Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) are a major public health concern with high and increasing global prevalence, and a significant disease burden owing to its progression to more severe forms of liver disease and the associated risk of cardiovascular disease. Treatment options, however, remain scarce, and a better understanding of the pathological and physiological processes involved could enable the development of new therapeutic strategies. One process implicated in the pathology of NAFLD and NASH is cellular oxygen sensing, coordinated largely by the hypoxia-inducible factor (HIF) family of transcription factors. Activation of HIFs has been demonstrated in patients and mouse models of NAFLD and NASH and studies of activation and inhibition of HIFs using pharmacological and genetic tools point toward important roles for these transcription factors in modulating central aspects of the disease. HIFs appear to act in several cell types in the liver to worsen steatosis, inflammation, and fibrosis, but may nevertheless improve insulin sensitivity. Moreover, in liver and other tissues, HIF activation alters mitochondrial respiratory function and metabolism, having an impact on energetic and redox homeostasis. This article aims to provide an overview of current understanding of the roles of HIFs in NAFLD, highlighting areas where further research is needed. Non-alcoholic fatty liver disease (NAFLD) is a progressive, widespread form of chronic liver disease with a large global burden. Worldwide, around 25% of the population have NAFLD and its prevalence is increasing . NAFLD iVegfa, encoding vascular endothelial growth factor, and genes encoding many glycolytic enzymes  for 8 weeks, though it remains unclear how this local hypoxia develops . The livRegulation of cellular metabolism is a major canonical function of HIFs. In order to maintain energy charge in hypoxia, HIFs increase the expression of genes encoding glycolytic enzymes such as lactate dehydrogenase , while rVhl-deficient mice, showed that HIF2α, but not HIF1α, is responsible for the suppression of FAO in these mice (Vhl-deficient mice had lower expression of peroxisome proliferator-activated receptor α (PPARα)-target genes, such as carnitine-palmitoyl transferase 1 (Cpt1) and acyl CoA oxidase (Acox), lowering fatty acid-supported oxidative phosphorylation  but not Hif1a deletion  , however, led to lower Lipin1 mediated PPARα/PGC1α pathway activation, which worsened steatosis relative to wild type mice , and this was normalised by HIF2α silencing (Fas and Scd1 relative to HFD fed mice not exposed to CIH (in vivo and in vitro models of hepatosteatosis (via Vhl disruption has been associated with decreased expression of lipogenic genes such as fatty acid synthase (Fas) (Vhl disruption (via genetic manipulation or hypoxia) can cause steatosis via inhibition of FAO and upregulation of lipid uptake, that liver hypoxia and HIF2α activation occur in NAFLD, and that HIF2α upregulates lipogenesis in diet-induced steatosis, which worsens lipid accumulation and can be prevented by treatment with HIF2α antagonists. However, whether HIF2α also impairs FAO in NAFLD remains unclear.Increased lipogenesis is an important feature of NAFLD in human patients –44. Agaiilencing . Similard to CIH . Conversteatosis . Thus, ise (Fas) , or, in sruption . These csruption . CD36 exsruption . Therefocd1, and CD36 in wild type  activation led to improved insulin tolerance and glucose handling ] and lipogenic  genes. Interestingly, unlike other models of liver specific HIF2α activation, Phd3 deletion was not associated with worsened steatosis. The authors observed that deletion of Phd1-3, which increased HIF2α stabilisation still further, did worsen steatosis, suggesting that lower level HIF2α activation may be predominantly beneficial via improved insulin sensitivity, while higher levels of stabilisation, as occurs with Phd1-3 and Vhl deletion  has a detrimental effect due to inhibition of FAO, leading to worsened steatosis.HIF2α also appears to be involved in hepatic insulin signalling, handling . HIF2α dsitivity . Again, via inhibition of FAO and upregulation of lipogenesis, though studies investigating the effect of Hif2a deletion in NAFLD on FAO are needed to confirm this. Meanwhile, low levels of HIF2α activation in metabolic diseases appear to have a beneficial effect on insulin sensitivity and glucose handling. Whether HIF1α activation is protective or harmful in the context of metabolic disease and hepatic steatosis remains less clear. There are conflicting results which may be the result of opposing roles in different cell types and tissues, although in hepatocytes specifically, HIF1α activation in obesity appears to improve insulin sensitivity and may be required to maintain FAO and prevent increased lipogenesis.Overall, significant evidence points toward a steatosis-promoting role for chronic HIF2α activation in liver, likely occurring 4 induced fibrosis  deletion  and hepatic stellate cells (HSCs)  see , and by SCs) see .in vitro and in vivo models. Isolated mouse hepatocytes exposed to hypoxia show increased expression of plasminogen activator-inhibitor 1 (PAI-1), and this is partially prevented by Hif1a deletion and completely prevented by Hif1b deletion, suggesting both HIF1α and HIF2α may be involved (Hif1a protects against collagen deposition and suppresses collagen crosslinking in the media of isolated hepatocytes exposed to hypoxia (Lox) expression, which requires Hif1a in vitro  and Acta2 (αSMA) mRNA. Collectively, these studies highlight that one role of HIF activation in liver fibrosis is the direct regulation of fibrogenic genes in hepatocytes and that this likely occurs in NAFLD. However, hepatocytes are not considered major sources of ECM deposition in vivo, and so it remains unclear how central this mechanism is to the pathology of NAFLD.HIF regulated expression of fibrogenic mediators in hepatocytes has been demonstrated in several relevant involved  -κB signalling. NF-κB is thought to be an important driver of fibrosis and inflammation in NAFLD  mRNA. Palmitic acid treatment also induced HIF1α in macrophages in vitro, and silencing of Hif1a suppressed the activation of NF-κB (via control of hepatocyte production of the cytokine histidine rich glycoprotein (HRGP)  . HRGP inn (HRGP) . CholineG2 cells . It therin vitro. This suggests HIF signalling in patients with NAFLD/NASH and OSA may induce or worsen inflammation, though more studies are needed to confirm this.HIF-signalling may also be involved in the link between OSA and NAFLD progression generally, and regarding inflammation in particular. Severity of nocturnal hypoxia in OSA correlates with NAFLD/NASH severity, including liver inflammation, independent of other risk factors in patients , and subThe evidence currently available highlights potential mechanisms by which HIF signalling may be involved in several key aspects of NAFLD, namely steatosis, inflammation and fibrosis. Further work is required to confirm many of these mechanisms and provide a more detailed understanding, and to determine whether targeting HIF signalling is a viable treatment strategy to improve these aspects of the pathology. It also remains uncertain what drives liver hypoxia and HIF activation in NAFLD in the first place.in vitro studies using ROS scavengers to investigate whether this prevents HIF activation in in vitro models of NAFLD. Investigation of SIRT4 in this context could also be valuable as reduced levels of this sirtuin have been demonstrated in patients with NAFLD  and other co-activated pathways. Further investigations into how closely linked HIF activation and OSA are in patients with NAFLD would be useful, and studies in animal models of NAFLD exposed to CIH—a way of mimicking OSA in rodents, which do not develop OSA spontaneously—could provide insight into whether and how these pathophysiological mechanisms differ.The ultimate goal of understanding the involvement of HIF signalling in NAFLD would be to attempt to treat the disease by targeting this pathway. Current evidence supports the use of animal studies to investigate this, and both HIF1α and HIF2α antagonists have been developed, largely with a view to treating cancers . Early sFinally, while this review has focussed on the role of HIF signalling in the liver, some studies point toward roles of HIFs in other tissues and organs that are likely to impact on NAFLD and outcomes in NAFLD. For example, HIF activation in adipocytes  and adipvia oxygen therapy or antagonism suggest that HIF2α drives lipogenesis. These mechanisms could explain the protective effect that Hif2α deletion has against steatosis in NAFLD. While some beneficial effects of HIF activation have been noted, such as a potential role in improving insulin sensitivity, on balance, HIF activation appears to be harmful in NAFLD, and may therefore be a useful therapeutic target. Further research is required to fully elucidate the mechanisms by which HIF activation contributes to NAFLD and NASH, in particular the effect on FAO, the signalling pathways involved in regulating the expression of lipogenic, fibrogenic, and pro-inflammatory genes, and the link between HIF signalling and OSA in NAFLD and NASH.In conclusion, considerable evidence points toward HIF activation occurring in NAFLD and NASH, and having widespread, predominantly harmful effects. Both HIF1α and HIF2α activation appear to worsen inflammation, though the mechanisms involved in this require further study. Further, evidence from studies of fibrosis shows important HIF mediated mechanisms, including control of profibrotic gene expression in hepatocytes and HSCs, regulation of HSC activation and HIF mediated pathological angiogenesis, though only control of profibrotic gene expression has been demonstrated to occur in animal models of NAFLD and NASH. Evidence also highlights a role for HIFs, in particular HIF2α, in driving steatosis. Studies of HIF activation under normoxic conditions suggest that HIF2α can inhibit FAO, while studies that interfere with HIF2α activation in NAFLD All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.LH was supported by a 4-year PhD studentship Program funded by the Wellcome Trust (Grant Number: 220033/Z/19/Z). AM was supported by the Research Councils UK (Grant Number: EP/E500552/1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition associated with type 2 diabetes (T2DM) and cardiovascular disease (CVD). Since systemic metabolic dysfunction underlies NAFLD, the current nomenclature has been revised, and the term metabolic-associated fatty liver disease (MAFLD) has been proposed. The new definition emphasizes the bidirectional relationships and increases awareness in looking for fatty liver disease among patients with T2DM and CVD or its risk factors, as well as looking for these diseases among patients with NAFLD. The most recommended treatment method of NAFLD is lifestyle changes, including dietary fructose limitation, although other treatment methods of NAFLD have recently emerged and are being studied. Given the focus on the liver–gut axis targeting, bacteria may also be a future aim of NAFLD treatment given the microbiome signatures discriminating healthy individuals from those with NAFLD. In this review article, we will provide an overview of the associations of fructose consumption, gut microbiota, diabetes, and CVD in patients with NAFLD. In 1986, the term nonalcoholic fatty liver disease (NAFLD) was proposed by Schaner and Thaler , and sooThe definition of NAFLD combines the presence of steatosis in more than 5% of hepatocytes and metabolic risk factors, especially obesity and T2DM, and exclusion of excessive alcohol consumption defined as ≥30 g per day for men and ≥20 g per day for women, or other chronic liver diseases . It shouThe latter definition of MAFLD is based on the presence of hepatic steatosis and at least one other condition such as