The vast majority of species that belong to the plant or animal kingdom evolved with two main strategies to counter tissue damage—scar formation and regeneration. Whereas scar formation provides a fast and cost-effective repair to exit life-threatening conditions, complete tissue regeneration is time-consuming and requires vast resources to reinstall functionality of affected organs or structures. Local environments in wound healing are widely studied and findings have provided important biomedical applications. Less well understood are organismic physiological parameters and signalling circuits essential to maintain effective tissue repair. Here, we review accumulated evidence that positions the interplay of local and systemic changes in metabolism as essential variables modulating the injury response. We particularly emphasise the role of lipids and lipid-like molecules as significant components long overlooked. Some animals, like salamanders, possess the unique ability to regenerate an entire limb in a few weeks. Other species like planarians and cnidarians even regenerate an entire body. In contrast, most vertebrates evolved to rapidly repair damages that expose the organism to external hazards. Such repair may result in non-functional tissue replacements and the formation of a fibrous matrix called a scar.2Regeneration in mammals is observed vastly in tissues with high proliferative potential and resident stem cell populations—the liver, blood or intestinal epithelium. Furthermore, decline or depletion of the resident stem cell pool , affects cellular turnover and the regenerative potential.While the extent of regeneration and the timing differs from organ to species, there is a clear need for (1) new building blocks: amino acids, sugars, lipids and nucleic acids, (2) signals to orchestrate the process and (3) energetic fuel to accomplish it. Despite this, little is known on how a regenerating organism regulates its metabolism. Furthermore, and often overlooked, regeneration should not be seen as an isolated process, but rather influenced by the circumstances of the organism at a particular time and place. For example, a salamander that spends at least 6-9 weeks re-making a whole limb, should rebalance its metabolic homeostasis to adapt to this long-term energy demand. However, it remains a mystery how a local injury shifts the energy resources and how the organism modulates it.7) and this evolutionary diversity underlines regeneration as a basic biological property in living organisms. One common initial mechanism is re-epithelization of the wound, to seal the organisms from the external environment. Some organisms go further and form a specialised wound epidermis (WE).8 The WE is a multi-layered epithelium, functioning as an important signalling centre during subsequent steps like the formation of a temporal structure called the blastema. The blastema is a highly proliferative cell mass, containing the dedifferentiated cells, as well as recruited resident stem cells which can give rise to all cell types needed for regeneration of the missing body part. Planarians, teleost fish, salamanders, reptiles and mammals among others, regenerate lost body parts through a blastema. In models where the blastema is absent, persistent active migration and proliferation are crucial for the re-formation of the missing structure. However, the requirement of energy to regenerate tissues and complex structures is a common ground.Animal and vegetal species regenerate through different mechanisms , the release of humoral factors into the bloodstream  and by the activity of dietary signal cues .10 Neural circuits sense and respond to the above-mentioned inputs, by commanding adjustments that will keep a balance between energy intake and energy expenditure. In the arcuate and paraventricular nuclei of the hypothalamus, neurons sitting in a highly vascularized area, directly sense nutrients and circulating hormones. In turn, proopiomelanocortin neurons (POMC) inhibit food intake and increase energy expenditure, or neuropeptide Y neurons (NPY) will direct food intake and inhibit energy expenditure . In addition, anabolic or catabolic turnover in dedicated storage organs  buffer the systemic availability of all essential cues required to allow optimal cellular functionality. These organs can also be regulated centrally, for example, sympathetic neurons stimulate the activity of adipocytes amplifying catabolic turnover to free fatty acids (FAs) from stores.14 While the storage of glucose (in the form of glycogen) is limited to small quantities, most energy resources are stored as fat in lipid droplets. Lastly, energy balance is influenced by other physiological systems such as the reproductive and stress axis (reviewed elsewhere9), and as proposed in this manuscript, by injury signals.To maintain energy availability in all tissues, a vast network of regulatory checkpoints ensures homeostasis. Upon food ingestion, the main role of the digestive system is to obtain lipids, carbohydrates, proteins, minerals and vitamins. Although there is a significant interest in sugar and amino acid traffic, more recent work has re-started to look into lipids and uncovering novel roles in metabolism and signalling.33.115 direct receptors that sense mechanical, thermal or chemical damage,16 or by directly regulating ion channels in nerve terminals. Specialised sensory neurons (nociceptors) detect stimuli that cause tissue damage and convey the information via the afferent neurons through the spinal cord, and then to the thalamus. The thalamus relays signals to the higher brain centres , where the information is processed and results in an action , pain modulation and stress reaction (reviewed elsewhere16).The nervous, immune and circulatory systems convey and process information from the injury site. These systems are well studied independently, however, the interaction of multiple systems and interorgan communication is gaining attention. The central nervous system collects injury-related information via soluble factors,17 Further immune system responds to damage-associated molecular patterns (DAMPs), released by damaged or dying cells, to ensure asepsis of the wound. Additionally, bacteria, pathogen-associated molecular patterns (PAMPs), metabolic by-products and cytokines also trigger an inflammatory response necessary to mobilise resources and restore homeostasis (reviewed elsewhere19). However, when an exacerbated response to a major injury is not controlled, secondary organ damage can occur even in locations remote from the injury, known as injury-associated systemic inflammatory response.20Injury-induced release of reactive oxygen species (ROS), triggers the first immune response recruiting inflammatory cells.21 However, it is likely that the interplay between the injured tissue, the immune system and the nervous system, is at least influenced by the various chemicals and metabolites produced locally.Other less explored elements are locally released factors  that enter the bloodstream. Together with DAMP signalling, such direct communication axes are able to convey injury signals into a systemic response and allow bidirectional communication between the site of injury and other body parts. Despite being well known that metabolites act as efficient signalling molecules, only little is known about the role of those locally released factors in wound repair and regeneration.3.223 In trauma patients with coma, hypermetabolism still occurs, suggesting that movement or awareness of the patient is not the cause. Moreover, metabolism after severe trauma is characterised by high cortisol, insulin resistance and hyperglycemia.25 Prolonged hyperglycemia and hypermetabolism has adverse effects for the healing process and could lead to further infections and even multiorgan failure. The type of injury is unlikely to be a determinant for a metabolic systemic response. For example, the resting metabolic rate is increased in patients with brain injury, burns and in skeletal trauma.26Data from patients with severe trauma supports a significant metabolic re-adjustment. After injury, the organism responds with a switch to a catabolic state and hypermetabolism (elevated resting energy expenditure). In this situation, skeletal muscle is catabolised to support protein synthesis and, interestingly, the administration of nutrients is not sufficient to revert the catabolic state.3.325 and likely to be the result of multiple factors. In patients with significant skin burns, the early phase post injury (hours) is characterised by high glucose levels and almost no response from the insulin signalling cascade. In a later phase (days and later) insulin levels rise, but blood glucose levels decrease only moderately, and heterotopic fat in various cell types including muscle and liver cells is observable. From the medical point of view, persistently high insulin reflects insulin resistance.27 In patients with brain trauma, metabolic dysfunction of brain cells adds to the damage, in the form of insulin resistance and decreased glucose uptake in undamaged brain areas,28 but also as a systemic effect.29 In a rat burn model, injury mimics the hyperglycemia and insulin resistance in patients. This response occurs as fast as 30-60 min after injury when trauma is combined with haemorrhage30 and helps to consolidate the response. In a rat model of surgical injury and haemorrhage, insulin signalling (IS) is affected in skeletal muscle as soon as 60 min post-surgery, reflected as a rapid decrease of AKT phosphorylation and insulin receptor function.31 Insulin resistance and hyperglycemia in turn, affect immune cells, favouring an inflammatory immune response.33 However, the interconnectivity and causation between insulin resistance, hyperglycemia and inflammation needs to be further investigated. In a tour de force study that mimics traumatic brain injury in Drosophila, Katzenberger et al., observe hyperglycemia in hemolymph and suggest that intestinal barrier dysfunction is a secondary trait triggered by injury.34It is difficult to predict or compare the severity of the triggered hypermetabolism, as there is no comprehensive study analysing different types of injury and normalising for the severity of the trauma. A common denominator is however an acute insulin resistance, which seems to be unique to injury35 Finally, diseases affecting metabolism (such as Diabetes) complicate the healing process including non-metabolic organs like brain, bones.36As dysregulation of IS has a direct impact on the patient’s recovery, there is a need to control alternations of insulin signalling resulting from injuries. Furthermore, the effects of IS on cellular decisions are evident. Glucose levels and IS are essential for stem cell division rates of Drosophila intestinal stem cells. The intestinal stem cell niche is formed by different cell types including visceral muscle cells. These cells produce the Drosophila insulin-like peptide 3 (DILP3) in response to intestinal damage and the availability of a calorie-rich diet. DILP3 is sufficient to promote stem cell division and its localised reduction minimises regenerative activity.3.437 The recovery of patients with traumatic brain injury (TBI), bone injury or concomitant TBI and bone injury was investigated. The group with combined injury showed accelerated bone healing, suggesting that humoral factors are involved. Indeed, an increase in growth hormone at 3 weeks after injury in the combined group is observed. In addition, an important systemic response to injury is the activation of the hypothalamic-pituitary-adrenal axis. Elevated corticosteroid levels were shown in patients of all groups. Moreover, within 24 h post injury in the group with the concomitant injury, a high concentration of the food intake and satiety regulator