate actual BF. Nevertheless, higher flow is still associated with higher concentrations of [15O]water in the tissues. However, when an arterial line is not available, the regional CBF (rCBF) can be reported relative to a normalization value, like the BF of another brain structure  or the mean global CBF (gCBF). With the latter, the rate of BF is normalized relative to the average CBF of the whole brain and assumes that gCBF remains consistent even when rCBF might be altered. Because [15O]water PET is based on the use of the short-lived positron-emitting radionuclide oxygen-15 , this radionuclide is ideal for measuring rCBF during cognitive or motor tasks that might be modulated by tDCS but is limited in its availability due to the need for a cyclotron to be in the immediate proximity of the PET scanner.The definitive gold standard for cerebral blood flow (CBF) determination is the injection of microscopic spheres, called microspheres, that travel in the blood and deposit into the capillaries. The deposition of the microspheres in a tissue is proportional to the blood flow (BF) that serves that tissue, and an understanding of the arterial input function and the concentration of deposited microspheres provides an accurate measure of tissue BF. However, microspheres cannot be used in humans because they permanently block the capillaries and determining their concentration is best done by “slice and dice” . The next best option for measuring CBF 15O]water PET imaging was validated as a measure of CBF in the early 1980’s by Raichle et al. [15O]water PET has been considered the “gold standard” neuroimaging technique for measuring CBF in humans and was the imaging method of choice for the major, multi-center carotid artery occlusion study  and tDCS polarity . Both anodal and cathodal tDCS resulted in diffuse increases and decreases in rCBF in cortical and subcortical areas compared to sham. The magnitude of rCBF changes from tDCS were similar to the task-related changes during finger movements and were unchanged during the 50-min PET scanning time. Paquette et al. [[e et al.  and Herse et al. . Since ty . [15O]wng , has a short half-life, and can be combined with other tracers. In addition, metabolism and blood perfusion are closely associated, with the oxidative phosphorylation of glucose as a leading energy source. Thus, CBF is considered an indirect measure of neuronal function and integrity [15O]water PET because this technique is considered the most “direct” measurement of CBF. The fMRI signal is often assumed to approximate the degree of local neural activation integrated over a spatial extent of several millimeters; however, this concept can be an oversimplification of the neurovascular coupling underlying many imaging sequences O2 (oxygen) gas with tDCS. [15O]O2 gas can be safely administered via inhalation and provides detailed information about tissue oxygen consumption and extraction from the blood. This radionuclide could provide valuable tDCS mechanistic information and future studies could use [15O]O2 gas, either alone or in combination with [15O]water, to investigate the neuronal effects of tDCS. Finally, it is noteworthy that, to-date, no studies have combined [11C]carfentanil and [11C]raclopride) have extended our knowledge of molecular mechanisms of pain management and cognitive function in the brain (11C]carfentanil is a strong and selective µ-opioid receptor (MOR) agonist. The µ-opioid system is the most important mechanism involved in the regulation of nociceptive signals and is a specific target of several opioid analgesics currently available for clinical use. Dos Santos et al. [11C]carfentanil to study the immediate effects of tDCS on MOR-mediated neurotransmission and heat-related pain thresholds in healthy subjects. Interestingly, they found that sham tDCS resulted in a decrease in MOR non-displaceable binding potential (BPND) in the periaqueductal gray matter (PAG), precuneus, and thalamus, which indicates activation of the endogenous µ-opioid system. Active tDCS (2 mA for 20 min) also prompted MOR activation in the PAG and precuneus, but additionally increased MOR activation in the left prefrontal cortex. In another study, Dos Santos et al. raclopride PET binding. These studies offer new insights for innovative uses of tDCS as a therapeutic aid for neuropsychiatric conditions involving dopamine transmission impairments, like the reward–motivation network, and suggest radiotracers for future tDCS work, particularly studies that target the DLPFC.Dopamine is the principle neurotransmitter for central motor control and is involved in cognitive processing  and moti18F]fallypride [18F]fluorodesmethoxy-fallypride [11C]FLB457 flllypride , or [11CC]FLB457 , all of Furthermore, since PET provides insight into the dopaminergic system the complimentary use of magnetic resonance spectroscopy (MRS) allows for the quantification of neurochemicals, such as glutamate and γ-aminobutyric acid (GABA). MRS is becoming an increasingly useful tool in studying physiological changes induced to tDCS .15O]water PET will serve as highly suitable biomarkers for testing whether tDCS counteracts cognitive impairments and motor dysfunction in the human brain. Additionally, receptor agents such as [11C]carfentanil and [11C]raclopride PET can be expected to play an important role in unraveling the neuroprotective and restorative effects of tDCS on the CNS. These latter radiotracers might have particularly interesting applications for refractory neurological symptoms  and dopaminergic disorders. It should be noted, that PET studies are expensive and PET scanners are often less available to researchers. However, PET