overweight/obesity, T2DM, or metabolic abnormalities with no additional exclusion criteria ,11. MetaDiabetes mellitus is a heterogeneous group of disorders that results in an increase in blood glucose concentration, yet the pathophysiological processes underlying type 1 and type 2 of the disease differ. Insulin resistance, which is tightly linked to T2DM, is not always present in type 1 of the disease, thereby explaining the different prevalence of NAFLD in those two subpopulations of patients .NAFLD has a high prevalence in the general population, varying from 13.5% in Africa to 31.8% in the Middle East ,15. LiveConsumption of fructose has increased over the last century mainly due to the use of high-fructose corn syrup . This phWhile there is the perception that NAFLD is a benign liver condition, it is the second most common cause of end-stage liver disease  and the Since MAFLD is a new term and has been seldom used in published studies, for this review article, we have summarized the current state of knowledge regarding NAFLD and its association with fructose consumption, microbiota, diabetes, and CVD.Fructose’s use in foods was limited until the late 1960s due to its high price . Since tFructose is a major dietary monosaccharide that occurs naturally in ripe fruits, honey, and in small amounts in some vegetables such as carrot, onion, paprika, and sweet potato. It also comes in industrially manufactured foods because it is a main ingredient in the most widely used sweeteners like disaccharide sucrose  and high fructose glucose syrup (mixture of fructose with sucrose or glucose) ,27.Fructose, in contrast to glucose, is almost totally cleared from circulation by the liver with the use of glucose transporter type—5 (GLUT 5). A large amount of acetyl-CoA is produced following fructose uptake because fructose clearance omits glycolysis, which is the rate-limiting step in acetyl-CoA production . Some acHowever, it is not only lipogenesis; other hepatotoxic effects are exerted by fructose, namely inducing an increase in oxidative stress . FructosNumerous animal and human studies have revealed the association of a close relationship between the consumption of fructose and the development of NAFLD .In a systematic review and meta-analysis of observational studies involving 4639 individuals, SSB consumption led to a 53% increased risk of developing NAFLD in comparison to participants who did not ingest SSB . Other sThe pioneering studies using germ-free mice and gut microbiota transfer related to the association of gut microbiome with metabolic diseases revealed a contribution of gut microbiota to weight gain and metabolic alterations . During Fecal microbiota transfer from patients with NASH to germ-free mice causes hepatic steatosis and inflammation in these animals . On the Human studies have been based on comparisons of gut microbiota between patients with NAFLD, NASH, and cirrhosis and individuals with a healthy liver performed to demonstrate gut microbiota signatures in these pathological conditions . Gut micThe gut–liver axis is an association between gut microbiota and the liver. The interaction is conducted through the portal vein, which transports products from the gut to the liver and, in return, bile and antibodies from the liver to the intestine . An impoAbnormalities of gut microbiota composition in stools of patients with NAFLD have been highlighted in a meta-analysis showing increased abundance of Escherichia, Prevotella, and Streptococcus and decreased abundance of Coprococcus, Feacalibacterium, and Ruminococcus . When paNAFLD also results in increased fecal ester volatile organic compounds (VOC) and the presence of higher concentrations of fecal propionate and isobutyric acid and serum 2-hydroxybutyrate and L-lactic acid ,66. VOC The aforementioned associations between NAFLD and gut microbiota have resulted in studies investigating the effects of microbiome alternation with probiotics, prebiotics, synbiotics, or fecal microbiota transplantation (FMT) on the clinical course of NAFLD with promising results ,70,71,72According to the definitions formulated by the WHO and Food and Agriculture Organization of the United Nations (FAO), probiotics are live strains of strictly selected microorganisms which, when administered in adequate amounts, confer a health benefit on the host . Prebiotbifidobacterium animalis and insulin [In this review article, we focus on the meta-analysis of RCT , showing that probiotic/synbiotic therapy results in improving liver enzymes’ activity and/or reduced steatosis/fibrosis in patients with NAFLD ,69,70,71 insulin . In anot insulin .clostridium difficile infection in 2013 [The role of FMT in patients with NAFLD has limited RCT evidence. FMT was first proven to be a good treatment method of antibiotic-resistant  in 2013  and soon in 2013 . In an R in 2013 . In the  in 2013 . The afoAccording to epidemiological data, the overall prevalence of NAFLD among patients with T2DM is 55.5%, and NASH is 37.3%; also, 17% of T2DM patients who underwent liver biopsy have advanced fibrosis . CurrentNAFLD and insulin resistance are interconnected, and therefore the development of prediabetes and diabetes is the most direct consequence of them at the extrahepatic level ,89. PartIn another cohort study of 10,141 participants, future diabetes mellitus risk could be modified with time by changes in NAFLD status where resolution of NAFLD could diminish the risk of diabetes onset. On the other hand, the development of NAFLD raised the risk of developing diabetes . CurrentIt is important to distinguish between simple steatosis, which does not progress to advanced fibrosis, and non-alcoholic steatohepatitis (NASH), which can lead to hepatocellular carcinoma. Indeed, NASH is characterized by fat accumulation, inflammation and necrosis, ballooning of the cells, and different stages of liver fibrosis, up to cirrhosis. The only method to differentiate between steatosis and NASH is liver biopsy. Masarone et al. performed this invasive procedure in 215 patients with elevated transaminases and metabolic syndrome or T2DM. The prevalence of NAFLD in patients with metabolic syndrome was 94.82% and was present in all the patients with T2DM. NASH was found in 58.52% of participants with metabolic syndrome and 96.82% of T2DM patients. According to the authors, one can assume that patients with T2DM have NASH. As insulin resistance is of crucial importance in the pathophysiology of both T2DM and NASH, NASH may be one of the early complications of T2DM [Some studies have reported that a reduction in the T2DM incidence  and the The strong link between T2DM and NAFLD was underlined in 2020 by an international panel of experts who proposed the new term MAFLD instead of NAFLD . The resCurrently, there is no single, independent of the presence or absence of T2DM, pharmaceutical treatment for NAFLD that has been approved by international guidelines . The freThe best documented direct beneficial effect remains assigned to pioglitazone ,102,103,Current guidelines state that pharmacotherapy is reserved for patients with NASH and for patients at high risk of disease progression. Among the antidiabetic drugs recommended, only pioglitazone is used for the treatment of NASH with insulin resistance  ,124. OldTwo relatively new antidiabetic drug classes, namely SGLT-2i (sodium-glucose cotransporter type-2 inhibitors) ,116,117 An upcoming drug of huge interest is a dual GIP and GLP-1 RA agent (tirzepatide), which is under investigation. It is being administered once a week in the therapy of T2DM patients with NASH and fibrosis . TreatmeBoth NAFLD and CVD are highly prevalent and associated with metabolic disturbances; hence they frequently coexist . This caNAFLD is linked with different manifestations of CVD, including subclinical atherosclerosis, overt atherosclerosis, and cardiovascular events and deaths ,130,131.One meta-analysis from the year 2021 deserves special attention because it was performed among people with histologically confirmed NAFLD who did not present with CVD at baseline  and were prospectively followed–up for a median of 13.6 years . This stThere are also studies that indicated that CVD risks in patients with NAFLD who are non-obese  or non-oOne retrospective, “real world” cohort study aimed to describe the CVD burden and mortality in patients with NAFLD during 14-years follow-up observation following hospital discharge, showing that in patients with non–cirrhotic NAFLD, the condition was associated with increased overall mortality . In contDue to the recent change in nomenclature of fatty liver disease (NAFLD to MAFLD), the prevalence of fatty liver disease and associated CVD risk required re-evaluation, taking into account each of these definitions separately . An analOn the other hand, another study concerning atherosclerotic lesions in T2DM patients found no significant difference in carotid IMT between T2DM patients with and without NAFLD . The samWhile an association between NAFLD and T2DM microvascular complications seems plausible, the number of studies on this topic is limited and inconclusive ,153,154.It is well established that patients with DM compared to people without carbohydrate disorders have a higher risk of heart failure and CVD. There is a term “diabetic cardiomyopathy”, deteriorating patient’s prognosis and being described as a form of heart disease occurring in diabetic patients, which causes significant structural as well as functional changes in the myocardium. There is a common pathophysiological mechanism of diabetic cardiomyopathy and NAFLD, namely insulin resistance . It resuMAFLD is a new clinical definition for fatty liver disease, which shifts NAFLD from a disease of exclusion to one of inclusion, where the pathogenic processes originate from underlying metabolic dysfunction. Because MAFLD is not widely used terminology in the scientific literature, most published data focus on NAFLD. The latter as an epidemic is tightly linked to T2DM, which are known to frequently coexist with and synergistically increase the CVD risk.Despite the high prevalence of NAFLD and many epidemiological studies showing correlations between NAFLD and CVD, it is still difficult to unequivocally identify a causal relationship between the two entities  and to sWe are still unclear whether the diagnosis of NAFLD can be used as a tool to improve cardiovascular risk and modify treatment . Lifesty