leptin was demonstrated. In support of a systemic effect, in a mouse model lacking leptin, the positive effect of the concomitant injury on bone healing is decreased.38Further evidence of a complex systemic response to injury is shown by Khallaf et al.39 and parathyroid hormone40 in circulation that could further explain the accelerated healing of bone. In addition, human and animal injury models have confirmed the consistent increase in cortisol in response to injury.41Others have shown that traumatic head injury increases the release of insulin-like growth factor 1 (IGF-1)25Why these humoral factors are secreted is not fully understood, however, the consequences are various. For example, it has been proposed that insulin resistance and hyperglycemia are cortisol-induced. Although recent evidence shows that blocking the increase of cortisol in a rat injury model, does not prevent insulin resistance in the liver.3.543 The role of mTOR in skeletal muscle injury has been described as necessary. In the absence, or by blocking mTOR, muscle stem cells (satellite cells) fail to activate and proliferate.44 Rodgers et al. showed that mouse muscle injury on one side of the body triggers quiescent muscle stem cells on the contralateral side to enter an alert state, via mTOR.45 In addition, mTOR is required during tissue regeneration in planarian and zebrafish by regulating stem cell activation and blastema outgrowth.47 In salamanders, a subset of cells in the contralateral limb from an amputated animal, also express mTOR in response to limb regeneration.48The target of rapamycin (mTOR) regulates cell growth and metabolism through phosphorylation of kinases such as protein kinase C (PKC) and protein kinase B (AKT), and interacting with other subunits, it forms two functional complexes, mTORC1 and mTORC2. In response to nutrient availability, mTOR is activated in different injured tissues.49 Moreover, activated AKT promotes the localization of the glucose transporter GLUT1 at the plasma membrane, ushering the cellular uptake of the sugar.50 Enhanced GLUT1-mediated glucose uptake is one prominent example of growth factor induced nutritional flow untethering cells from the regulation of whole-body glucose homeostasis.51One possibility to explain a systemic response via mTOR is the fact that mTOR is merged with insulin signalling via the AKT-Ser473. In fact, mTORC1 is an effector of the insulin/PI3K pathway that promotes lipid synthesis, glycolysis and nucleotide synthesis.To fill local metabolic needs of healing tissues, a rather complex local and systemic regulatory network sets off to coordinate energy expenditure and availability. We are now starting to understand how a sustained healing, and highly proliferative process such as epimorphic regeneration, can maintain an active source of available fuel.44.1Xenopus tadpole tail regeneration, blastema cells increase glycolysis and hexose monophosphate pathways.53 Similarly, in zebrafish metabolic reprogramming to high glucose uptake and lactate production (known as Warburg effect) is necessary for tail regeneration, and it is associated with the hexosamine biosynthetic pathway HBP and N-linked glycosylation.54Epimorphic regeneration is characterised by anabolic metabolism; it requires building blocks and local metabolic adaptations to regrow the lost appendage. The highly proliferative cells of the forming blastema are associated with a metabolic switch to a more embryonic-like metabolic state. In lizard and Xenopus and axolotl.55 The regenerative environment is generally characterised by a local increase in reactive oxygen species (ROS) within hours post-amputation.54 ROS are produced as a byproduct of mitochondrial oxidative phosphorylation causing cell damage or by NADPH oxidases, particularly NOX2, enhancing immune defence and cell signalling functions.56 ROS are speculated to mediate glucose entry into glycolysis and the pentose phosphate pathway metabolism and to play a role in mammalian axonal regeneration through the NOX2-PI3K-p-AKT signalling pathway.57Proteomic analysis has shown a decrease of several enzymes of the citric acid cycle and oxidative phosphorylation in both regenerating 58 HIF1α is a transcriptional master regulator that among many functions, induces the expression of VEGF, a key regulator of vasculogenesis and angiogenesis. In zebrafish and other vertebrates, vascularization of damaged heart tissue is one prerequisite to induce regeneration.59 This underlines how the vascular distribution, and thus oxygen and nutrient supply, during regeneration is intertwined with metabolism and stem cell function. Other less understood examples of ROS-induced tissue remodelling in response to wounding are reported in plants and flies. Plant leaves reconnect the wounded area shortly after tissue damage and an intense ROS production has been reported.61 Similarly, in the regenerating Drosophila gut, ROS induces vascular remodelling.62 Epimorphic regeneration also requires long-range factors like hormones that are locally upregulated. Notably, a local increase in the orexigen leptin and its receptor have been shown to enhance bone formation and healing of skin wounds.65 Although the role of circulating factors on leptin expression in the wound is not fully understood, there is evidence that cultured human fibroblasts secrete leptin in response to insulin.66 A further crosstalk with insulin signalling is observed intracellularly: leptin induces PI3K/AKT signalling, sharing the molecular cascade with the insulin receptor. At the injury site however, leptin is increased within early stages of Xenopus tail blastema and axolotl limb regeneration.68 It remains to be defined how the locally secreted leptin impacts neighbouring tissues or systemic leptin function.These findings are supported by an increase in hypoxiainducible factor 1-alpha (HIF1α) activity induced by high ROS levels.70 Insulin, proinsulin and insulin-like growth factor (IGF) are upregulated in tail regeneration and are required for blastema cell proliferation71 and heart regeneration in zebrafish.72 Insulin/IGF-1 signalling activates downstream pathways that enhance cell survival, proliferation and differentiation through MAPK signalling, and PI3K-AKT signalling. This leads to lower injury-induced hyperglycemia, protection against elevated ROS and angiogenesis.70 Thus, insulin/IGF axis not only regulates energy balance, it also plays an important local role activating growth, osteogenesis, angiogenesis and stem cell survival.Earlier studies have shown that salamander blastema cell proliferation and survival in vitro and in vivo depend on the combined presence of several hormones, like insulin or growth hormone (GH).Xenopus limb regeneration, that α-MSH/MC4R were increased in blastema in a nerve dependent manner, and blocking the signalling of MC4R impairs regeneration.73 Using a crude hypothalamic injury, the authors observe a similar impact on regeneration, arguing for a local and systemic role for MC4R. Local α-MSH/MC4R signalling regulates ROS levels, ensures mitochondrial stress balancing the rate of glucose metabolism and promotes neurotrophic functions within blastema cells. Furthermore, MC4R knockout mice fail to regenerate the digit tip, while systemic α-MSH administration rescues regeneration in heterozygous mice. When applied in a proximal and regeneration incompetent digit amputation, α-MSH stimulates regeneration.74 Additional important factors linking metabolism and regeneration can be found in Alpha melanocyte stimulating hormone (α-MSH) and its receptor melanocortin receptor 4 (MC4R) impact epimorphic regeneration both locally and systemically. α-MSH, a cleaved peptide from the precursor POMC, is released into circulation from neurons in the hypothalamus. Recently, it was shown in 55.197 The production of lipoprotein-lipid carriers is limited in type and location. Cells facilitate the uptake of lipoproteins or other lipid carriers initiating receptor mediated transport.99 These receptors belong to conserved protein families and their expression patterns are regulated by many factors including cell type, circadian rhythm and nutritional state.100 Stem cells are known to express different lipoprotein and albumin receptors (another major lipidated protein in circulation). It is likely that dividing cells in regenerating tissues require adequate lipids to facilitate their proliferation rates.101 Although most cell types are capable of producing lipid building blocks, such as FAs, to preserve time and energy lipids are absorbed from circulation. Animals absorb many dietary lipids; however, blood serum lipids represent a mixture of food lipids and endogenous lipids. The following sections are focused on bioactive lipids and lipid-like molecules of different origin.In many animal species the bulk of endogenous lipids which enter circulation are produced by cells functionally analogous to intestinal cells, adipocytes or hepatocytes. All these cell types are able to secrete lipoprotein particles, systemic lipid carriers, or lipidated proteins destined to spread their lipid loads within the organism.5.2103 However, plants are characterised by indeterminate post-embryonic development and the source for (re) generation of new organs are shoot or root stem cell niches—the so-called apical meristems. It is debated if these meristem cells are equivalent to stem cells in animals and if (re)growth of plants can be compared to animal regeneration.105 However, processes like the complete restoration of root-tips astonishingly mimic in many aspects the limb regeneration process in animals . RA has many targets including genes involved in mediating canonical antioxidant responses, or nuclear retinoid receptors involved in the regulation of many systemic parameters including the glucose-lipid homeostasis.114 Moreover, it is known that RA is essential for patterning during salamander limb regeneration115 mediating the proximodistal specification in salamander limb regeneration.117 Furthermore, antagonising the retinoic acid receptors disrupts skeletal patterning and skeletogenesis during regeneration.118 In zebrafish, RA regulates proliferation and is required for blastema formation,119 as well as in heart regeneration, where injury-triggered RA controls cardiomyocyte proliferation.120 Not surprisingly, blocking the rate-limiting enzyme Raldh2, inhibits regeneration in zebrafish.121In the past years, metabolic research retook the focus on the metabolic activity of dietary lipid-like molecules and lipids. Lipid-like vitamins, such as A, D and E, are essential to many animals and their biochemical properties allow such compounds to integrate into cellular membranes . As such122 PUFAs reduce ROS species efficiently, limiting the range of such signalling.123 Hence, vitamin E indirectly modulates metabolic rates since membrane integrated PUFAs shift the signalling capacity of many signalling pathways and the local lipid composition likely modulates vascularization. Studies suggest an inhibitory effect of vitamin E on liver regeneration in rats by altering the lipid peroxidation in the cytosol and plasma membranes.124 In contrast, vitamin D promotes regeneration of zebrafish fin125 and heart126 tissues as well as human skeletal muscle repair, and is necessary for skeletal tissue integration in salamander limb regeneration.127Vitamin E is an important antioxidant and protects polyunsaturated fatty acids (PUFAs) in cellular membranes from oxidative destruction.128 Essential fatty acids (EFAs) are precursors for the generation of endogenous complex lipids acting as signalling molecules , or can directly induce metabolic signals.129 In humans, EFAs need to be absorbed from food. Most studies focus on plant derived n-3 and n-6 omega FAs with a carbon chain length of 18-20 atoms, such as alpha-linolenic