is a standard for cancer diagnosis and monitoring in most hospitals and clinics throughout the nation and additional usage for research is potentially viable worthwhile.tDCS is currently being promoted as an inexpensive and effective tool to enhance cognitive and behavioral function. However, the research exploring the efficacy of tDCS is far from conclusive. This review highlighted the potential role of PET as a molecular imaging technique for tDCS research. PET imaging provides distinct benefits and will be an important tool in the arsenal of different imaging techniques that seek to determine the mechanistic effects of tDCS. FDG-PET and water is equivalent to approximately 25% of the annual natural background  radiation exposure and a 5 mCi/dose of [18F]-FDG is equivalent to approximately 11% of the annual radiation limit for a medical worker. These levels of radiation exposure are considered to be ethically appropriate for healthy subjects and routinely employed in research uses.Lastly, some critical points should not be left out. The risk/benefit ratio is different for treating and investigating symptoms with PET tDCS in diseased patients versus increasing motor and cognitive function in healthy subjects. While tDCS studies in clinical populations often require detailed risk assessments, the level of acceptable risk is different for healthy subjects and they might have a different risk/benefit ratio . PET uses radiotracers and subjects receive a dose of radiation during participation. For example, the amount of radiation from 15 mCi/dose of [tDCS effects are highly variable among human subjects. Age ,66, gend


The hypothesis that host adaptation in virulent Newcastle disease viruses (NDV) has been accompanied by virulence modulation is reviewed here. Historical records, experimental data, and phylogenetic analyses from available GenBank sequences suggest that currently circulating NDVs emerged in the 1920–1940′s from low virulence viruses by mutation at the fusion protein cleavage site. These viruses later gave rise to multiple virulent genotypes by modulating virulence in opposite directions. Phylogenetic and pathotyping studies demonstrate that older virulent NDVs further evolved into chicken-adapted genotypes by increasing virulence , or into cormorant-adapted NDVs by moderating virulence (velogenic–neurotropic pathotypes with ICPIs of 1.4 to 1.6), or into pigeon-adapted viruses by further attenuating virulence (mesogenic pathotypes with ICPIs of 0.9 to 1.4). Pathogenesis and transmission experiments on adult chickens demonstrate that chicken-adapted velogenic-viscerotropic viruses are more capable of causing disease than older velogenic-neurotropic viruses. Currently circulating velogenic–viscerotropic viruses are also more capable of replicating and of being transmitted in naïve chickens than viruses from cormorants and pigeons. These evolutionary virulence changes are consistent with theories that predict that virulence may evolve in many directions in order to achieve maximum fitness, as determined by genetic and ecologic constraints. Orthoavulavirus, species Avian orthoavulavirus 1 in the family Paramyxoviridae. They are negative-sense, single-stranded, non-fragmented RNA viruses, distributed worldwide, and they are able to infect wild, peridomestic and domestic avian species. NDVs are highly adaptable viruses, capable of infecting over 200 avian species and of causing a diverse range of clinical outcomes in birds [Newcastle disease viruses (NDVs), also known as avian paramyxoviruses type 1, are members of the genus in birds .Two accessory proteins (V and W) with a possible role in virulence have been described for NDV. These proteins are generated by RNA editing of the genomic region corresponding to the phosphoprotein gene region. Although the role of the W protein is still controversial, the role of the V protein in modulating virulence through a process that involves targeting the phospho-STAT1 degradation to block IFN-α signaling has been demonstrated ,5,6,7,8.Although every protein of the virus is likely to interact with the host in one manner or another, none have been studied more deeply than the fusion protein ,13,14,15There are a large variety of pathogenicity tests to evaluate NDV virulence, and these are used to determine if NDVs are capable of producing disease in naïve chickens. These tests range from evaluation of the pathogenicity in eggs , or in one-day-old naïve chicks , to an evaluation in adults  ,16,18,19NDVs display large genetic diversity, which determines the existence of two distinct classes of viruses (class I and II) ,22. WhilThe hypothesis that the large capacity of NDVs to infect and replicate among multiple avian species is accompanied by a capacity to develop genomic changes that modulate and fine tune virulence in order to achieve host adaptation, is analyzed here. NDVs are unique examples in the study of the relationship between virulence and host adaptation for the following reasons: (1) Most NDVs are known to be able to infect more than one different avian host species; (2) NDVs’ genetic changes associated with virulence have been identified; (3) NDVs’ lineages associated with specific hosts have been identified, ; (4) There are very good tests to determine virulence; (5) The virulence of a large number of viruses of different origin has already been characterized; (6) There are existing databases of genomic sequences accompanied by clinical data from the 1930s through present day.Low virulent forms of NDV likely existed and evolved over centuries in wild birds; however, science was only able to document evidence of the presence of NDV when large flocks of