Non alcoholic steatohepatitis (NASH) is the inflammatory reaction of the liver to excessive accumulation of lipids in the hepatocytes. NASH can progress to cirrhosis and hepatocellular carcinoma (HCC). Fatty liver is the hepatic manifestation of metabolic syndrome. A subclinical inflammatory state is present in patients with metabolic alterations like insulin resistance, type-2 diabetes, obesity, hyperlipidemia, and hypertension. Platelets participate in immune cells recruitment and cytokines-induced liver damage. It is hypothesized that lipid toxicity cause accumulation of platelets in the liver, platelet adhesion and activation, which primes the immunoinflammatory reaction and activation of stellate cells. Recent data suggest that antiplatelet drugs may interrupt this cascade and prevent/improve NASH. They may also improve some metabolic alterations. The pathophysiology of inflammatory liver disease and the implication of platelets are discussed in details. The term non-alcoholic fatty liver disease (NAFLD) was coined by Ludwig and colleagues  to descrNAFLD is histologically characterized by macrovesicular steatosis and further categorized into non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) a more severe and evolutive disease, including inflammation and balooning.The definition by EASL Guidelines for the management of non-alcoholic fatty liver disease is the following: NAFLD characteristic is the excessive hepatic fat accumulation, which is associated with insulin resistance. NAFLD is also defined by the presence of steatosis in more than 5% of hepatocytes in histological analysis or by density fat fraction exceeding 5.6% as assessed by proton magnetic resonance spectroscopy or quantitative fat/water selective magnetic resonance imaging. The term NAFLD includes two distinct conditions with different prognosis: non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). While NAFL is a milder condition, NASH covers a wide spectrum of disease severity, including fibrosis, cirrhosis and hepatocellular carcinoma (HCC). .Now the term metabolic associated fatty liver disease (MAFLD) has been adopted, instead of NAFLD/NASH, chosen by a group of experts . Three cAnother condition, MAFLD-related cirrhosis, is characterized by cirrhosis with past or present evidence of metabolic risk factors that meet the criteria to diagnose MAFLD, with at least one of the following: 1) Documentation of MAFLD on a previous liver biopsy. 2) Historical documentation of steatosis by hepatic imaging . Being aRisk factors for the development of NAFLD, or its evolution to NASH, are the same of metabolic syndrome, a disorder consisting by definition of obesity, arterial hypertension, impaired glucose metabolism and atherogenic dyslipidaemia, a clinical condition with high prevalence in the adult population worldwide, particularly in industrialized countries .In some countries, NAFLD represents the primary cause of cirrhosis , the maiIn the United States the prevalence of NAFLD, as assessed using ultrasound associated with transaminases increases or scores like fatty liver index/NAFLD score, reaches 19 to 46 percent in the adult population, with most biopsy-based studies reporting a prevalence of NASH of 3–5 percent . WorldwiEstimates of prevalence of NAFLD in Asia-Pacific regions range from 5 to 30 percent, depending upon the population studied  while inWhile NASH is considered a condition that promotes fibrosis progression, longitudinal studies have demonstrated that the liver-related prognosis of patients with NAFLD is also mostly related to the extent of liver fibrosis  as obserThe hepatic steatosis is characterized by high accumulation of lipids , cholesterol, and ceramides) and this aberrant accumulation results in liver toxicity .de novo lipogenesis process) contribute to the accumulation of hepatic and lipoprotein fat in NAFLD , has also been observed in NAFLD . Furtherin NAFLD .de novo lipogenesis exceed the oxidation rate of FFA and the export of VLDL; the impaired metabolism of lipids is also associated with the progression of NAFLD to NASH. Changes in hepatic and serum lipidomic signatures have been proposed as index of the development and progression of fatty liver disease  disease is the main cause of morbidity and mortality in patients with NAFLD  where thper se on CV events and death, might be questionable. However, the bidirectional relationship between NAFLD and hypertension seems to be independent of other components of the metabolic syndrome (MetS) (Trying to dissect the impact of NAFLD e (MetS) .Insulin resistance is considered the main determinant of hepatic steatosis and steatohepatitis. . Also otNAFLD patients are often obese and/or affected by type 2 diabetes mellitus, two conditions associated with peripheral insulin resistance. Nevertheless, insulin resistance has also been observed in non-obese NASH patients and in those who have normal glycemic levels, thus suggesting a strong association between insulin resistance and lipid accumulation . SubjectVisceral fat accumulation is considered an independent risk factor in NASH patients, as it has been suggested that a higher visceral fat level in these patients leads to higher liver fibrosis and inflammation: this could be linked to proinflammatory cytokines activity, like interleukin-6 (IL-6) , or actiSeveral studies have investigated the possible genetic polymorphisms present in patients with NAFLD/NASH and the results obtained have suggested a certain role of IL-6, adiponutrin apolipoprotein C3, and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) , Sookoi. RecentlIt has been suggested that the phenotype of manifestations of NAFLD and the progression of the disease are the result of complex interactions between the environment and the genetic pool of the subject. Some studies highlight a strong heritability of lipid content in the liver  and famiThere are many genes associated with insulin signaling and lipid metabolism, which are involved in the development of NAFLD, and it is not our aim to give them a full discussion here. However it is important to list at least the genetic polymorphisms of greatest interest in this pathology. Notably, patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M variant is the commonest inherited determinant of NAFLD as it is associated with progression of NAFLD, NASH, and NAFLD-related HCC . The PNP. A well-known genetic polymorphism studied in NAFLD subjects is the Membrane bound O-acyltransferase domain-containing 7 (MBOAT7) downregulation: MBOAT7 is a gene implicated in phosphatidylinositol (and other phospholipids) remodeling via the incorporation of arachidonic acid and other unsaturated fatty acids into lyso-phospholipids. The common genetic variant leads to a downregulation of MBOAT7 activity and consequantely to accumulation of lyso-phosphatidyl-inositol in hepatocytes; this in the end leads to a higher sinthesis of triglycerids in the liver and NAFLD. (The Transmembrane 6 superfamily member 2 (TM6SF2) gene E167K variant promotes a reduction in VLDL secretion from the liver, inducing higher triglycerides levels in the liver and a lower capacity of LDL secretion from hepatocytes, while patients with TM6SF2 E167K polimorfism show a reduced cardiovascular risk . A well-d NAFLD. .de novo lipogenesis in the liver; the GCKR P446L is a missens variant that causes a protein loss of function, in the end resulting in a constitutively active glucose inflow in the hepatocytes (Glucokinase regulator (GCKR) controls the glucose inflow in hepatocytes thus regulating the atocytes , (Choudhatocytes ..The protein phosphatase 1 regulatory subunit 3B (PPP1R3B) encodes for a protein involved in glycogen synthesis. PPP1R3B rs4841132 variant has been suggested to protect against hepatic fat accumulation and liver fibrosis in NAFLD subjects : this variant increases the lipid oxidation and downregulates some lipid metabolism and inflammation pathways .via the modulation of phagocytes and hepatic stellate cells activity, and a specific variant (MERTK rs4374383) exerts a protective role, reducing the MERTK expression in the liver (Interferon lambda 4 (IFNL4) rs368234815 variant is associated to higher inflammation and fibrosis in the liver; Mer T kinase (MERTK) variants could alter the hepatic inflammation and fibrosis he liver .HSD17B13 gene encodes for an enzyme that concentrates lipid droplets in hepatocytes: loss-of-function variants in HSD17B13 result in higher protection against liver inflammation, cirrhosis, and HCC .Another important pathogenetic factor is represented by the accumulation of iron, which can contribute to the development of NASH and promote oxidative stress, thus producing free oxygen species which lead to liver damage and fibrosis, and in the end NASH .Nevertheless, studying hemochromatosis gene (HFE) mutations, no significant role in the development of insulin resistance-associated liver siderosis was seen, apart from compound heterozygosity .Unexplained hepatic iron overload is frequently associated with the insulin-resistance syndrome irrespective of liver damage. This insulin-associated iron overload is characterized by a mild to moderate iron excess with hyperferritinemia and normal to mildly increased transferrin saturation .via bile salts deconjugation, and inactivation of hepatic lipotropic molecules (such as choline)  .The production of endogenous alcohol and acetaldehyde  is another suggested mechanism : colonicOther factors and mechanisms have been studied relating to the pathogenesis of NAFLD. The cholesterol intake with diet has been proposed as an independent factor in developing of NASH . It has Platelets are involved in different models of liver damage .Apart from the well-defined interaction between CV risk factors and NAFLD/NASH, a pro-thrombotic condition may derive from altered endothelial and vascular function and platelet activation and interaction with blood and liver cells.via intralobular hypoxia and local ischemia, . Lipid species cause inflammation and activation of both infiltrating and resident immune cells. How are platelets involved in this process? Platelets are involved in pathological processes such as chronic inflammation and atherothrombosis and possibly fibrogenesis .Platelets contain granules that are released in response to activatory stimuli (platelet release reaction). Alpha and delta (dense) granules may release in the microenvironment the proaggregatory factors ADP, serotonin and thrombin (the amplificatory process) along with appreciable amounts of inflammatory cytokines, chemokines and growth factors such as platelet-derived growth factor (PDGF), endothelial growth factor (EGF), insulin-like growth factor 1 (IGF-1), transforming growth factor beta (TGFβ), tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), chemokine ligand 4 (CXCL4), vascular endothelial growth factor A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor (FGF). . PlatelePlatelets release factors that change gene expression in endothelial cells, leukocytes, stromal cells and fibroblasts thus directly participating to inflammation . Platelein vivo platelet activation , mean platelet volume (MPV) values, and platelets/lymphocyte ratio have been associated with hyperleptinemia and hypoadiponectinemia . In obestivation . In NAFLtivation .via activation of the classic complement pathway  production  and altered downstream cGMP/cGMP-dependent protein kinase (PKG) signalling system. Similarly, the inhibitory activity of prostacylin (PGI2) towards platelet activation and the engagement of the cAMP/cAMP-dependent protein-kinase (PKA) pathways are impaired . This is2 signalling, which catalyses arachidonic acid release and TXA2 generation. Activation of the aldose reductase pathway is implicated in oxidative stress-induced TXA2 biosynthesis amplified by exposure to collagen, indicating that when the vascular endothelium is damaged thromboembolic events are promoted  expression are upregulated in NAFLD (W. via proteinase activated receptor 1–4 (PAR1–4) signalling, leading to platelet hyperreactivity. While experimental models confirm thrombin generation in NAFLD clinical evidence, is lacking (TXANAFLD W. . NAFLD f lacking .Patients and mice with NAFLD have increased blood levels of molecules present in granules from platelets. Thrombospondin (TSP-1), present in platelets , but synOne mechanism of interaction between platelets and leukocytes is through CD40L. CD40L belongs to the TNF superfamily, and is increased in NAFLD platelet surface, signalling leukocytes expressing CD40 . It has Glycosaminoglycans and CXCR3, a chemokine receptor, bind to CXCL4, a protein secreted by platelets . In expevia platelet-derived microparticles to hepatocytes has also been demonstrated. PMP carrying miR-25-3p promoted hepatocyte proliferation modifying gene expression  through mediators with both pro- and anti-fibrotic effects . AdenineThe profibrotic effects of activated platelets  are due The real role of platelets in increasing liver fibrosis is underlined by the positive effect of aspirin , documenRegarding immune response, both innate and adaptive, platelets play a very important role in its stimulation. Activated platelets attract immune cells and modulate inflammation through the expression of specific receptors and release of chemokines and cytokines  in the lAccording to Malehmir et al., platelets are involved only in pathophysiology of NASH, while they do not play any role in steatosis . In expe+CD8+ T cells, CD11b + MHCII + myeloid cells and Ly6G + granulocytes (In patients with NASH and in mice with choline deficient high fat diet (CDHFD) induced NASH there is increased hepatic infiltration of CD3ulocytes . Treatmeulocytes . Aspirinulocytes .The demonstration that the improvement of NASH with the combined treatment with aspirin-clopidogrel is not COX-dependent comes from the evidence that in experimental NASH, sulindac, only a COX-inhibitor, does not modify obesity, liver/body weight ratio, hepatic triglycerides and glucose tolerance, liver damage .Considering the role of platelets receptors in NASH, it has been shown that the GPIIb subunit of the platelet fibrinogen receptor GPIIb/IIIa is not involved, confirming that platelets aggregation is not responsible for NASH , as wellDevelopment of diet induced NASH was not affectd by deletion of P-selectin (Selp–/–), , von-WilRecently, the contribution of platelets to liver inflammation was confirmed by immunohistochemical staining on liver biopsies showing accumulation of platelet and neutrophil extracellular traps (NET) in liver, with a correlation with NAFLD activity score. Circulating platelets from patients with NAFLD were shown to have significant increase of inflammatory transcripts, while leukocytes did not  Figure .Few studies have addressed the effect of antiplatelet therapy in NAFLD/NASH, as previously anticipated.via intercepting signal transduction from Akt to c-Raf  and proportion of CD4+/CD8+ and NKT cells, CD11b+F4/80hi, Kupffer cells, Kupffer cell activation, inflammatory myeloid cell infiltration were reduced. Thus, aspirin-clopidogrel prevented NASH and reduced NASH-related increase of platelet interaction with the liver endothelium, T cells and innate immune cells.The most striking evidence of the benefit of platelet inhibition is shown by Malehmir et al. . They shFinally, antiplatelet treatment seems to be effective only in the liver, affecting interactions of GPIbα+ platelets with Kupffer cells, in mouse and human NASH .Scientific evidence supports the hypothesis that platelets are implicated in the pathophysiology of NAFLD/NASH, mostly by exerting proinflammatory and profibrotic activities, rather than exerting their thrombogenic activities. The recently discovered interaction of platelets with liver cells and the immune system introduces new models of inflammation and fibrogenesis in the setting of chronic liver diseases, anticipating the potential efficacy of antiplatelet agents to prevent the progression of NAFLD towards NASH and the eventual liver cancer. Further research is required to identify detailed mechanisms and potential specific target of pharmacological intervention. Clinical trials in selected patients, who may benefit of antiplatelet intervention, are warranted.


Non-alcoholic fatty liver disease (NAFLD), which affects about a quarter of the global population, poses a substantial health and economic burden in all countries, yet there is no approved pharmacotherapy to treat this entity, nor well-established strategies for its diagnosis. Its prevalence has been rapidly driven by increased physical inactivity, in addition to excessive calorie intake compared to energy expenditure, affecting both adults and children. The increase in the number of cases, together with the higher morbimortality that this disease entails with respect to the general population, makes NAFLD a serious public health problem. Closely related to the development of this disease, there is a hormone derived from adipocytes, leptin, which is involved in energy homeostasis and lipid metabolism. Numerous studies have verified the relationship between persistent hyperleptinemia and the development of steatosis, fibrinogenesis and liver carcinogenesis. Therefore, further studies of the role of leptin in the NAFLD spectrum could represent an advance in the management of this set of diseases. Lepti as the p similar .Leptin is characterized by having pleiotropic effects due to the great variety of leptin receptors (known as Ob-R or LEPR), thus being able to affect many biological processes at different levels. The six existing spliced Ob-R forms are called Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, Ob-Re and Ob-Rf, and belong to the class I cytokine superfamily ,7 but diThis adipokine, leptin, is mostly recognized for playing a key role in the central control of both energy metabolism  and obesToday, several problems are associated with NAFLD. This entity is the leading cause of liver disease worldwide and its prevalence is increasing , affecti2) and 25% of lean patients (BMI 20.0–24.9 kg/m2) and can be affected by other factors  [NAFLD is a clinicopathologic entity comprising a broad spectrum of liver diseases ranging from simple steatosis to NASH, a more aggressive form of NAFLD and associated with varying degrees of hepatic fibrosis, cirrhosis, and HCC . NAFLD hnd race) . The hignd race) . This mand race) ,27.NAFLD is also considered the hepatic component of the metabolic syndrome, whose prevalence is increasing worldwide at the same time as obesity and type 2 diabetes-mellitus (T2DM) ,30. In fThe pathogenesis of NAFLD entail a complex interplay between environmental factors, obesity, changes in the microbiota and predisposing genetic variants that result in altered lipid homeostasis and hepatocyte triglyceride accumulation . In turnLeptin acts by binding to its receptors. Specifically, Ob-Rb isoform is the main leptin receptor as it provokes signaling cascades . After tLeptin and other inflammatory adipokines such as IL-6 or TNF-α (Tumor nechrosis factor) promote insulin resistance, which has been extensively described in the pathophysiology of NAFLD during the last few decades ,53,54, aMoreover, some pathologies can cause NAFLD. This is the case of congenital or acquired lipodystrophy, which is characterized by the total or partial absence of subcutaneous adipose tissue and promotes ectopic accumulation of fat in other locations, including the liver, which leads to severe insulin resistance and the development of NAFLD ,58. PatiNAFLD comprises a set of liver diseases, some of them irreversible. NAFLD development is divided into three main steps: simple steatosis, NASH, and liver cirrhosis. However, NAFLD can eventually trigger in HCC. Simple steatosis is caused by factors such as high-fat and/or high sugar diet, obesity, T2DM, and other metabolic diseases, while NASH can be developed by inflammation and hepatocyte apoptosis. If liver fibrosis is provoked in this step, cirrhosis (and possibly HCC) will be also developed . In the Hepatic steatosis has different degrees of severity related to liver damage in NAFLD: from simple steatosis to NASH, which is the most important disease in the NAFLD spectrum, since its prevalence is estimated to be approximately 1.5–6.5% in the general population, and considerably increasing this percentage in obese individuals . AlthougHepatic steatosis can be caused by both aberrant lipid and glucose metabolism. One of leptin functions is to limit the storage of triglycerides in adipocytes and non-adipose tissues including the liver, thereby preventing lipotoxicity. Under normoleptinemia conditions, leptin exerts an anti-steatotic effect and improves insulin sensitivity by suppressing hepatic glucose production and lipogenesis ,65. ThisLeptin has also been suggested to have a synergistic effect when used together with insulin, probably inhibiting the production of very low-density lipoproteins (VLDL) ,67,69. ABy contrast, high leptin levels have also been associated with hepatic steatosis and NAFLD pathogenesis since a high percentage of NAFLD patients have been observed to suffer obesity, which is closely related with hyperleptinemia ,50,74. TIn NAFLD, most patients have simple steatosis, but those with NASH can advance to the next step of the disease, which is fibrosis. The mechanisms of progression from simple steatosis to NASH are not entirely clear, but some factors are known to be involved in the process , includiAdvanced fibrosis implies an increased risk for developing other NAFLD-related complications, such as cirrhosis and HCC. For that reason, an early diagnosis of patients with advanced fibrosis is crucial . In this+CD57+ T cells, found in NASH progression [Activated HSC also contribute to increase inflammation and liver fibrosis by releasing TGF-β1, angiopoietin-1, VEGF , and collagen-I. In addition, HSC appear to produce leptin, and have also been proposed to express Ob-Rb, which establishes a vicious cycle by stimulating proliferation and preventing apoptosis of HSC and thus affecting hepatic inflammation and fibrosis . KC can gression . Also, pgression .According to several studies with small sample sizes, progression from NASH to liver cirrhosis can occur in up to 25% of patients. This high disease burden has led to an increase in the number of NASH-related transplants, possibly becoming in the leading cause of liver transplantation worldwide in coming decades, displacing the hepatitis C virus . Up to nThere are references in other types of liver cirrhosis about leptin, in which this hormone has been demonstrated to be in both high  and low Obesity and T2DM are cancer promoters and, in coexistence with NAFLD, the aggressive potential can be underestimated. HCC is the neoplasm most closely related to obesity in men. In this regard, HCC incidence increased by 3% per year in the last decade, unlike other malignancies also associated with obesity, such as breast or colon cancer, whose incidences remained stable or decreased. In part, this fact may be explained by the increase in the prevalence of NASH . NAFLD pIn HCC there are some established risk factors, including chronic hepatitis B, chronic hepatitis C, alcohol consumption, and NAFLD, all of them potentially linked to leptin . As withHowever, Elinav et al. (2006) suggested a beneficial role of leptin in HCC murine models since this hormone decreased tumor size and improved survival . In the NAFLD is a worldwide health problem due to its increasing prevalence, so the research on its diagnosis, follow-up, and subsequent treatment has become essential. Moreover, NAFLD requires a multidisciplinary approach given its high risk of cardiovascular morbidity and mortality. In this sense, there is an urgent need for non-invasive diagnostic methods to replace liver biopsy, so that early diagnosis and treatment monitoring is possible in a large part of the population. Leptin, due to its direct relationship with body fat levels and insulin resistance, has been shown to be an independent predictor of the presence or development of NAFLD. This adipokine has been shown to have antisteatotic effects, although it has also been associated with hepatic steatosis and may promote more advanced stages of NAFLD that include NASH and liver fibrosis. The role of leptin in both NAFLD-related cirrhosis and HCC has never been studied. Its functions in other liver cirrhosis remains controversial. However, there is much evidence to establish the protumoral role of this hormone in HCC derived from other liver diseases.Treatment with leptin has proven to be effective in patients with congenital leptin deficiency; however, its use in the rest of the affected subjects remains controversial, which highlights the importance of continuing the line of research on the development of leptin analogues that conserve the antisteatotic effect and lack proinflammatory and profibrogenic action, as well as leptin sensitizers, or their synergistic effect when associated with different drugs. While further observational studies and large clinical trials with long-term follow-up are needed to fully evaluate the efficiency of the use of this adipokine, leptin could be used as an interesting biomarker in the diagnosis and follow-up of NAFLD, including the combination of leptin level measurement together with metabolic analyses, lipid profile, and glucose levels.