acid or linoleic acid  produced by intestinal microbes.138 Absorbed by host organisms, SSFAs are suggested to play many roles including regulating insulin signalling.139 The ratio between dietary SFAs/PUFAs changes the absorption rate of nutritional cues and metabolic rates, impacting on many parameters including lipid turnover . Some PAHSA-lipid species promote insulin release into the blood system.146 However, dietary supplementation of these lipids had no effect on IS.147 Fasting also changes the activity of neuronal subsets wired with organs responsible for the redistribution of nutritional molecules.148 For example, free FAs that induce signalling in hypothalamic neurons or glia.149 Starving cells dismantle endogenous complex lipids to fill energy demands and produce free FAs fit for mitochondrial reduction. Free FAs activate neurons wired with hepatocytes and in response, the liver cells start to release hepatic glucose into the blood system, essential to maintain basic blood-glucose levels.150A less studied set of molecules in the context of repair are endogenous lipids regulating metabolic signalling cascades. However, what we currently know poses them as an important player. Starving organisms lower their metabolic rates and many enter dormancy-like states to endure long periods of food deprivation. Nevertheless, some species maintain their regenerative capacity despite none or insufficient food consumption.151  or microbial treatments aiming to optimise nutritional absorption, potentiate the immune response or provide microbial metabolites catalyzing metabolic signalling. In addition, ongoing research on insulin signalling and the metabolic shift of immune and stem cells close to tissue damage may result in therapeutic strategies including patients suffering from metabolic diseases like Diabetes.6.5In this review we have investigated the possibility that systemic metabolism is one key variable to promote regenerative tissue repair. We propose that regenerating cells do not simply rely on local signalling cues and sufficient nutritional provision, but actively influence other tissues responsible for the mobilisation of molecular building blocks, such as lipids. Future coordinated studies into the topic across different species will reveal conserved molecular mechanisms key to formulate biomedical strategies aiming to improve wound healing.


Though gray matter deficits have been consistently revealed in chronic treated schizophrenia, it is still not clear whether there are different brain alterations between chronic never treated and treated patients. To explore the different patterns of gray matter alterations among chronic never treated patients and those treated with monotherapy, we recruited 35 never-treated chronic schizophrenia patients with illness durations ranging from 5 to 48 years, 20 illness duration-matched risperidone monotherapy and 20 clozapine monotherapy patients, and 55 healthy controls. GM  measures were extracted and compared using ANCOVA across the four groups followed by post hoc tests. Relative to controls, both treated and never-treated chronic schizophrenia patients showed reduced GM mainly involving the bilateral medial and rostral middle frontal, left banks superior temporal sulcus, left fusiform, and left pericalcarine cortex and increased in the left cuneus. Compared with the untreated patient group, the two treated groups showed reductions mainly in the bilateral prefrontal, temporal, and left inferior parietal lobules. The clozapine monotherapy patients demonstrated more severe decreases in the bilateral prefrontal cortex and left cuneus and less severe decreases in the left ventral temporal lobe than risperidone monotherapy patients. These findings provide new insights into the long-term effects of antipsychotic treatment on gray matter alterations in schizophrenia patients. Furthermore, the characteristic findings of reductions in the inferior parietal lobule might be specific for long-term antipsychotic treatment, which could be a possible target for medication development in the future. Previous structural MRI studies9 have demonstrated that first-episode drug-naive schizophrenia patients showed GM reductions in the prefrontal and temporal lobes, and chronic schizophrenia patients have abnormalities in the frontal, temporal, superior parietal, and occipital lobes15. Although the above studies have reported inconsistent findings in patients with chronic and first-episode schizophrenia, it is still unclear whether the findings are due to neuroprogressive mechanisms or stage-specific characteristics.Recent advances in psychoradiology15, but these studies have mostly been during the middle course of the illness. However, only two studies17 reported GM alterations during a relatively later course of the illness. A cross-sectional brain structural study16 found that, relative to healthy controls, schizophrenia patients with an untreated illness course of more than 20 years demonstrated less cortical thickness in the bilateral ventromedial prefrontal cortices, left superior temporal gyrus, and right pars triangularis and greater cortical thickness in the left superior parietal lobe. However, there has been a lack of direct comparisons between long-term treated and untreated patients, which is crucial for understanding the neurobiological consequences of long-term treatment.Previous studies have shown GM abnormalities in chronic schizophrenia20 and have found progressive GM loss in the frontal, temporal and occipital lobes after 2–3 years of treatment. However, only a few studies have explored longer duration treatment effects, but the findings have been inconsistent22. Beng-Choon Ho et al.21 reported that parietal lobe reductions in schizophrenia were associated with higher doses of non-clozapine atypical antipsychotics in patients over an average follow-up of 7.2 years. However, this finding was not replicated by Veijola et al.22. These inconsistent findings may have resulted from different types/doses of antipsychotics. Furthermore, it is still not clear whether there are different characteristic changes in brain structure after long-term antipsychotic medication use in schizophrenia patients. Therefore, the study of monotherapy may shed more light on the effects of long-term treatment on brain structure in schizophrenia patients.In fact, the short-term effects of antipsychotic treatment have been well studied by a number of longitudinal MRI studiesTherefore, to explore the long-term effects of antipsychotic treatment on brain morphology, we recruited 35 never-treated chronic schizophrenia patients with a mean illness duration of 20 years, 20 illness-duration-matched risperidone monotherapy patients, 20 illness-duration-matched clozapine monotherapy patients, and 55 healthy controls. We hypothesize that characteristic or specific GM change patterns would be found both in schizophrenia with risperidone monotherapy and clozapine monotherapy relative to never-treated patients and that brain structures differentially respond to different kinds of antipsychotics.23 with the information provided by the patients, other family members, and other sources. Patients with a duration of illness longer than 5 years were defined as “chronic”25. Symptom severity assessment was carried out using the Positive and Negative Syndrome Scale (PANSS)26 on the same day as MRI scanning. This study was approved by the Institutional Review Board, and written informed consent was obtained from each participant.Thirty-five never-treated chronic schizophrenia patients with long-term illness (ranging from 5 to 48 years), 20 illness-duration-matched risperidone monotherapy patients, 20 illness-duration-matched clozapine monotherapy patients and 55 healthy controls (HC) were included and assessed using a single GE MR scanner. The diagnosis of schizophrenia was determined with the consensus of two psychiatrists using the Structured Interview for the DSM-IV (SCID). Illness onset was evaluated by the Nottingham Onset ScheduleNever-treated chronic schizophrenia patients were identified from the community Mental Health Screening Program, aiming to identify and provide psychiatric care to individuals with serious but untreated mental illness. Most of these never-treated patients are over 40 years old and were typically recruited from rural or suburban areas within 35 km of downtown Chengdu and Zigong city in Western China. Based on available retrospective information, 9 of these patients had developed acute psychosis after significant life trauma, such as the loss of a close relative or parental marital difficulties. The other 26 patients had a gradual and insidious onset of symptoms. Twenty-one had no involvement in any employment. Fourteen were able to care for themselves in many activities of daily living. Most of the never-treated patients had 6 years of primary education, while the antipsychotic-treated patients had 9 years of education in China. These never-treated patients had not previously received any antipsychotic drug treatment for a variety of reasons, but all of the patients had been well taken care of by their families since the onset of their illness.27.The treated patients were recruited from the same or nearby communities and had received antipsychotic treatment relatively consistently beginning early in the course of their illness. The patients had been treated with risperidone or clozapine, and no patient had switched to another antipsychotic in the previous 5 years. The dose of antipsychotic medication was converted to chlorpromazine (CPZ) equivalentsn = 55) were recruited to match the patient groups from poster advertisements in the local area of patients, and they had similar socioeconomic and educational backgrounds. All controls were screened using the SCID-Non-Patient Version to confirm the lifetime absence of psychiatric and neurological illnesses, and all participants were right-handed.Healthy control subjects  contraindication for MRI examination, (2) another axis I psychiatric disorder, (3) any neurological disorder, (4) lifetime drug or alcohol abuse or dependency, and (5) pregnancy or significant systemic illness, such as hepatitis or cardiovascular disease. MR images of all participants were inspected by an experienced neuroradiologist to exclude subjects with visible brain abnormalities.o, voxel size = 0.47 × 0.47 × 1 mm3, the field of view = 240 × 240 mm2, matrix = 256 × 256, 156 axial slices, and slice thickness = 1 mm. All participants were told to lie still and keep their eyes closed without thinking anything during scanning. Images were immediately checked by a neuroradiologist, and participants with substantial motion or other artifacts were subjected to reimaging.All MRI data were acquired on a 3.0 T GE Signa EXCITE Scanner  with an 8-channel phase array head coil. All participants went through a uniform protocol that comprised an axial high-resolution T1-weighted image with a volumetric three-dimensional spoiled gradient-echo sequence with the following parameters: repetition time = 8.5 ms, echo time = 3.4 ms, flip angle = 12https://surfer.nmr.mgh.harvard.edu/) package was used to perform cortical modeling and volumetric segmentation and to measure cortical volume and subcortical volume by using image intensities and continuity information from the entire MR volume to construct representations of the gray/white matter boundary and pial surface30. The processing steps, including skull stripping, segmentation of the gray matter, white matter and CSF, cortical surface reconstruction, registration, and parcellation, were initialized with common information from the within-subject template31. The above procedure generated average cortical volumes for 68 regions and the subcortical volumes for 14 regions of interest  using the Desikan–Killiany