chickens started to be raised for their meat in the 1920s. There is evidence that poultry domestication may have started over 3000 years ago when Red Jungle Fowl, a bird whose native territory stretches from east India to Malaysia, dispersed to India China, Egypt, Greece, and Rome . HoweverAs with other RNA viruses, NDVs are expected to adapt to evolutionary constraints by developing genomic changes that facilitate their replication and maintenance ,46. WithChanges in virulence at the cleavage site are rapid, punctual changes in a genomic region that is no longer than 15 nucleotides but is capable of causing drastic changes in virulence . VirulenThe hypothesis that lentogenic viruses from wild birds were the ancestral origin of some older virulent viruses is fairly well supported by evidence. The origin of older virulent NDVs genotypes could be explained in two different ways: (1) multiple separate changes in the fusion virulence cleavage sites occurred on different existing lentogenic genotypes circulating during the 1920s to the 1940s, (2) a change in virulence at the fusion site in a single unknown lineage, occurring further back in time was followed by genetic diversification of this lineage into other old and new virulent genotypes.The first possibility is supported by the presence of lentogenic viruses that are very closely related to virulent viruses. For class II genotype II viruses circulating in the United States, the lentogenic LaSota and Hitchner/B1 in 1946 and 1947, respectively, present only minor nucleotide differences when compared with old contemporary virulent viruses of class II genotype II such as Texas/GB/1948, Beaudette C/1945, and others virulent viruses that were collected in the 1940s and the 1950s in the United States . These mAnother well-documented piece of evidence of the change in virulence at the cleavage site is represented by the outbreak of NDVs of genotype I in Australia during 1998–2001 ,53. ReseAs the origin of other older virulent lineages  is not as well documented, the second hypothesis is only indirectly supported by the evidence that other more recent virulent viruses appear to have originated by evolution from older virulent viruses . The exiThe second type of genomic changes are progressive, cumulative nucleotide changes leading to different types of host fitness adaptations at the protein level, including neutralization escapes, among others . GenotypThe third type of genomic changes are codon usage adaptations defined as the selective use of codons by the viral genome to better adapt to the availability of amino acids produced by the host. It is known that codon usage is not identical among animal species. Thus, during the process of host adaptation, viruses may improve their fitness to replicate in a particular host by changing the use of codons necessary to translate proteins in order to utilize efficiently the host transfer RNAs . NonrandA study by Taylor and others  includedIn order to estimate the time to the most recent common ancestor of class I and class II viruses and between best-represented genotypes within class II, a Bayesian Inference approach was implemented using the BEAST package  by TayloIn contrast with previous results, a Pearson’s r correlation coefficient of 0.8 was observed when analyzing the relative synonymous codon usage values among different viruses within class II. Class II viral fusion gene sequences higher positive correlation suggested that fewer codon usage changes have occurred among recently evolved viruses. Among virulent viruses normally expected to be maintained in chickens (genotype VII) versus mesogenic viruses that are normally maintained in pigeons (genotype VI), there was an evolutionary estimate time to the most recent common ancestor of approximately 69–73 years; thus, in agreement with the idea of a slow process of codon usage adaptation, the observed differences in codon usage these among class II viruses were not as significant as the differences observed between classes. The analysis concluded that codon usage adaptation is likely to be active, but it is a slow process that is not easily measured in short evolutionary periods.P-M, F-HN, and P) for class I (n = 29) and class II (n = 259) viruses, was determined using Gallus gallus as the reference genome. The class II F and HN are surface proteins that have already been shown to be affected by positive selection and by antibody-selective pressures and encode genes with clear functional differences with genes encoded in other genomic regions  inter-host transmission; (2) origin of recent virulent viruses from older virulent viruses; (3) genetic changes consistent with adaptation to maintenance in different hosts; and (4) demonstration of changes in virulence-associated with these genetic changes. Modern techniques of phylogenetic analysis provide evidence for events 1, 2 and 3, and comparison among the pathogenicity and virulence of viruses adapted to different hosts provides evidence for type 4 events. Although many reports support the capacity of NDV to infect multiple hosts, a recent global phylodynamic analysis of avian paramyxovirus-1 done by J. T. Hicks using the complete fusion protein coding sequence has provided sufficient statistical evidence supporting NDVs capacity for inter-host transmission . Hicks’ This nucleotide-based analysis demonstrated the capacity for NDV to transmit among divergent host taxa over time . The hign = 284) and class II (n = 1672). Detailed tables of all available sequences, including genotypes, host, date and place of isolation, are available in Supplemental Tables S1 and S2 of Dimitrov 2019 manuscript [Demonstration of the