Pkd1−/− and 786‐0 cells. Furthermore, its administration curbed cystogenesis in Pkd2 zebrafish and early‐onset Pkd1‐deficient mouse models. In conclusion, NS398 is a potential therapeutic agent for ADPKD.Autosomal‐dominant polycystic kidney disease (ADPKD) is characterized by uncontrolled renal cyst formation, and few treatment options are available. There are many parallels between ADPKD and clear‐cell renal cell carcinoma (ccRCC); however, few studies have addressed the mechanisms linking them. In this study, we aimed to investigate their convergences and divergences based on bioinformatics and explore the potential of compounds commonly used in cancer research to be repurposed for ADPKD. We analysed gene expression datasets of ADPKD and ccRCC to identify the common and disease‐specific differentially expressed genes (DEGs). We then mapped them to the Connectivity Map database to identify small molecular compounds with therapeutic potential. A total of 117 significant DEGs were identified, and enrichment analyses results revealed that they are mainly enriched in arachidonic acid metabolism, p53 signalling pathway and metabolic pathways. In addition, 127 ccRCC‐specific up‐regulated genes were identified as related to the survival of patients with cancer. We focused on the compound NS398 as it targeted DEGs and found that it inhibited the proliferation of  When PC1 or PC2 is missing, the levels of intracellular calcium ions decrease, whereas those of cyclic AMP increase. This affects downstream signalling pathways, cell proliferation and the secretion of cyst fluid, ultimately leading to the formation and growth of cysts.ADPKD is mainly caused by mutations in Previous studies have found that ADPKD and tumours share many mechanisms and signalling pathways; thus, ADPKD has been called ‘neoplasia in disguise’.We have previously developed the Renal Gene Expression Database (RGED), a bioinformatics database comprising comprehensive gene expression datasets of studies on renal diseases published in the NCBI Gene Expression Omnibus (GEO) database.To clarify the similarities and dissimilarities between ADPKD and ccRCC and screen and identify candidates for ADPKD therapy, we designed an integrated bioinformatics approach based on the aforementioned rationale and conducted corresponding experiments for validation. Thus, our results will provide insights into the relationship between ADPKD and ccRCC, as well as a new therapeutic strategy for ADPKD.22.1http://rged.wall‐eva.net) and the high‐throughput gene expression datasets from GEO (http://www.ncbi.nlm.nih.gov/geo/). The two datasets retrieved were GSE7869 and GSE53757, both constructed based on the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array). GSE7869 included cystic tissues of different sizes from 5 Pkd1 polycystic kidneys and non‐cancerous renal cortical tissues from 3 nephrectomized kidneys, whereas GSE53757 included 72 ccRCC tumour tissues of all disease stages and matched kidneys.We collected the gene expression profiles of renal cystic tissues of patients with ADPKD and tumour tissues of patients with ccRCC from RGED  were used for background correction, standardization and expression value calculation of the original datasets GSE7869 and GSE53737. Fold‐change and adjusted p‐values were used to screen DEGs. The annotate package was utilized to annotate DEGs. Volcano maps of DEGs were constructed using ggplot2. Subsequently, we identified intersecting DEGs between the datasets using the Venn package in R.R packages simpleaffy, affyPLM, affy, gcrma and limma (2.3https://david.ncifcrf.gov) to investigate the function of the DEGs. Gene ontology (GO) annotation includes three categories: biological process (BP), cellular component (CC), and molecular function (MF). Significant GO terms and KEGG pathways were defined as p < 0.05. The interaction between proteins was identified using the String database . Kaplan‐Meier plots and log‐rank test results were retrieved using the cgdsr, survival and survminer package in R. The high‐expression group was defined as those with expression value > 75% quantile, whereas the low‐expression group was defined as those with expression value < 25% quantile. p < 0.01 was set as the cut‐off value.Function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery 6.8  provides gene expression profiles from cultured human cells treated with bioactive small molecules and can be used to discover functional connections among diseases, genetic perturbation and drug action.P value < 0.05.The CMap  were a kind gift from Yiqiang Cai  and cultured as described by Shibazaki et al.ADPKD cyst‐lining epithelial mouse cells  for 4 h, DMSO was added to dissolve the crystallized products. Absorbance at 490 nm was measured. The measurement was repeated in six duplicate wells.2.7Cell cycle staining was performed using a propidium iodide (PI) cell cycle kit according to the manufacturer's instructions (Multi Sciences Biotech). Briefly, cells were harvested, washed twice with phosphate‐buffered saline and stained with 50 µg/ml PI. Cells were then counted in a flow cytofluorometer based on red emissions at 630 nm, and data were analysed using a Beckman Coulter CyAn ADP analyzer.2.8Pkd1−/− or 37°C for 786–0 cells) for 4 h. After fixing with 4% formaldehyde, the click reaction cocktail (0.2 ml) was added to each well, and the DNA was stained with Hoechst. The samples were imaged using Olympus BX‐51 fluorescence microscopy.EdU assays were conducted following the manufacturer's instructions (Beyotime). After adding 2× EdU working solution to each well, 24‐well plates were placed in the incubator (33°C for 2.92O (ddH2O) and 5× protein loading buffer (Yamei) was added to the protein sample. The solution was boiled in a 95°C dry heater for 15 min. An equal amount (20 μl) of protein was loaded onto an SDS‐PAGE gel and electrophoresed, and separated proteins were transferred onto a polyvinylidene fluoride membrane. After incubation with the appropriate primary and secondary antibodies, development was performed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific). Densitometric analysis for the determination of relative protein expression was performed using Image lab system (Bio‐Rad Laboratory), with GAPDH as a loading control.A column tissue and cell protein extraction kit (Minute) was used to extract the proteins of cultured and processed cells and mouse kidney tissues. Protein was quantified using a BCA kit (Thermo Fisher Scientific) following the manufacturer's instructions. The protein concentration of each sample was balanced by double‐distilled H2.10Pkd2 morpholino (Pkd2 MO): 5ʹ‐AGGACGAACGCGACTGGAGCTCATC‐3ʹ was constructed using Gene Tools (Philomath) and injected at the 1‐cell stage.Pkd2 morphants  were divided into two groups 24 h post‐fertilization. One group was treated with NS398 (10 μM), whereas the other was treated with DMSO as the vehicle‐treated control group. The embryos were anaesthetized and immobilized in methylcellulose 72 h post‐fertilization to visualize the pronephric cyst formation and measure the rate of cyst formation in all embryos. The experiment was independently conducted three times.Wild‐type zebrafish (Nüsslein‐Volhard Laboratory) were maintained, mated, and staged as described previously.Pkd1fl/fl: tamoxifen‐Cre mice at postnatal day 10 (P10) to induce Pkd1 deficiency. NS398 was administered every other day via intraperitoneal injection from P13 at 20 μg/g body weight. Mice were killed by isoflurane using a rodent gas anaesthesia machine (Yuyan Machine) at P30; blood was collected by retro‐orbital puncture and kidneys were harvested. The serum blood urea nitrogen (BUN) concentration was measured with a urea assay kit (BioAssay Systems). The kidneys were weighted and collected for morphological analysis and Western blot analysis.The early‐onset PKD mouse model was generated as described previously.The study protocol was approved by the Second Military Medical University's Animal Care and Use Committee .2.11Kidney sections were fixed in 4% paraformaldehyde buffered solution and embedded in paraffin. Then kidneys were sliced into 2‐μm‐thick sections and stained with haematoxylin and eosin (HE). Aperio XT and Leica Aperio (Leica Biosystems) were applied to scan all specimens. The size and shape of kidney cysts were quantitatively measured from the sagittal section of the kidney. The cyst index (CI) was determined as previously described by Yang et al.2.12t test. p < 0.05 was considered statistically significant.Data are presented as the mean ± standard error of at least triplicates or as a representative of three separate experiments. Significance was determined using a two‐sided Student’s 33.12FC| ≥ 2 and p < 0.001. From the Venn diagrams, 22 up‐regulated and 95 down‐regulated DEGs were found to be common to both datasets . The top 6 genes were PPP1R18, PLAUR, TMEM44, JAK3, PTTG and ENTPD1 ranked by the hazard ratio, and their Kaplan‐Meier survival curves are displayed in Figure Because the main difference between ADPKD and ccRCC is the malignant and metastatic nature of ccRCC, which determines the prognosis of patients, we have been suggested that ccRCC‐specific DEGs are closely related to the prognosis of patients with ccRCC. Of the 880 genes, 127 were identified to be significantly related to the survival of patients with kidney cancer . Consistent with the results of the MTT assays, the percentage of EdU‐positive cells was lower in Pkd1−/− cells treated with 50 μM NS398 than that in cells treated with DMSO . The EdU proliferation assay showed a similar result—the number of EdU‐positive 786‐0 cells was remarkably reduced after treatment with 100 μM NS398  reduced the levels of phospho‐ and total Akt and ERK in s Figure . NS398 i3.5Pkd2 morphants administered DMSO or NS398 are shown in Figure Pkd2 morphants significantly decreased after treatment with 10 μM NS398  and renal clear‐cell carcinoma (786‐0) cells. Furthermore, NS398 significantly inhibited cyst formation in zebrafish and mouse animal models of PKD. Thus, our results indicate the value of NS398 in the treatment of ADPKD and/or tumours. Basic and clinical studies are warranted to validate our results.In summary, we applied an integrated bioinformatics approach based on the high‐throughput data of ADPKD and ccRCC and verified these results The authors confirm that there are no conflicts of interest.Sixiu Chen: Data curation ; Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Visualization ; Writing‐original draft (lead). Linxi Huang: Data curation ; Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Visualization (lead); Writing‐original draft . Shoulian Zhou: Data curation ; Formal analysis ; Investigation ; Methodology ; Validation (lead); Writing‐original draft . Qingzhou Zhang: Data curation ; Formal analysis ; Software (lead). Mengna Ruan: Formal analysis ; Investigation ; Methodology ; Validation . Lili Fu: Data curation ; Formal analysis ; Visualization . Bo Yang: Data curation ; Formal analysis ; Methodology . Dechao Xu: Data curation ; Formal analysis ; Methodology ; Resources (supporting); Writing‐original draft (supporting). Changlin Mei: Conceptualization ; Formal analysis (supporting); Funding acquisition ; Project administration ; Resources ; Supervision ; Writing‐review & editing . Zhiguo Mao: Conceptualization ; Formal analysis (supporting); Funding acquisition ; Project administration ; Resources ; Supervision ; Writing‐review & editing .Fig S1Click here for additional data file.Supinfo S1Click here for additional data file.