atlas template32. Postprocessing visual inspection for the quality of both imaging processes was reviewed without knowledge of subject characteristics. Cortical thickness and surface area were also investigated, using similar methods.The FreeSurfer  and the subcortical volumes of 14 regions using the Desikan–Killiany atlas template were extracted. Analysis of covariance (ANCOVA) was conducted using a general linear model (GLM) followed by the least significant difference (LSD) post hoc pairwise comparisons after controlling for age and sex. The statistical significance level was set at p = 0.05 with two-tailed, controlled for multiple comparisons using a false discovery rate (FDR) criterion for each of the 68 cortical measurements and 14 subcortical volumes. Pearson correlation analyses among the anatomical measures and drug dose, PANSS scores, and illness duration were performed.All statistical analyses were performed using SPSS . Demographic variables  and clinical characteristics  were compared among the groups via one-way analysis of variance (ANOVA), two-sample p > 0.05). The sex ratio was significantly different among the groups (p < 0.01), with the RT-SCZ group having more males than the other groups. Both treated patient groups showed symptom remission relative to the NT-SCZ group . No significant difference in symptoms was found between the RT-SCZ group and the CT-SCZ group or in the daily dosage of antipsychotic medications based on CPZ equivalents .The demographic and clinical characteristics of the study participants are listed in Table p < 0.05; see details in Supplementary Table p > 0.05). The GM characteristics in cortical volumes, thicknesses, and surface areas of all regions are listed in the supplementary materials  demonstrated a significant main effect among the four groups by analysis of covariance (ANCOVA) after multiple comparisons, involving 46 brain regions, but excluding the bilateral caudal middle frontal, lateral occipital, insula, cingulate, motor and sensory cortex , left inferior temporal volume , left middle temporal volume , and left inferior parietal volume  , PANSS total scores , negative syndrome scores , and general psychopathology scores . The left parahippocampal thickness was related to positive syndrome scores . In the CT-SCZ group, the illness duration was negatively correlated with the right fusiform thickness  and right inferior temporal volume . The left frontal pole thickness was related to the PANSS total scores  and negative syndrome scores . The alterations in GM were not associated with illness duration or clinical symptoms in the NT-SCZ group (p > 0.05).In the RT-SCZ group, the illness duration was negatively correlated with the left inferior temporal thickness (To our knowledge, this is the first study investigating brain gray matter morphometric alterations between never-treated and long-term monotherapy-treated chronic schizophrenia patients. In general, both treated and never-treated chronic schizophrenia patients, relative to controls, showed cortical GM reductions and increases in similar brain regions, mainly involving bilateral medial and rostral middle frontal, left banks superior temporal sulcus, and left medial occipital cortex regions. Relative to never-treated patients, the two treated chronic patient groups showed widespread reductions mainly in the bilateral prefrontal, temporal, and left inferior parietal lobules. Within the treated group, the clozapine monotherapy group had a more severe reduction mainly in the bilateral prefrontal cortex than the risperidone monotherapy group. In addition, GM reduction was negatively correlated with dosage, duration and symptom severity among treated patients. These findings provide novel insights into the influences of antipsychotic medications on GM abnormalities over the long-term course of illness.34, which showed decreases predominantly in the middle and inferior frontal, superior and middle temporal, and anterior cingulate cortex in both treated and untreated patients. This suggests that specific brain regions involved in schizophrenia may be affected in the same way by short- and long-term antipsychotic treatment. In addition, we also found increased GM in the left cuneus in both never-treated and treated chronic patients, which agreed with the previous study of cortical thickness increases in antipsychotic-naïve, first-episode schizophrenia9. This might represent a compensatory response related to increased activation in the cuneus cortex to support visual processing in patients with schizophrenia36. Taken together, the reductions in the bilateral superior temporal lobe and rostral middle frontal lobe cortex and increase in the left cuneus cortex were found both in untreated and treated chronic and first-episode schizophrenia patients. Therefore, these common alterations in brain regions both in chronic and first-episode schizophrenia patients may be relatively stable throughout the course of illness.One of the main findings of the current study was the reductions mainly involving bilateral medial and rostral middle frontal, left banks superior temporal sulcus, and left fusiform cortex and increases in the left cuneus in both long-term treated and untreated chronic patients compared to healthy controls. These findings were consistent with previous studies of first-episode schizophrenia before and after short-term treatment38, i.e., mainly reductions in the prefrontal and temporal lobes. Furthermore, the prefrontal and temporal cortices have been reported to be associated with the treatment dosage during short-term treatment38, are within the dopamine pathway4 and show dopaminergic dysregulation41 in schizophrenia. Therefore, our study further demonstrates that the prefrontal and temporal cortices may be not only short-term treatment targets but also targets for long-term treatment.By comparing the never-treated with treated chronic schizophrenia patients, it was remarkable that although the whole brain was examined, the reductions were mainly in the bilateral prefrontal and temporal cortex, and left inferior parietal lobule in the treated patients. These findings were partly consistent with previous brain structural studies of short-term treatment in schizophrenia patients42. In contrast, some schizophrenia studies with long-term treatment45 have also shown parietal lobe decreases in treated patients45 and in animal models44. It is possible that there are differences between short- and long-term antipsychotic treatment effects on the inferior parietal lobule. Therefore, the parietal lobe might be the key target for long-term antipsychotic treatment, which is considered the key area regarding several cognitive and motor functions and tasks involving the integration of different stimuli47. Furthermore, the inferior parietal lobule has been shown to be involved in various neuropsychological functions affected by schizophrenia and supports the frontal lobe in storing and retrieving verbal information46. The reduction in the inferior parietal lobule cortex may be characteristic of schizophrenia patients with long-term treatment, and it could also be a specific target for long-term treatment. Therefore, our findings not only provide evidence for similar brain alterations with short-term and long-term treatment but also the specific alterations of long-term treatment for schizophrenia patients.Another interesting finding was that the inferior parietal lobule decreased in chronic patients after long-term treatment, which has rarely been reported in previous studies with short-term treatment17 and functional networks48 than treated patients, our findings revealed more regions with reduced GM in treated patients than untreated patients. One of the possible explanations is that the mechanism of antipsychotics may be associated with GM reductions and functional performance improvements. Neuroinflammation is one of the pathophysiological mechanisms of schizophrenia49, including an increase in microglial activation50 and extracellular volume51 in GM and white matter. Antipsychotic treatment could promote decreases in activated glia and extracellular volume and improve neuronal function, which has been associated with an anti-inflammatory effect52. The different principles underlying brain structural and functional MRI may result in different findings. The second possible explanation is that it is mainly gray matter loss that progresses over time, whereas white matter deficits are stable or may even improve over the course of the illness49. GM loss progresses further after the onset of schizophrenia53, and antipsychotics may not counteract the pathophysiologic processes leading to the GM loss underlying schizophrenia21. Taken together, these findings suggest the possibility of different effects of treatment on GM and white matter, which may be the focus of significant future work.It should be noted that unlike our previous findings that long-term untreated patients showed more severe deficits in the white matter54; for example, cortical thinning of the pars triangularis was found in first-episode schizophrenia patients with 2 years of clozapine treatment, and no significant differences were found in patients with risperidone treatment. However, our study suggests that the CT-SCZ group had more severe reductions mainly in the bilateral prefrontal cortex and left cuneus and fewer decreases in the left ventral temporal lobe than the RT-SCZ group. One possible interpretation is that the effects of antipsychotic treatment may be cumulative over the years of the illness course or more pronounced later in the course of the illness17. Although it is nearly impossible to have chronically treated patients with monotherapy during the whole illness, we recruited stably treated patients with monotherapy for at least the most recent 5 years, which could represent the long-term treatment effect on the brain. Therefore, our findings may suggest a specific and different effect of long-term risperidone and clozapine treatment on this pathway.Another contribution of the current study is that the patients with long-term clozapine monotherapy displayed more severe and extensive GM loss than the patients with risperidone monotherapy. These findings were similar to those of a previous study56. Therefore, the findings indicate that cumulative antipsychotic exposure, illness duration, and symptom severity may be potentially contributing factors influencing the progression of GM deficits in chronic schizophrenia patients.The current study also revealed that GM reductions were negatively correlated with dosage, duration and symptom severity among the treated patients, involving the inferior and middle temporal lobes, inferior parietal lobe, and fusiform cortex regions. This finding suggests that widespread cortical deficits occurring in the later stages of schizophrenia may be crucial to the pathogenesis of the disease. The anatomical deficits in the inferior parietal lobe may represent the core pathology during the later course of schizophrenia. Previous studies have shown that the above cortical regions were associated with symptoms of schizophrenia59 explored the impact of sex differences on the brain features of schizophrenia, but the results are inconsistent, even a study58 reported no significant differential sex effects in schizophrenia for either GM cortical thickness or subcortical volume development. In this study, we took age and sex as covariates by ANCOVA analysis to control for potential confounding effects and found that sex has no interactive effect on the results. Therefore, the results of the differences between groups may result from long-term antipsychotic treatment, rather than sex differences. Although all patients were well matched on variables such as age, duration, age at onset, and