origin of recent virulent viruses from older virulent viruses (2) and evidence of genetic changes consistent with adaptation to maintenance in different hosts (3) can be obtained by phylogenetic analysis of large databases. The genetic diversity of current NDV class II virulent genotype viruses was recently studied by an international consortium . For thinuscript . Conclusnuscript . Phylogen = 1672). In the phylogenetic trees produced by Dimitrov’s study, the origin of current virulent viruses can be traced back to the oldest characterized virulent genotypes circulating during the 1930s and 1940s. These include the genotype III virus identified as Australia 1932 and chicken Mukteswar 1940 as the oldest ancestors in Asia. The genotype IV oldest viruses in Europe were the Herts isolated in chickens in the United Kingdom in 1933 and the Italien isolated in Italy in 1944  ancestral relationship of with genotype VIII chicken viruses (1960–1970) for viruses representing nearly all of the most recent cormorant, pigeons, and vvNDV genotypes of chickens . Pigeon origin viruses have been a part of the panzootic in Columbiform birds that emerged more than four decades ago, with these viruses still circulating worldwide ,39,40,66vnNDV and mNDV that infect cormorants and other species of the genus Phalacrocorax) in North America represent the second group of wild bird viruses that may have originated from a common ancestral virus from chickens and adapted by reducing virulence . VirusesThe factors important for the apparent virulence adaptation seen in pigeons and cormorants’ viruses are unknown. Although speculative, one would expect that host species that form long-term associations, or high-density colonies in which proximity is granted would favor certain replicative features of virulent viruses that have previously adapted to replication in poultry, such as a capacity to transmit only in close proximity with long term contact periods. Mass nesting is common in pigeons, with dozens of birds sharing buildings in which nests and droppings tend to stay clustered and remain dry when out of the weather. Cormorants are generally gregarious, nesting in colonies, gathering in flocks and often hunting together in groups, which sometimes numbers up to 4000 birds. Other species that congregate in larger groups, such as parrots, have also been reported to carry virulent viruses. The isolation of highly virulent viruses of genotype V from psittacine in the past has suggested a possible role as wild bird reservoirs for highly virulent viruses. However, the persistence of those psittacine isolations over time has not been demonstrated. It remains to be seen if herons, egrets, flamingos, albatrosses, penguins, swallows, oropendolas, and other colonial birds may also be receptive to the maintenance of virulent viruses currently circulating in chickens. In infections of wild avian populations (such as those of pigeons and cormorants) with lower or non-existent levels of immunity in non-infected birds, it may not be efficient to maintain highly lethal viruses, and further evolution toward a lower level of virulence may be a more effective viral strategy for achieving fitness. However, those theories need to be further investigated.Although an initial inference of changes in virulence during evolution can be indirectly achieved by analyzing the prevailing ICPI of viruses representing different genotypes, the ICPI test often lacks the resolution to distinguish changes in virulence affecting adult birds. Consequently, changes in virulence as an evolutionary host adaptation mechanism are best studied doing pathogenicity studies in adult birds. Despite the ICPI values shown in Pathogenicity studies in adult-specific pathogen-free chickens were used to demonstrate that viruses classified as vvNDV have increased virulence in comparison with vnNDV viruses. The studies by Brown and Piacenti demonstrate that the viruses from both pathotypes can target cells of the brain and cause mortality. However, a key difference between vvNDV and vnNDV was more rapid mortality in chickens infected with vvNDV (4–6 days) as one of the factors that prevent the development of neurogenic damage . The increased virulence of vvNDV in CA 1083, 90-14698, and 93-28710 isolates was characterized by acute systemic illness, extensive necrosis of lymphoid areas in the spleen and intestine, neurological damage with rapid mortality A,B. InstAdditional evidence for changes in virulence during adaptation to different hosts was demonstrated by Ferreira and others who compared virulence and transmission of NDV of different origins . In thesMortality rates, clinical signs, and gross lesions observed in birds infected with the chicken-origin vvNDV were similar to those reported previously for other vvNDV ,75. ChicThe role of host-associated evolutionary pressures is a neglected area of research that needs to be further investigated. The need to increase the frequency of poultry vaccination against NDV in recent years in endemic countries to control the constant presence of vvNDV suggests that evolution toward increased virulence in chickens may be a byproduct of current poultry production systems . ImmunizThe role of virulence on host adaptation (or vice versa) during more than ninety years of NDV evolution was analyzed. The data suggests that recent virulent viruses from wild birds originated from older chicken virulent viruses and indicates that the maintenance of virulent NDV in chickens or in wild birds may have led to the modulation of virulence in opposite directions. In evolutionary terms, the evolution of virulence in different directions is expected to occur ,83,84. R