The cyclic tensile behavior of steel-reinforced high strain-hardening ultrahigh-performance concrete (HSHUHPC) was investigated in this paper. In the experimental program, 12 HSHUHPC specimens concentrically placed in a single steel reinforcement under cyclic uniaxial tension were tested, accompanied by acoustic emission (AE) source locating technology, and 4 identical specimens under monotonic uniaxial tension were tested as references. The experimental variables mainly include the loading pattern, the diameter of the embedded steel rebar, and the level of target strain at each cycle. The tensile responses of the steel-reinforced HSHUHPC specimens were evaluated using multiple performance measures, including the failure pattern, load–strain response, residual strain, stiffness degradation, and the tension-stiffening behavior. The test results showed that the enhanced bond strength due to the inclusion of steel fibers transformed the failure pattern of the steel-reinforced HSHUHPC into a single, localized macro-crack in conjunction with a sprinkling of narrow and closely spaced micro-cracks, which intensified the strain concentration in the embedded steel rebar. Besides, it was observed that the larger the diameter of the embedded steel rebar, the smaller the maximum accumulative tensile strain under cyclic tension, which indicated that the larger the diameter of the embedded steel rebar, the greater the contribution to the tensile stiffness of steel-reinforced HSHUHPC specimens in the elastic–plastic stage. In addition, it was found that a larger embedded steel rebar appeared to reduce the tension-stiffening effect (peak tensile strength) of the HSHUHPC. Moreover, the residual strain and the stiffness of the steel-reinforced HSHUHPC were reduced by increasing the number of cycles and finally tended toward stability. Nevertheless, different target strain rates in each cycle resulted in different eventual cumulative tensile strain rates; hence the rules about failure pattern, residual strain, and loading stiffness were divergent. Finally, the relationship between the accumulative tensile strain and the loading stiffness degradation ratio under cyclic tension was proposed and the tension-stiffening effect was analyzed. Ultrahigh-performance concrete (UHPC), as one of the current advanced high-performance cementitious composites, exhibits eminent mechanical properties: comparatively high compressive strength ≥120 MPa) and tensile strength (≥10 MPa), strain-hardening characteristics under tensile load for a given volume of discontinuous internal fibers, and excellent durability due to an optimized dense matrix as well 20 MPa an. BesidesThe intensified properties of UHPC have urged a wide range of researchers and engineers to apply it in various types of innovative civil engineering, where bending prevails . NeverthRecently, the rehabilitation of a concrete bridge deck or orthotropic steel deck was widely conducted by employing a thin reinforced UHPC overlay, instead of a relatively thicker reinforced concrete layer, because the demand to upgrade the load-bearing capacity of bridge structures is rising ,11. In tThe objective of the present study was to investigate the behavior of steel-reinforced HSHUHPC members under cyclic axial tension at the serviceability-limit state. Twelve steel-reinforced HSHUHPC dog-bone-shaped specimens under cyclic uniaxial tension and four identical specimens under monotonic uniaxial tension were fabricated and tested, supplemented with crack width detection and AE source locating technology as well. AE technology provides support in detecting the internal damages of reinforced UHPC from the microcosmic point of view. AE source locating is a method to obtain AE source distribution in three-dimensional space. The experimental variables included the loading pattern, diameter of embedded steel rebar, and the level of target strain at each cycle. All of the specimens were loaded for 10 cycles. The evolution of mechanical properties, damage mode, crack spacing, distribution of damage points, residual strain, tension-stiffening response, and stiffness degradation mechanism of steel-reinforced HSHUHPC members under cyclic tension were investigated and analyzed.A total of 12 steel-reinforced HSHUHPC dog-bone-shaped specimens under cyclic uniaxial tension and four specimens under monotonic uniaxial tension as reference specimens, with identical dimensions, were fabricated and tested. The detailed dimensions of all specimens are shown in The HSHUHPC materials used in this research consist of UHPC premixed powder, steel fibers, and water. For steel-reinforced HSHUHPC specimens, HRB400 deformed steel rebars with different diameters and a length of 500 mm were used for longitudinal reinforcement. Three bare steel rebars were tested to obtain the tensile stress–strain characteristics. The average tensile stress–strain relationship of the steel rebars, which were tested in a universal testing machine, was obtained as shown in A direct tensile test was carried out through a universal testing machine (WDW-300 servo-controlled testing system) running in a displacement control manner. The configuration, dimensions, and test setup of the steel-reinforced HSHUHPC dog-bone specimens are presented in i + 1 cycle; the total strain amplitude of the i + 1 cycle is the superposition of the cumulative residual strain generated on the i cycle and the target strain value. Moreover, the loading rate remained at a speed of 0.3 mm/min throughout the whole loading process, whether for the loading part or the unloading part. All the specimens were loaded for 10 cycles.Four loading scenarios were performed in this study. Before each test, a preloading of 0.5 kN was applied and was then unloaded to zero in order to stabilize the testing system and obtain the complete stress–strain curve originating from zero point. For the monotonic loading scheme, the specimens were subjected to a displacement control load with a speed of 0.3 mm/min until the specified strain. For cyclic loading schemes, three different loading histories were used to investigate the damage evolutions of steel-reinforced HSHUHPC under cyclic tension with three levels of target strains  corresponding to the different service states of reinforced UHPC structures. In the case of a given target strain, the loading part of each cycle was performed when the preloading force reached 0.5 kN, while after attaining the target strain, the unloading part was started and finished until 0.5 kN. What calls for special attention is that for the The test results implied that despite significantly different reinforcement ratios and loading patterns, the level of target strain in the case of cyclic tension led to a distinct difference in the failure pattern of steel-reinforced HSHUHPC members caused by various eventual accumulative ultimate strains. When the level of target strain was 1000 με, after 10 cycles, all the specimens exhibited multiple micro-cracks invisible to the naked eye. It is worth noting that the boundary of the crack width is 0.05 mm between the invisible micro-crack and the visible macro-crack. This also demonstrates that the HSHUHPC possesses good control ability in terms of crack width.When the levels of target strain for the tested specimens are 2000 με and 2500 με, after 10 cycles, the maximum tensile strain generally has exceeded the yield strain of the reinforcement and ultimate tensile strain of HSHUHPC. The damage pattern is transformed into a single macro-crack along with a series of narrow and closely spaced micro-cracks. The main reason is that the tensile strain-hardening characteristics of the HSHUHPC provide the steel-reinforced HSHUHPC members with an effective crack width control ability for the initially opened multiple micro-cracks prior to the yielding of the embedded steel rebar. Once the embedded steel rebar begins to yield, the gradually increased demand for deformation on the specimen progressively results in the occurrence and propagation of a localized macro-crack at the weakest cross section, possibly where the least effective number of fibers are distributed in the direction parallel to the tension loading; however, the local strain in the other segment is still below the ultimate strain. Eventually, there is a pronounced post-yield localized macro-crack. It is worth noting that the occurrence of the visible macro-crack was between the yielding strain of the steel rebar and the ultimate tensile strain of HSHUHPC, that is, around 2700–2840 με. This may be due to the fact that as the steel rebar yields, the steel fibers at the crack are motivated to debond and pull out gradually and the synergistic effect between the steel rebar and HSHUHPC weakens. Besides, the steel fibers at the crack cannot balance the large deformation demands of the steel rebar after yielding, despite the presence of the bridging effect of the steel fibers. As shown in As shown in When the level of the target strain was 2000 με and 2500 με, the residual strain of steel-reinforced HSHUHPC specimens gradually remained stable once the steel rebar yielded as the number of cycles increased. Upon the yielding of the steel rebar, the crack pattern of the steel-reinforced HSHUHPC transformed the uniformly distributed micro-cracks into a localized single macro-crack. Meanwhile, once the crack width progressed beyond a certain threshold, the role of the bridge effect of fibers in the cracks was enhanced and gradually undermined due to the debonding and pull-out of the fibers.i-th cycle to the initial loading stiffness, and in the same way, the normalized unloading stiffness of each cycle refers to the ratio of the unloading stiffness at the i-th cycle to the initial unloading stiffness. These reflect the stiffness degradation degree of the members. Regardless of the level of the target strain, the normalized loading stiffness and unloading stiffness were considerably reduced during the 2nd or 3rd cycle and remained stable with some fluctuations as the number of cycles increased. Compared with For all the steel-reinforced HSHUHPC specimens, it could be observed that the size of the embedded rebars had a positive effect on the tensile stiffness of the specimens in the elastic stage. Besides, in the case of the elastic–plastic stage, increasing the diameter of the steel rebar upgrades the tensile stiffness effectively. AE analysis technology could effectively monitor and detect the internal damages of UHPC under cyclic tensile loading on the microcosmic level. As mentioned above, the diameter of the embedded steel rebar has little influence on the damage pattern; thus a typical specimen for each level of target strain was discussed herein. Additionally, in order to simplify the analysis, the research took 8 cycles for in-depth analysis due to the fact that the experimental phenomena showed that after the 4th cycle, the loading and unloading curves would tend to be stable for all the specimens. The total tensile bearing capacity of steel-reinforced HSHUHPC consists of steel rebar and HSHUHPC. By getting rid of the pure steel rebar response from the total tensile load–strain envelope curve, the contributions of HSHUHPC in the steel-reinforced HSHUHPC member, usually called the tension-stiffening effect, are obtained as shown in In addition, it is hypothesized that steel yields when the concrete crushes in traditional reinforced concrete design methods based on the mechanics of slender beams. It is observed experimentally that an early strain hardening effect in the steel occurs due to the high bond strength between HSHUHPC and steel rebar and the tension-stiffening effect. Therefore, traditional methods based on the mechanics of slender beams might typically underestimate the strength of reinforced HSHUHPC components subjected to bending, even when accounting for the tensile capacity of the HSHUHPC material. Further experimental research, including the effect of reinforcement ratio, layout