dosage, these issues need to be considered when interpreting our findings. Third, there were no significant differences in subcortical volumes after multiple comparisons. Subcortical structures may not have been accurately segmented60 and may not significantly change as a whole. Therefore, it is necessary to adjust the segmentation and perform further subarea analysis. Lastly, to clarify mechanisms of these effects, animal model research will be needed to examine longitudinal long-term differential antipsychotic effects on GM to experimentally confirm a relationship between antipsychotics and GM.Several limitations should be considered in interpreting our findings. First, the GM differences among the three patient groups and their relationships with treatment history are constrained by the lack of random assignment to long-term treatment versus no treatment conditions. However, such a study would not be feasible or ethically justified. Therefore, our cross-sectional approach may be the only feasible strategy for addressing questions about the long-term course of schizophrenia and antipsychotic effects on brain structure. Second, we note that the sex ratio is significantly different among the 4 groups. Previous studiesIn conclusion, our findings provide new insights into the understanding of the long-term treatment of chronic schizophrenia and the different brain structural manifestations after different medications. These findings of long-term treatment were similar to those in short-term studies in treated schizophrenia, mainly involving the temporal lobe and prefrontal lobe. Both treated and never-treated chronic schizophrenia patients showed cortical gray matter reductions and increases in similar brain regions, and the reductions in the inferior parietal lobule may be characteristic of schizophrenia patients with long-term treatment. Our study also suggests that patients with long-term clozapine monotherapy displayed more severe and extensive GM loss in the bilateral prefrontal and left cuneus cortex than patients with risperidone monotherapy. These findings deepen our understanding of the effects of long-term treatment and may provide evidence for the development of new medications or potential treatment targets for long-term treatment in schizophrenia patients.Supplementary table S1


Acanthurus nigrofuscus. The spatial component of our analyses revealed that 1) A. nigrofuscus were phenotypically similar between marine reserves and fished reefs during their early life history; 2) marine reserve and fished reef populations diverged into significantly different length-at-age morphs between ages three and six, in which protected fish were predominantly larger than conspecifics in fished reefs; and 3) A. nigrofuscus returned to a state of general phenotypic similarity during later life. The temporal component of our analyses revealed that younger generations of A. nigrofuscus exhibited significant, positive year effects that were maintained until age eight, indicating that, within the significant age cohorts, younger generations were significantly larger than older generations.Quantitative assessments of the capacity of marine reserves to restore historical fish body-size distributions require extensive repeated sampling to map the phenotypic responses of target populations to protection. However, the “no take” status of marine reserves oftentimes precludes repeated sampling within their borders and, as a result, our current understanding of the capacity of marine reserves to restore historical body-size distributions remains almost entirely reliant on independent, static visual surveys. To overcome this challenge, we promote the application of a traditional fisheries tool known as a “back-calculation”, which allows for the estimation of fish body lengths from otolith annuli distances. This practical application was pursued in this study, using data collected in five marine reserves and adjacent fished reefs in the Philippines, to investigate spatiotemporal disparities in length-at-age of the brown surgeonfish,  Reductions in length-at-age are well known to diminish population fitness and the sustainability of fisheries stocks through consequential effects on reproduction and survival –5. In moA back-calculation allows for the estimation of fish body lengths from sagittal otolith (ear stone) annuli distances, thereby providing a method by which length-at-age data from earlier time periods in the life history of a fish can be obtained from a single sampling effort . TraditiAcanthurus nigrofuscus , was used as a representative species of the Acanthuridae family that has critical anthropogenic and ecological importance in the Coral Triangle region. Investigations estimate that near-shore fisheries, of which acanthurids comprise a major component, sustain the primary source of dietary protein for approximately one billion people in Asia mapview [ [sp [sf , ggplot2[ggplot2 , ggmap [2 [ggmap , ggsn [4ap [ggsn , ggspatigspatial , patchwoatchwork , rnaturaralearth  and rnatrthhires  packagesApprovals from the Department of Agriculture of the Philippines, the Bureau of Fisheries and Aquatic Resources of the Philippines and the local governmental units of Candelaria, Masinloc and Santa Cruz were obtained. All vertebrate work was independently reviewed and approved by the Institutional Animal Care and Use Committees of the Florida Institute of Technology and the University of the Philippines. Sampling procedures were additionally reviewed and approved as part of obtaining the Philippines, Department of Agriculture Gratuitous Permit GP-0096-15 issued on June 01, 2015.Sampling was performed via scuba with spearfishing equipment. Following collection, specimens were euthanized via ice immersion and transported back to a laboratory where standard length (mm) measurements were taken. Sagittal otoliths were removed and prepared for ageing using the protocol described in Fidler et al. . Ageing L) of a fish at age (i) was calculated as:Lcpt is the length of a fish at capture, Rcpt is the radius of a fish’s otolith at capture, a is the biological intercept between Lcpt and Rcpt, L0P is the length of a fish at increment formation, Ri is the otolith radius at age i, and R0P is the mean radius of the first annulus in the otolith.Various independent validation assessments have determined that the modified fry back-calculation (MFBC) model used in this study operates as an efficient and conservative model for the back-calculation of body lengths from annuli distances , 48. In FSA [openxlsx [ImageJ1  was usedFSA  and openopenxlsx . The totpwr [car [userfriendlyscience [ggplot2 [Similarities and disparities in length-at-age were statistically analyzed via two-way analyses of variance (ANOVAs), which allowed for examination of (1) age-specific differences between MR and FR populations (referred to as “status effects”), as well as (2) age-specific differences between year classes (referred to as “year effects”). A posteriori power analysis was additionally performed to determine the statistical power of our spatial analyses (status effects). Results are presented as locally estimated scatterplot smoothing regressions (LOESS: a robust non-parametric regression method) with 95% confidence intervals. LOESS span values, which determine the degree of regression smoothing, were standardized at 0.95 to focus and emphasize visualization of the large-scale phenotypic shifts examined in this study. All statistics and figures were computed using R v. 3.5.0. . Packagepwr , car [54yscience  and ggpl[ggplot2 .Significant disparities in the body lengths of MR and FR populations were found for five age cohorts (two-way ANOVA). No significant status effect was found at ages one and two  although an effect appeared at ages three, four, five and six .It is conceivable that the observed pattern of early phenotypic similarity followed by divergence and late convergence between MR and FR sites reflects the complexity of the main and interactive effects of factors that underlie the impacts that MRs have on the biology and ecology of exploited fish populations on coral reefs. These statistical results must be interpreted with caution given that a posteriori power analysis, used to determine the ability of our statistical analyses to detect significant status effects, identified low statistical power. This indicates reduced ability to determine whether a lack of significant status effect at any given age is, in fact, true or a result of low statistical power, which could stem from small sample sizes, high variance, and/or the given effect size.A. nigrofuscus is a broadcast spawner [Notwithstanding, phenotypic homogeneity during the early life-history stage may reflect larval connectivity, a fitting hypothesis given that  spawner  and that spawner , 57–59. A. nigrofuscus, whose characteristically fast growth rates place them well within the target range of fishers from an early age [Exposure to differing size-selective pressures (e.g. size-selective fishing) between MRs and FRs suggests that individuals inhabiting protected areas are likely to experience heightened survival probabilities while similar sized individuals inhabiting FRs are more likely to experience heightened mortality probabilities. This dichotomy is emphasized for arly age , 61. WhiA. nigrofuscus exhibit relatively large home ranges that peak at an approximate linear distance of three kilometers. The home ranges of a broad suite of coral-resident fishes, however, have been shown to correlate positively with body size [In a meta-analytical investigation of the home ranges and movement patterns of coral reef fishes, Green et al.  determinody size , suggestody size  might onA. nigrofuscus.In addition to the status effects presented above, significant year effects were observed between ages one and eight, all of which were positive and indicate that younger generations were significantly larger than older generations. If MR populations were exporting individuals with favored life histories, which is highly plausible given the observed spatial results and life history theory of teleosts, then both protected and fished populations could be expected to exhibit gradual enlargements in length-at-age over time as larger size confers greater reproductive output and offspring viability , 7. HoweA. nigrofuscus to protection. A broad range of biological and environmental variables have been determined to influence MR performance, illustrated by the variable success of MRs worldwide. As a result, further spatiotemporal analyses across taxa that experience different life histories and across sub-regions that experience different degrees of exploitation are required to determine the ubiquity of these findings. Continued studies confirming the positive conservation and fishery benefits of MRs will remain critical in garnering support for the establishment of MRs globally in the coming decades.In conclusion, the longitudinal data procured via the application of the MFBC model provided critical insight into the regional phenotypic responses of S1 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S2 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S3 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S4 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S5 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S6 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S7 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S8 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S9 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S10 FigTwo-way ANOVA results are shown in the lower left, with significant disparities between year classes (year effects) indicated via year numbers above the regressions.(TIF)Click here for additional data file.S1 Data(CSV)Click here for additional data file.


Acute myocarditis represents a challenging diagnosis as there is no pathognomonic clinical presentation. It is rare to see heart block as the first-and-only presentation of infectious myocarditis. We report the case of a young healthy patient who presented with syncope secondary to a complete heart block. It was caused by acute presumed viral myocarditis. The diagnosis was confirmed with cardiac magnetic resonance imaging. With close monitoring, the EKG abnormalities resolved over the following 5 days. In this case report, we present the importance of several imaging tools to diagnose a rare and reversible cause of conduction disturbances. In at-risk individuals, clinicians should rule out a treatable cause of heart block before proceeding with permanent pacemaker implantation due to enormous clinical and cost implications involved. Acute myocarditis represents a challenging diagnosis as there is no pathognomonic clinical presentation . It rang3 with 48% lymphocytes, and elevated C-reactive protein at 90mg/L. Serum electrolytes, kidney and liver functions were within normal limits. A chest X-ray showed an appropriate cardiac size. The initial transthoracic echocardiography (TTE) showed a normal global left ventricular function (left ventricular ejection fraction (LVEF) 60%) and wall motion abnormalities , with reduced left ventricular global longitudinal strain (LV GLS= -14.1%)  showed grade III atrioventricular (AV) bloc with a ventricular rate of 38 beats per minute . Laborat -14.1%) .Cardiac magnetic resonance (CMR) showed increased signal intensity at the mid-lateral wall on T2-weighted images. Late enhancement revealed a patchy subepicardial hyperenhancement of the medio-distal segments of the anterior and anterolateral walls which confirm the diagnosis of myocarditis . The patAcute myocarditis represents a challenging diagnosis as there is no pathognomonic clinical presentation . The truCommon causes of acquired AV block that need to be ruled out when considering a diagnosis of myocarditis in young patient include ischemic heart disease and acute myocardial infarction, cardiomyopathies including hypertrophic obstructive cardiomyopathy, infiltrative myocardial processes , hyperkalemia, collagen vascular disease , increased vagal tone , thyroid disease, and drugs  . Most ofHigh-degree AV blocks have been related to higher morbidity and mortality in all patients with myocarditis . The patEchocardiography is useful as an initial diagnostic tool to detect the presence and severity of regional or global ventricular dysfunction and chamber dilation in children with acute viral myocarditis. Speckle tracking echocardiography is a quantitative, objective technique for intrinsic cardiac deformation in real time, providing accurate assessment of abnormal regional wall contractility as in myocarditis. Several studies have shown the superiority of strain imaging over the conventional methods used for assessment of regional myocardial dysfunction . CMR imaComplete heart block is usually an ominous finding that might require placement of a permanent pacemaker. Infectious myocarditis is a reversible cause of cardiac conduction abnormalities that should definitely be considered in the differential diagnosis of heart block before resorting to permanent pacemaker implantation, especially in young healthy patients. A suggestive history and physical exam, coupled with abnormal echocardiographic findings and positive CMR helps in the diagnosis.


The aim of this review is to outline emerging biomarkers that can serve as diagnostic tools to identify non-cirrhotic chronic hepatitis B (CHB) patients who could safely discontinue nucleos(t)ide analogues (NAs) before HBsAg loss. Regarding possible predictors of post-NAs outcomes, a number of studies have evaluated numerous factors, which can be categorised in markers of hepatitis B virus (HBV) activity, markers of host immune response and markers of other patient characteristics. In clinical practice, the most important question for patients who discontinue NAs is to differentiate those who will benefit by achieving HBsAg loss or at least by remaining in remission and those who will relapse requiring retreatment. Most of the discontinuation studies so far came from Asian and only few from European populations and examined the rates and predictors of post-NA virological and/or combined relapses or HBsAg loss. To date, there is still controversy about predictors of post-NA relapses, while only HBsAg serum levels at NA discontinuation seem to be the most robust predictive marker of the probability of subsequent off-treatment HBsAg seroclearance. Newer viral markers such as HBV RNA and hepatitis B core-related antigen seem promising, but further research is required. Chronic hepatitis B (CHB) remains a major health problem worldwide  and, desHowever, the latest recommendations from European, US and Asian liver associations  suggest that NAs cessation may be considered in selected CHB patients before HBsAg loss ,4,5,6. RCHB patients with compensated cirrhosis are considered a patient subgroup at high risk for liver decompensation and hepatic failure in case of HBV flares following NAs cessation. Thus, NA discontinuation may be considered in patients with cirrhosis who will remain under strict surveillance only in the Asian recommendations and mainly for countries with very limited resources , but notEven for the non-cirrhotic CHB patients in whom NA discontinuation is considered safe under the aforementioned conditions , clinicaThis review aims to summarize such emerging biomarkers that could serve as useful diagnostic tools to safely guide non-cirrhotic CHB patients’ selection for NAs discontinuation before HBsAg loss.The most useful and well known biomarkers that guide clinical decisions regarding the management of CHB patients are the markers of HBV activity. To begin with, HBeAg is used to define the stages of chronic HBV infection but it also contributes in defining an endpoint of NAs discontinuation ,5. Therep = 0.064) [p = 0.01) [Still after NA cessation, these patient subgroups seem to have distinct outcomes. The results from a systematic review showed that the pooled durable rates of virological remission at 12 and 24 months after NA discontinuation were 63% and 53% in pretreatment HBeAg-positive and 44% and 31% in HBeAg-negative CHB patients (= 0.064) . On the  = 0.01) . Given t = 0.01) . TherefoThe second most important and valuable viral marker is HBsAg. To date, HBsAg quantification (qHBsAg) in serum is performed on widely available instruments used for routine serology with an automated method which is considered highly reproducible ,41.HBeAg and HBsAg represent the main tools which define the subgroups of patients who could stop NAs. First, it is quite clearly stated in all currently available international guidelines ,4,5 thatRegarding HBsAg-positive patients’ outcomes after NAs discontinuation, serum HBsAg levels are a tool of great importance . A numbePretreatment qHBsAg levels have not been associated with patient outcomes in any ,30,33,44Serum viral load, which is measured via serum HBV DNA quantification, has been assessed in various settings as a tool for treatment decisions in CHB patients, but in respect to patient management after NAs discontinuation, only few studies showed that there is association between higher pretreatment HBV DNA levels and post-NAs relapses ,29,30,32One study showed that delayed suppression of HBV DNA during treatment was an independent predictor of post-NAs relapse , but thiSimilarly, low clinical value seems to be offered by the identification of HBV genotype in patients who stop NAs, since there is no proven association between any specific genotype and post-NA outcomes in almost any study so far. Only, in the initially HBeAg-positive patients of the cohort of Chen et al. genotype C was associated with increased risk of virological and/or clinical relapses . Howeverp < 0.001) [Interestingly, very recent data from two cohorts highlighted the importance of serum titers of antibody against hepatitis B core antigen (anti-HBc) as a predictive marker for clinical relapses. Tseng et al.  showed t< 0.001) . ApparenEmerging viral markers, such as hepatitis B core-related Antigen (HBcrAg) and serum HBV RNA levels have drawn researchers’ attention lately. Particularly, HBcrAg was reported to be strongly correlated with intrahepatic HBV cccDNA and was considered as a tool that could be used to monitor disease progression  as well Serum HBV RNA levels, which seem to correlate with intrahepatic HBV cccDNA transcriptional activity , have beMoving one step further from the viral markers, studying the mechanisms of host immune activity in patients who discontinue NAs seems to be of great value, although immune biomarkers might be used for research purposes only for the time being.To this end, a small yet very interesting study of Kranidioti et al. found a distinct immune signature in HBeAg-negative CHB patients who remained in off-treatment remission for 3 years after NAs cessation and suggested that RNA levels of interferon gamma (IFNg), interleukin 8 (IL-8), Fas Ligand (FASLG) and carbon tetrachloride (CCL4) from peripheral blood mononuclear cells (PBMCs) might be used as potential biomarkers in this setting .The study of Rivino et al. also supported that a distinct subpopulation of HBV-specific cells might serve as a biomarker for NAs discontinuation, since they showed that post-NAs remission correlated with the presence of HBV core and polymerase-specific T cells that were contained within the ex vivo programmed death 1 (PD-1) population during NAs viral suppression. According to the authors, this also underlines the value of T-cell exhaustion markers that should be further evaluated in this setting .On the other side, distinct T-cell phenotype was detected in CHB patients under long-term NA therapy and was only slightly altered after treatment discontinuation, according to Rinker et al. . In thatThe same team suggested that other cell types might also be involved in the immune response after NAs discontinuation in CHB patients. Special natural killer (NK) cell compartment has been revealed by unsupervised stochastic neighbour embedding analysis; increased activity of NK cells correlated with post-NAs biochemical flares and was particularly enhanced in patients experiencing subsequent HBsAg loss .Furthermore, various plasma cytokines and growth factors have been assessed as biomarkers indicating the activation of immune responses. A small prospective study that included 15 HBeAg-negative CHB patients showed that off treatment virological flares were associated with induction of plasma tumour necrosis factor α (TNF-α), interleukin 10 (IL-10), interleukin 12p70 (IL12p70) and interferon inducible protein 10 (IP10). These markers significantly increased in the first month after NAs discontinuation, while IP10 peaked at month 2 . We alsoDriven by the concept that off-treatment immunological