of reinforcement, and evaluation of the flexural capacity of the UHPC slender beam considering the tension-stiffening effect, need to be done in combination with numerical simulation, theoretical calculation, and so on.2) of 0.902, 0.869, 0.776, and 0.913 for different diameters of the embedded steel rebar. The presence of some large discreteness is due to the great difference in stiffness degradation rate at the elastic stage and the strain-hardening stage, respectively. The relationship model needs to be studied further using more tests.As mentioned above, it could be supposed that there was hardly a slip generated between HSHUHPC and steel rebar prior to the yielding of the steel rebar. Hence the steel-reinforced HSHUHPC could be regarded as the isotropic material and undergo linear elastic deformation in coordination between steel rebar and HSHUHPC until reached at the ultimate tensile load. According to the research study by Guo Jun-Yuan et al. , it is aTwelve steel-reinforced HSHUHPC specimens under cyclic axial tension, accompanied with AE sources locating technology, were tested in this paper. The effects of the loading pattern, the diameter of the embedded rebar, and the level of target strain in each cycle on the tensile behavior of steel-reinforced HSHUHPC were studied. The following conclusions were drawn:(1)For a target strain of 1000 με in each cycle, the typical failure of steel-reinforced HSHUHPC exhibited uniformly distributed multiple micro-cracks due to the excellent crack width control ability of HSHUHPC, while for the target strain of 2000 and 2500 με in each cycle, the typical failure pattern exhibited a single, localized macro-crack in conjunction with a sprinkling of narrow and closely spaced micro-cracks due to the inclusion of steel fibers, which intensified the strain concentration in the embedded steel rebar. AE source locating technology provides strong evidence for the damage distribution of the whole loading process.(2)In terms of load–strain response and envelope curves, the larger the diameter of embedded steel rebar, the smaller the maximum accumulative tensile strain under cyclic tension, which indicated that the larger the diameter of embedded steel rebar, the greater the contribution to the stiffness of the R-HSHUHPC specimens in the elastic–plastic stage.(3)For the target strain levels of 2000 με and 2500 με in each cycle, once the embedded steel rebar yielded and the crack width progressed beyond a certain threshold, fibers began to pull out, and the ability of the fibers to bridge the cracks began to decrease. Hence the residual strain and stiffness degradation rate in each cycle remained stable.(4)According to the tension-stiffening response of steel-reinforced HSHUHPC members, the larger the embedded steel rebar, the lower the peak tensile strength of the HSHUHPC after deducting the steel rebar. This is most likely due to the fact that the larger diameter of the steel rebar results in a worse discontinuous distribution of the steel fiber, although the contact area between steel rebar and UHPC increases, and due to the presence of autogenous shrinkage, the bond interaction at the interface between steel rebar and UHPC is weaker.(5)The relationship model between the loading stiffness degradation ratio and the cumulative residual strain for steel-reinforced HSHUHPC members was proposed. The relationships can be employed to evaluate the stiffness of steel-reinforced HSHUHPC under cyclic tension to some extent.Further studies are needed to include different fiber volumes, steel rebar diameter-to-cover ratios, and reinforcement ratios to better understand how the mutual contribution of steel fibers and rebars, splitting cracks, and so on influence the damage progression and deformation capacity of the reinforced UHPC. Additional tests are necessary to evaluate the flexural capacity of reinforced UHPC members considering the tension-stiffening effect.


The declaration of the Mediterranean Diet as Intangible Cultural Heritage of Humanity in order to preserve a cultural and gastronomical legacy included the protection of lifestyles, knowledge, sociability, and environmental relationships. However, the patrimonialization, popularization, and globalization of a certain conception of this diet have turned it into a de-territorialized global phenomenon. As a consequence of this process, it has been necessary to notably increase the production of its ingredients to satisfy its growing demand, which, in turn, has generated “secondary effects” in some Mediterranean environments of Southeastern Spain. If, on the one hand, their wealth has increased and population has been established, on the other hand, the continuity of certain cultural landscapes linked to local knowledge and particular lifestyles has been broken, replacing them with agro-industrial landscapes exclusively at the service of production. This, at the same time, has caused social and environmental inequalities It has become commonplace to bring up a certain saying attributed to Josep Pla—“the cuisine of a country is its landscape set in a pot”—to refer to the relationship between diet and landscape. As if, by necessity, the landscape of a particular space limited and conditioned its dominant diet, establishing a relationship of dependence between environmental conditions and the possibilities of nourishment. In a way, this long-held idea underlies, as well, the Resolution approved by the European Parliament  on 12 MaIn this paper, I present some concrete examples of the environmental, social, or cultural impacts that the rise of this diet is having, or at least a certain way of conceiving it, in some specific areas of the Spanish Mediterranean. Due to the mobility restrictions inherent to the current pandemic, these pages cannot include recent information produced by field work and are based on the analysis of the existing literature on these places, completed with a follow-up of the documentation generated by the administrations public and collected in the specialized magazines. Thus, in the first place, I tell how the conception of the Mediterranean Diet promoted by UNESCO and other organizations has been gradually replaced by a biomedical consideration that, instead of emphasizing landscapes and lifestyles, highlights the value of products regardless of their origin and how they were produced. Below are the specific effects that this conception is having on certain cultural landscapes that are being replaced by new agro-industrial landscapes that, in turn, are changing the ways of life that the UNESCO declaration said must be protected. With this, it is exposed how the declaration of the Mediterranean Diet as a World Heritage Site and its promotion, in addition to the positive effects on the health of those who consume it, may also have had negative effects in some environmental and social contexts.Sweetness and Power. He states that, generally speaking, human groups hardly ever eat every foodstuff found in their surroundings, and, while some are cherished, others are rejected. Likewise, Jesús Contreras, Antoni Riera, and F. Xavier Medina [https://www.alimentosdespana.es/es/campanas/ultimas-campanas/alimentos-de-espana/el-pais-mas-rico-del-mundo/default.aspx (accessed on 29 January 2021)).The intimate connection between landscape and diet was already questioned in 1985, by Sidney Mintz , when anr Medina  have poir Medina  (p. 18).r Medina  (p. 46),r Medina  or consuHowever, if the relationship between landscape, product, and diet is no longer a direct one, but one that comes mediated by other elements , it is worth questioning if the foodstuff that include in their nomenclature the name of a place must have been produced fully in said location or if it is enough for this place to be present in some phase of its manufacturing. For instance, when making wine coming from any of the Designation of Origin (DO) areas, must the grapes have been harvested in said DO, or can they have been harvested far away and processed in the DO? Or, considered from across the pond, can we consider a “Mediterranean diet” one that employs products with similar characteristics and features to those of the heterogeneous Mediterranean landscapes when these products have been grown in their totality in California or Mexico? Ultimately, does a Mediterranean Diet require a Mediterranean landscape? Although, apparently, the answer to these questions seems simple, in practice it is conditioned by the existence of different conceptions about what the Mediterranean Diet is.As part of the defense prepared by the permanent representatives of Spain, Greece, Italy, and Morocco to the UNESCO to promote the inscription of the Mediterranean Diet in the Representative List of the Intangible Cultural Heritage of Humanity, it is explicitly stated that “The Mediterranean Diet is the set of skills, knowledge, rituals, symbols and traditions, ranging from the landscape to the table, which in the Mediterranean basin concerns the crops, harvesting, picking, fishing, animal husbandry, conservation, processing, cooking, and particularly sharing and consuming the cuisine”  (p. 6), https://ich.unesco.org/es/RL/la-dieta-mediterranea-00884 (accessed on 22 January 2021)) will be able to confirm how, even though this list includes knowledge, skills, traditions, agriculture-related symbols, ways of managing food, commensality, fellowship, communal living, intercultural dialogue, craftsmanship, holidays, markets, etc., there is no reference to Mediterranean landscapes. Likewise, any similar mention is missing from the inventory that supports the Intangilbe Cultural Heritage candidacy presented by Spain (https://ich.unesco.org/doc/src/19700.pdf (accessed on 22 January 2021)). Landscape also fails to play a fundamental part in the candidacy of the set of institutions who joined the proposal, whose consent is included in https://ich.unesco.org/doc/src/16813.pdf (accessed on 22 January 2021). In fact, half of the associations or institutions that supported this candidacy present it both generically and vacuously, as an indispensable addendum that needs to be named in order to fulfil bureaucratic requirements. Furthermore, references to the landscape appears in a vague text—the same one in almost every case, merely changing the name of the signing institution—that states the inclusion of the Mediterranean Diet in the aforementioned list must be contemplated due to the negative effects that “globalization and socio-cultural changes” are having on “the health and welfare of people, the stability of the rural population, and the protection of the environment, landscape and culture of Mediterranean communities” . At the same time, a significant number of the signers do not even explicitly mention the relationship between landscape and diet, or they present a variation on the stated formula.However, in spite of these considerations, those engaging the information offered by the UNESCO in regards to the 2013 inscription of the Mediterranean Diet in the Representative List of the Intangible Cultural Heritage of Humanity (https://ich.unesco.org/doc/src/16813.pdf (accessed on 22 January 2021). Annexe 4).Something similar takes place with the 41 proves of consent by the associations or institutions for their knowledge, practices, or activities to be disseminated in order to support the presentation of the candidacy of the Mediterranean Diet to UNESCO, linked to the city and province of Soria. While most of them mention landscape in passing, only the document signed by the Sorian Association for the Defence and Study of Nature—Asociación Soriana para la Defensa y Estudio de la Naturaleza ASDEN (Ecologistas en acción)—points out the importance of this inclusion, “in order to better the health of citizens, as well as their quality of life, collaborating with the conservation of peculiar ecosystems with centennial traditions, and the conservation of peculiar landscapes” ). That is, apparently, the Sorian