response may lead to immune control and functional cure, some studies have supported that patients who suffered virological rebounds and/or biochemical flares and were not retreated had higher probability of subsequent HBsAg loss . In thisThere are suggestions that some basic patient characteristics may serve as markers for the physician to select the CHB patients who would be eligible for safe treatment discontinuation, even before they start the long-term NA therapy. However, there is no concordance among the various studies’ results  and therThere are five studies suggesting that older age is associated with post-NAs virological relapses ,17,18,29In one study , male seAmong the liver function tests, levels of alanine aminotransferase (ALT) before treatment were the most commonly examined marker as possible predictor of post-NA relapses or HBsAg loss . NeverthIn addition, there are interesting recent findings from few studies suggesting that the choice of NA might affect the risk of relapse after treatment discontinuation. A study from Germany  and two Finally, even if cirrhotic patients are excluded from the “stopping-NAs” strategy, since this patient subgroup is more likely to face an imminent peril with uncontrolled HBV flares after treatment discontinuation ,7, the sLong-term, perhaps indefinite, NA therapy is usually recommended for CHB patients, particularly for HBeAg-negative cases treated in Western countries. Thus, discontinuation of NAs in patients with HBeAg-positive and especially HBeAg-negative CHB who remain HBsAg positive is still considered a controversial approach by many, at least Western, experts, although it has been included in the Asian guidelines for >7 years  and in tIn clinical practice, the most important decision for patients who discontinue NAs is to distinguish between those who will have a benefit by achieving HBsAg loss or at least remaining in remission and those who will relapse and require retreatment. Although the first relevant studies were published almost a decade ago, post-NAs remission has been used as an endpoint in only two old studies ,43, whilRegarding HBsAg loss, there has been only one reliable predictor, EOT qHBsAg levels, which were significantly associated with the probability of subsequent HBsAg clearance in all nine relevant studies. The most commonly proposed HBsAg levels cut-off for optimal predictability was 100 IU/mL, as shown in In the 24 studies evaluating predictors of post-NAs virological relapses , the mosSince the majority of CHB patients who discontinue NAs develop virological relapses and a proportion of them experience biochemical relapses and occasionally ALT flares, it is reassuring that prompt retreatment can reintroduce biochemical and virological remission . At the In conclusion, the identification of factors that can reliably predict the outcomes of CHB patients who discontinue NAs is quite important. Unfortunately, although there have been many studies trying to identify predictors of post-NA relapses, accurate predictors have not been reliably identified so far. The probability of HBsAg loss after NA discontinuation is high in patients with qHBsAg levels ≤ 100 IU/mL and much lower in patients who have qHBsAg levels > 100 and particularly > 1000 IU/mL, but large proportions of these patients are not going to have biochemical relapses and will not require retreatment. Newer markers like serum HBcrAg and HBV RNA levels are currently under investigation as predictors of post-NAs outcomes and seem to be the most promising ones for optimizing prediction in this setting. However, the outcome of patients who discontinue NAs cannot be reliably predicted today.


We examined the incidence of emotional distress in women with newly diagnosed breast cancer to determine whether the degree of emotional distress affected their choice of breast-conserving surgery (BCS) or mastectomy and evaluated how the patient's preferred role in decision-making influenced her choice of surgical method.  This prospective study included 85 patients newly diagnosed with in situ or invasive breast cancer eligible for BCS. Their degree of depression/anxiety and attitude toward the decision-making process were measured using the Hospital Anxiety and Depression Scale (HADS) and Control Preference Scale (CPS), respectively. After receiving information on both surgical methods, the patients indicated their preferred surgical method and completed the CPS at their initial and second visits before surgery. p < 0.001). However, the presence of depression or anxiety was not associated with patients' surgical choices . Patients who preferred a more active role in the decision-making process were more likely to choose mastectomy over BCS, while those who were passive or collaborative chose BCS more frequently (p=0.001).  After the diagnosis of breast cancer, 75.3% of patients showed abnormal or borderline HADS scores for depression and 41.2% for anxiety. Patients with borderline or abnormal degrees of depression were more likely to have coexisting abnormal degrees of anxiety ( Although many patients with newly diagnosed breast cancer experience depression and anxiety before surgery, these do not affect the choice of surgical method; however, their attitudes toward the decision-making process do. Breast-conserving surgery (BCS) is a well-established treatment option for early breast cancer patients with a comparable survival to that of mastectomy , 2. ReceThe decision-making process regarding the type of breast cancer surgery is affected by a wide array of factors, including the clinicopathological factors of the tumor, attitudes of the individual patients, and advice from the treating surgeon. According to the recent systematic review by Gu et al. , patientThe decision for breast conservation or mastectomy is often further complicated by the emotional stress that newly diagnosed breast cancer patients undergo. Being diagnosed with breast cancer is experienced as a major challenge and constitutes an important stressor in a women's life that may cause emotional distress, particularly anxiety and depression , whose pIn the present prospective trial, we investigated the incidence of emotional distress in Korean women with newly diagnosed breast malignancies and assessed their attitude toward the treatment decision-making process during the discussion of different types of breast cancer surgery to determine how their degree of emotional distress and attitude toward the decision-making process affected the final choice of surgery type.https://cris.nih.go.kr) with the identification number KCT0004736. All participants provided written informed consent.Between August and December 2018, we invited a total of 107 patients to participate in our study. The inclusion criteria were patients newly diagnosed with invasive breast carcinoma or in situ carcinoma, older than 20 years of age, not candidates for neoadjuvant systemic therapy, and eligible for breast conservation surgery based on their clinical tumor size at initial presentation. This prospective study was approved by the Institutional Review Board (IRB no. 1807-054-957) and was conducted according to the Declaration of Helsinki. This trial was registered on the CRIS website  and Anxiety subscale (HADS-A) of the HADS questionnaire. Patients were classified into three groups based on their subscale scores  . InformaA total of 85 patients diagnosed with ductal carcinoma in situ or invasive breast cancer who were eligible for breast conservation between August 8 and December 31, 2018, participated in this study. The baseline clinical information is listed in n = 50, 58.8%) or borderline  scores for depression, while only 21 patients (24.7%) displayed normal ranges. Regarding anxiety, 13 (15.3%) and 22 patients (25.9%) showed abnormal and borderline HADS scores, respectively. Patients with borderline or abnormal degrees of depression after the diagnosis of breast malignancy were more likely to have coexisting abnormal degrees of anxiety (p < 0.001). Although most demographic or clinical features were not associated with emotional disturbance (p=0.031).The degree of depression and anxiety was measured using the HADS questionnaire  on theirturbance , patientp=0.394 and 0.530, respectively; Next, we investigated whether the levels of depression or anxiety in these newly diagnosed patients affected their choice of type of breast surgery. After providing information on both mastectomy and breast conservation using decision aids, each patient's preference for surgical method was recorded on the initial and second visit, when the surgical methods were discussed again and finalized. Seventeen patients (20%) had difficulty in deciding their surgical choice at the first visit, which decreased to six (7.1%) at the second visit. The presence of borderline or abnormal degrees of depression or anxiety was not significantly associated with the patients' surgical choices . Patients were asked about their preferred role in the decision-making process using the modified CPS, as suggested by Chiu et al. , before p=0.001, Furthermore, we observed that the patients' preferred role in the decision-making process was significantly associated with their choice of breast surgery. Patients who preferred a more active role in the decision-making process were more likely to choose mastectomy over breast conservation, while patients who were either passive or collaborative chose breast conservation more frequently p=0.001, . The mosBeing diagnosed with breast cancer constitutes a major challenge and an important stressor in a woman's life that may cause various emotional distresses, particularly anxiety and depression . Causes Studies have thoroughly investigated whether the breast surgery type significantly influences patients' emotional status, and although some reported a modest advantage for breast conservation, most found that mastectomy and breast conservation have similar effects on emotional outcomes in breast cancer patients –28. In tInterestingly, we observed that patients' attitudes in the decision-making process were significantly associated with their choice of mastectomy in the present study. Patients in the “active” decision-making group (more patient-controlled) were more likely to choose mastectomy than other groups. Hawley et al.  also repOur study has several limitations. First, the decision-making process of breast cancer patients is a complex process affected by multiple factors. Molenaar et al.  reportedIn conclusion, this prospective trial demonstrates that there is a high incidence of emotional distress in newly diagnosed Korean breast cancer patients. Emotional distress did not influence the decision-making process on breast surgery type; however, their attitude toward the decision-making process showed a significant association with their preferred surgery type. Patients with more active involvement in the decision-making process were more likely to undergo mastectomy over breast conservation.


The ongoing revolution in laparoscopic liver surgery has delivered scientific evidence of improved short-term outcomes and uncompromised long-term oncologic results for patients with resectable malignant liver disease, including cholangiocarcinoma (CCA) cases.2In the article by Sucher et al., we share our strategy of a total laparoscopic approach for pCCA type 3b resection. The hepaticojejunostomy, in our opinion the most challenging part of the procedure, which is still commonly performed through a larger service incision at the end of a minimally invasive liver resection, even by highly decorated hepato-pancreato-biliary (HPB) surgical teams, was furthermore intended to be performed using a minimally invasive approach. To accomplish our goal, we successfully applied a ‘parachute’ running suture technique for biliary-enteric reconstruction, which facilitated an improved view on both components of the anastomosis, the posterior wall of the hepatic duct, and the corresponding jejunal segment. Additionally, short biliary drains were placed, aiming to splint the anastomosis and hence prevent cholestasis in the early postoperative period. Using the laparoscopic approach for pCCA surgery, hepatobiliary surgeons are urged not to compromise on radical lymphadenectomy, which has recently been shown to be equally effective and accurate when compared with open surgery.3Every new technology that brings advantages also comes with inconveniences. In the case of laparoscopic pCCA surgery, a standardized procedure for intraoperative bile leakage testing is still in demand. Enhanced intraoperative visualization techniques such as near infrared indocyanine green staining methods


Metamaterials provide compelling capabilities to manipulate electromagnetic waves beyond the natural materials and can dramatically enhance both their electric and magnetic fields. The enhanced magnetic fields, however, are far less utilized than the electric counterparts, despite their great potential in spintronics. In this work, we propose and experimentally demonstrate a hybrid perfect metamaterial absorbers which combine the artificial metal/insulator/metal (MIM) metamaterial with the natural ferromagnetic material permalloy (Py) and realize remarkably larger spin rectification effect. Magnetic hot spot of the MIM metamaterial improves considerably electromagnetic coupling with spins in the embedded Py stripes. With the whole hybridized structure being optimized based on coupled-mode theory, perfect absorption condition is approached and an approximately 190-fold enhancement of spin-rectifying photovoltage is experimentally demonstrated at the ferromagnetic resonance at 7.1 GHz. Our work provides an innovative solution to harvest microwave energy for spintronic applications, and opens the door to hybridized magnetism from artificial and natural magnetic materials for emergent applications such as efficient optospintronics, magnonic metamaterials and wireless energy transfer. A typical example of optimized light-matter interaction is perfect absorption of electromagnetic wave with unity energy absorption efficiency and hence facilitating numerous high performance functional devices including plasmonic sensor13, filter15, bolometer17, electromagnetic wave cloaking19, multi-band/broad-band perfect absorber24, photodetectors27 and solar cells28. In most of reported perfect metamaterial absorbers (PMAs) so far, however, the enhanced absorption is mainly based on enhanced electric field components and the electric response of the constitution materials including metal ohmic loss and/or dielectric absorption. The metal ohmic loss eventually leads to heat generation which can thereby be utilized in hot carrier related solar-thermal and chemical processes29, while the improved dielectric absorption can be implemented in optoelectronic applications such as photodetectors, lasers30 and photovoltaic solar cells31. Nevertheless, the counterpart magnetic field components are far less addressed and utilized32, despite urgent demands in spintronics due to intrinsically weak light-spin interaction strength35.Metamaterials offer a great avenue to control the absorption, reflection and transmission of the electromagnetic wave over a wide spectrum range from microwave to visible light39. The high frequent spintronic applications therefore require tremendous efforts to overcome this limitation. As an example of high frequent spintronic devices, spin dynamo40 has aroused considerable interest due to its unique ability to transform microwave electromagnetic energy to direct current (DC)41 and therefore provides a potential solution to realize distributed power system for future nanoelectronics. The reported works43, however, relied on coaxial cable connection with mandatory transmission line design and demonstrated rather low conversion efficiencies from microwave power to DC power. Therefore, enhanced light-spin interaction is highly demanded to boost the microwave excitation efficiency of spins dynamics and even enable wireless version for real application. As mentioned above, designing artificial metamaterials specifically for spintronic device may be a good choice to satisfy this demand.In the field of spintronics, manipulation of spin degree of freedom offers tremendous opportunities for future information processing and nonvolatile information storage. The energy utilization of spintronic devices, however, is severely restricted by weak interaction between electromagnetic waves with spins in natural magnetic materials46, and dramatically increase their energy conversion efficiency. More functional spintronic devices can therefore be triggered by the hybrid magnetism from artificial and natural magnetic materials, as demonstrated here.In this work, we propose a hybrid perfect metamaterial absorber (HPMA) embedded with ferromagnetic permalloy (Py) to enhance considerably light-spin interaction. The metal/insulator/metal (MIM) tri-layer structure of the HPMA harvests microwave energy and thereby enhances the magnetic fields to excite the spin precession in Py. By optimizing the parameters of the MIM structure to equalize the absorptive and radiative factors of the HPMA, a perfect absorption condition is approached and spin-rectifying DC photovoltage from the embedded Py is substantially enhanced. Without losing generality, our work can be extended to more spintronic applications such giant magnetoresistance (GMR), colossal magnetoresistance (CMR), spin pumping and spin-hall effectsThe HPMA consists of a top copper metal layer with split-ring resonator (SRR) array, a bottom continuous aluminum (Al) foil mirror layer and a sandwiched dielectric layer in which Py stripe is inserted.47, this symmetric SRR structure will function as a localized particle which construct a purely electrical resonant response48, as a result the microwave magnetic field can be confined around the central line of the SRR and converted to DC power through the under placed spin dynamo. The sandwiched dielectric layer is a glass or polyethylene terephthalate (PET) substrate with the thickness S on which the Py stripe (act as the spin dynamo) with the length 49. The radiative loss of this configuration can be controllable by changing S, thus realizing the perfect absorption conditions, on which the energy that should be dissipated into heat can be almost completely converted into electrical energy in spin dynamo.Figure mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimz-direction with its electric/magnetic-field polarization being along x/y-direction, as schematically shown in Fig. y-component enhancement of microwave magnetic field distribution . This static magnetic field breaks the symmetry of the two electrodes for each Py stripe such that anisotropic magnetoresistance (AMR) effect50 comes into play and the motion of spin-polarized charges becomes asymmetric along 50. Using the experiment setup as shown in Fig. j is the microwave current induced by the microwave electric field in Py stripe, and m is the non-equilibrium magnetization driven by the microwave h-field, and r| with different S of the HPMA are displayed in Fig. 35.Under the guidance of simulation, we experimentally placed Py stripes under the top SRRs structure with the distance of d and each stripe is aligned to the central arm of SRR, which enables adequately utilization for the magnetic field component of the HPMA resonant structures. The magnetic field induced by the resonantly oscillating current in the central arm of each SRR excites the spin precession in underneath Py stripe. Begin to measure the spin rectifying signal, we applied a static magnetic field in the entclass1pt{minimay-direction. Further more, the relative phase between the microwave magnetic field and the microwave current .All the samples in this experiment are fabricated by standard optical lithography and magnetron sputtering methods.x-direction). A standard microwave generator (Agilent E8257D) whose amplitude was modulated by a square wave with a period 120 x-direction. The photovoltage generated by Py spin dynamo was detected by a lock-in amplifier (Stanford SR830) with sweeping the external DC magnetic field in the range of To measure the spin rectification photovoltage, we applied a static magnetic field in the