support for the Mediterranean Diet would assume that one of the defining elements of a local diet—every association endorsing the project showed the connection between their local or provincial activities and this local diet—is a product that requires importation from other places, since the province’s environmental conditions preclude its manufacture. In this case, therefore, the knowledge, traditions, symbols, food management, etc., presented by the UNESCO declaration are necessarily extra-local and, in consequence, point to a disconnection between diet and landscape.In spite of its continental features and its lack of proximity to the Mediterranean Sea, Soria’s shows of consent proved decisive in the process of making the Mediterranean Diet Intangible Heritage, since Soria was the first Spanish town to support the candidacy through a long process where diverse interest came into conjunction . During This separation becomes possible because in the particular case of the Mediterranean Diet, this crucial role in the patrimonialization process has been played by agents linked in one way or the other to the medical profession and food science. Within the 53 backers of the inscription of the Mediterranean Diet presented by the permanent representatives of Spain, Greece, Italy, and Morocco, we find, among others, the Spanish Academy of Nutrition and Food Science, the University of Barcelona’s Research Group for Communitarian Nutrition (Grupo de Recerca en Nutrició Comunitària), the Spanish Federation of Food and Wine Guilds, the Mediterranean Diet Foundation, the Mediterranean Agronomical Institute of Zaragoza, the Department of “Food Systems, Culture and Society” of the Open University of Catalonia, the NGO Nutrition without Borders, Foundations for Nutritional Research, the UNITWIN-UNESCO Chair of Research, Planning and Development of Local Sys-tems of health of the University of Las Palmas de Gran Canaria, the UNESCO Chair of Visual Health and Development of the UNESCO chairs network of the Polytechnic University of Catalonia, the Foundation for the Promotion of health, etc.In consequence, its relevance is supported by the positive effects following this diet has, and not on its connection with social or cultural processes in any particular region. However, as an “ideal model of a healthy diet”  (p. 19),If there ever was such a thing as the customs, styles, and values that classical social anthropology in the 20th century identified as prototypically Mediterranean in regards to food consumption, the actual identification of the Mediterranean Diet and its bio-medical justification has superseded them for good. This process involves a “substitution of traditional and socially regulated reasons that recommended eating this or that, in a particular state, for other nutritional and scientifically legitimated reasons, in such a way that the hierarchy of goals of the act of eating—pleasure, socialization, health—varies”  (p. 201)https://cruceroland.com/recetas-sanas-faciles-gastronomia-mediterranea/ (accessed on 20 January 2021)), while both the ecosystems and coast-adjacent residents see themselves as being highly affected by the environmental—among other kinds—pollution these ships generate. In fact, even though it is difficult to establish a linear causal relationship, the emphasis on the products without a focus on the environmental contexts where they are grown sometimes generates very negative effects on the second of these.In this setting, it is not surprising, for instance, that the hundreds of cruise ships that would, before the emergence of this devastating pandemic, cross the waters of the Mediterranean, carrying thousands of tourists from port to port, could offer in their many restaurants “Mediterranean gastronomy” or “Mediterranean Diet” .Olimerca  (p. 21).Furthermore, of a drastic nature are the changes generated in the landscape by the importance the Mediterranean Diet gives to horticulture. According to the World Vegetable Map 2018 report, developed by RaboResearch Food & Agribusiness, there is a “growing importance of production in greenhouses and vertical farms” . AccordiThe price paid in exchange has been an increase in social inequality up to hard-to-conceive levels and the depletion of aquifers that the greenhouse owners want to recover by expanding saltwater desalination and reducing underground water consumption . In the On top of the substitution of heterogeneous rural landscapes associated with local production by others, homogenous and agro-industrial in nature, the emphasis on the product itself can also affect the ethno-ecologies and put “bio-cultural memory” at risk . As thesIn other occasions, these risks to the upkeep of local know-how is related to the attempts to achieve heritage status for a particular product through a certification of quality. Thus, Cantero and Ruiz Ballesteros  counter Productive homogeneity allows for the easy identification of a product, and, in consequence, the achievement of heritage status for itself or any other aspect tied to it—an area, a landscape, a custom, a dish, etc. This status, however, may generate discomfort and resistance. For instance, some wineries are abandoning the Rioja Qualified Denomination of Origin , “in order to offer the consumer the opportunity to discover the diversity of our land”  However,On the other hand, this move toward the “ecological” or “sustainable” being identified with the “local” or “traditional” may have quite diverse consequences: Some defend this trend as being a result of an exercise in awareness of the limitations of the planet and the effects of climate change; others, however, set their eyes on economic factors and see this as an alternative to the industrial Common Agricultural Policy (CAP) that, they believe, has generated negative consequences in the rural sphere. There can also be found some who understand agro-ecology as a part of the defense of food sovereignty or linked to the love of the land. For others, finally, it is a simple fad that is largely related to “the association of its consumption to a particular status and social class” that turns its products into “referents of exclusivity, sybaritism, and elitism”  (p. 67).The conversion of the multiple particular diets associated with the different Mediterranean landscapes in which they were produced and consumed into a single global diet is having very negatives effects on some of the Spanish landscapes associated with the products that medical rationality considers better for health. That is, by focusing the diet on the products, regardless of how and where they are produced, some of the landscapes, far from being protected as the UNESCO declaration foresaw when it was incorporated into the Representative List of the Intangible Cultural Heritage of Humanity, are suffering irreversible transformations that distance them from the complex cultural landscapes stereotypically characterized as “Mediterranean”, becoming new and uniform agro-industrial landscapes. These new landscapes, subordinated to market logics, by breaking with the historical processes that have originated them, produce the “Mediterranean”, but they are no longer “Mediterranean”.The transformation of cultural landscapes into a commodity destined exclusively to produce merchandise in a process of de-historization that causes an emptying of the meanings linked to local identities and connections, allows us to evoke the image of the Mediterranean within a transnational political economy of feelings ,71 that No Logo, suggested that “we were so busy analyzing the pictures being projected on the wall to notice that the wall itself had been sold” [Naomi Klein, in her renowned work en sold”  (p. 161)In accordance with this market logic, food, landscapes, customs, etc., have been selected as referents of heritage in a process that, equally, disregards everything that does not fit within these parameters and that makes it difficult to consume the image they evoke. Mediterranean landscapes and diet meet in the global space of the tourist who, while consuming both of them at the same time, buys, above all, their image.In any case, this de-territorialized and de-historized image is continuously reformulated due to tensions between local and global dynamics. On the one hand, Mediterranean gastronomy, on which the diet is based, renovates itself constantly though the transformations  that take place within local and regional cuisines. On the other hand, landscapes must continuously adjust to settled processes of socialization and to multi-levelled normative that incorporate local and regional demands linked to the processes that reconfigure them.


Pseudorhaetus sinicus is a stag beetle common to China and Vietnam, but whose distribution is limited within China. Little is known about the molecular biological characteristics of this species, so we characterized its complete mitochondrial genome (GenBank accession number MZ504793.1). The mitogenome consists of a circular DNA molecule of 18,126 bp, with 67.693% AT content. It contains 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. The PCGs have typical ATN (Met) start codons and TAN stop codons. Phylogenetic analysis suggests that P. sinicus is closely related to Prosopocoilus confucius. This newly described mitochondrial genome provides a valuable resource for the phylogenetic analysis of Lucanidae beetles. Pseudorhaetus sinicus, which belongs to order Coleoptera, family Lucanidae, is distributed mainly in Vietnam and China. It is commonly found in Fujian, Zhejiang, Jiangxi, and Guizhou Provinces, China. The partial mitochondrial genome (KP987575.1) of P. sinicus from Daming Mountain in Guangxi Province, China, has previously been sequenced , Fujian Tianbaoyan National Nature Reserve, Yong’an City, Sanming City, China, on 24 September 2020, and deposited by Yu Cao (Email: yucaosuccess@126.com) in the animal specimen room of Guiyang University (specimen accession number: GYU-20200924-001). Total genomic DNA was isolated using an Aidlab Genomic DNA Extraction Kit . Universal primers were designed (Supplementary Table 1) to match generally conserved regions to amplify short fragments from 12S and 16S rRNA, cox1, cox2, nad1, nad2, and nad5. PCR products were cloned into a pMD18-T vector  and then sequenced, or sequenced directly by the dideoxy nucleotide procedure, using an ABI 3730 automatic sequencer . Thirty-six short sequences were obtained which range in length from 253 bp to 1081 bp.A specimen of adult P. sinicus (GenBank accession number: MZ504793.1) was assembled manually. It is a circular DNA molecule that is 18,126 bp long . Using Perna and Kocher’s formula (Perna and Kocher The complete mitogenome of P. sinicus mitogenome contains 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes, which were annotated using the MITOS web server (http://mitos.bioinf.uni-leipzig.de/)  from Daming Mountain, Tenebrio obscurus  had ATA start codons, one (nad2) had an ATC start codon, five  had ATG start codons, and four  had ATT start codons. All 13 PCGs had typical TAN stop codons. Four genes  had TAA stop codons, four  had TAG stop codons, and five  had incomplete stop codons that were completed by the addition of A nucleotides at the 3′ ends of the encoded mRNAs. The 22 tRNA-encoding genes were interspersed throughout the coding region and ranged from 61 bp (tRNA-Cys) to 71 bp (tRNA-Lys) long. The genes encoding large rRNA and srRNA were 1269 bp and 760 bp long, respectively.The P. sinicus, its mitochondrial PCGs and those of 16 other species in class Insecta were used to construct a maximum-likelihood phylogenetic tree with 1000 replicates using MEGA X software (Kumar et al. T. obscurus (Bai et al. Z. atratus (Bai et al. P. sinicus is closely related to Prosopocoilus confucius (Lin et al. P. sinicus, and also provides essential genetic and molecular data for further phylogenetic and evolutionary analyses of family Lucanidae.To validate the phylogenetic position of