Idiosyncratic drug-induced liver injury (DILI) caused by xenobiotics  presents with a range of both phenotypes and severity, from acute hepatitis indistinguishable of viral hepatitis to autoimmune syndromes, steatosis or rare chronic vascular syndromes, and from asymptomatic liver test abnormalities to acute liver failure. DILI pathogenesis is complex, depending on the interaction of drug physicochemical properties and host factors. The awareness of risk factors for DILI is arising from the analysis of large databases of DILI cases included in Registries and Consortia networks around the world. These networks are also enabling in-depth phenotyping with the identification of predictors for severe outcome, including acute liver failure and mortality/liver transplantation. Genome wide association studies taking advantage of these large cohorts have identified several alleles from the major histocompatibility complex system indicating a fundamental role of the adaptive immune system in DILI pathogenesis. Correct case definition and characterization is crucial for appropriate phenotyping, which in turn will strengthen sample collection for genotypic and future biomarkers studies. Drug-induced liver injury (DILI) is probably one of the most intriguing and complex liver diseases because unlike other safety problems with the use of drugs, the facets of hepatotoxicity are multiple, difficult to approach and with great potential impact in clinical drug development. DILI is a broad condition that symptomatically can mimic most kinds of acute and chronic liver conditions. Despite stringent requirements for drug development imposed by regulatory agencies, DILI is an increasing health problem and a significant cause for failure to approve drugs, market withdrawal of commercialized medications and adoption of regulatory measures. Toxic liver disease is a challenging differential diagnosis for the doctors, not only because of its potential severity, but also by the inability to establish a definitive diagnosis in most cases. Acute DILI has around a 10% probability of evolving into chronic and severe forms, and even be fulminant . Gastroewww.spanishdili.uma.es) and the Drug-Induced Liver Injury Network (http://dilin.duke.edu) are collaborative multicenter networks with large databases and biosample collections of prospectively recorded DILI cases in Spain and the US, respectively, and subsequently important resources for hepatotoxicity studies. In population based studies there are a greater proportion of asymptomatic cases in comparison with the cases included in the DILI Registries probably because of the selection bias of reporting to Registries only the more severe cases [Data on DILI incidence in the general population are very limited. In a prospective study of a population of 80,000 inhabitants of northern France, conducted during 1997–2000, the incidence of DILI was 13.9 cases per 100,000 patients-year, a rate 16 times higher than those reported to the French National Agency . More reIn this review, we addressed aspects on phenotype characterization, clinical presentation and outcomes, as well as the main risk factors both from the drug and the host known to be involved in idiosyncratic drug induced liver injury.DILI has a very broad spectrum of presentation, both in phenotype and severity this last ranging from asymptomatic elevations of liver aminotransferases to acute liver failure. DILI can mimic almost every other liver disorder including rare syndromes such as vascular disorders and liver tumors although the most common presentation is an acute viral hepatitis-like syndrome . Despitei.e., statins) can occasionally induce minor and transient alterations in liver tests. Hence, a consensus group proposed to meet one of the following criteria as a prerequisite to consider a case like DILI [In the clinical setting, case characterization usually does not rely on liver histopathological manifestations because liver biopsy is not routinely performed in suspected cases of hepatotoxicity. Instead, liver biochemistry is used to define liver injury. The analytical determinants for DILI has changed over the last 20 years and today discrete elevations of aminotransferases have an uncertain meaning as modest elevations in the liver profile is now more frequently seen possibly due to the growing prevalence of non-alcoholic fatty liver disease in general population and the fact that some drugs (ike DILI : (a) alaR = (ALT patient/ULN)/(ALP patient/ULN). The resultant pattern is classified as hepatocellular (R ≥ 5), cholestatic (R ≤ 2) and mixed cases (R > 2 and <5)  .R-value scores as hepatocellular [R-value during disease progression [nR), (ALT or aspartate-aminotransferase (AST), whichever was highest, /ULN/(ALP/ULN) at DILI onset. The rationale for assessing this nR was based on the finding that AST level was independently associated with the development of acute liver failure/orthotopic liver transplantation (OLT) at all-time points being most predictive at DILI recognition [The international consensus also recommended that AST substitute ALT when the latter is unavailable . On the cellular . The firgression . In the gression  the pattgression  , while bile plugs and duct paucity appeared more commonly in cholestatic cases (R ≤ 2) [Nevertheless, tentative correlations between histological findings and biochemical classifications found lobular disarray and rosette formation to be more prevalent in hepatocellular cases ( (R ≤ 2) . However: Elevated ALT/ALP values reaching criteria for DILI and TB ≥ 2 × ULN, or symptomatic hepatitis; (c) Severe: Elevated ALT/ALP values reaching criteria for DILI, T ≥ 2 × ULN, and one of the following: (1) International normalized ratio (INR) ≥1.5; (2) ascites and/or encephalopathy, disease duration <26 weeks, and absence of underlying cirrhosis or (3) other organ failure considered to be due to DILI; and (d) Fatal: Death or transplantation due to DILI. Similarly, the DILIN group developed an operational procedure system for severity classification [The degree of elevation of enzyme levels alone may not reflect the severity of liver injury because these values do not accurately predict specific clinical outcomes. Hence, a consensus group graded severity taking into consideration clinical and laboratory features  as follofication . The disWith regard to the prediction of severe outcomes in DILI, in the late 1960s it was recognized by Zimmerman, the pioneer of modern liver toxicology, that a patient who presents with jaundice as a result of a drug-induced hepatocellular injury (without a significant obstructive component and once other causes for increased bilirubin levels such as hemolysis or Gilbert syndrome are excluded) had at least a 10%  chance of fatal liver failure . This isR or nR ≥ 5) accompanied by hyperbilirubinemia, without excluding cases based on ALP level. This group also developed a composite algorithm to predict an ALF outcome in idiosyncratic DILI cases which showed improved specificity (82%) and sensitivity (80%). This algorithm, which still requires further validation in a larger prospective DILI cohort, is based on analytical parameters  from the first available blood test after DILI initiation [et al. [Recently, the Spanish DILI Group  has reasitiation  and is i [et al.  study; n [et al. . Interes [et al. . This fiThe most common form of presentation of DILI is an acute viral “hepatitis-like” syndrome, with jaundice, nausea, fatigue and abdominal discomfort or pain . HoweverR ≥ 5. Sometimes there are features of hypersensitivity, such as fever, rash or peripheral eosinophilia, suggesting drug allergy. This phenotype is more frequent in young females  in the DILIN analysis [versus 1% ALF/OLT in cholestatic group).Acute hepatocellular injury is defined as ALT ≥ 5 × ULN or analysis ,26. The analysis . In hepaR < 2, and there are two subtypes: pure cholestasis  and acute cholestasis or hepatocanalicular hepatitis. The probability of a cholestatic phenotype increases with advancing age and is more common in males [Acute cholestatic injury is defined as ALP > 2 × ULN or in males . Its usuin males .Pure cholestasis is characterized by increased serum levels of conjugated bilirubin, ALP and G-glutamyl transpeptidase (GGT) with little or no alteration of transaminases. Histologically, it presents with hepatocyte cholestasis and dilated biliary canaliculi with bile plugs, without evidence of necrosis or inflammation. This type of lesion is characteristic of contraceptives and anabolic steroids .In Hepatocanalicular hepatitis there may be fever and abdominal pain simulating acute biliary obstruction, and often associated hypersensitivity features. Pathological findings include, portal and ductal inflammation as well as hepatocyte necrosis with marked predominance centrilobular cholestasis predominance .Among the drugs involved in this subtype are amoxicillin-clavulanate, macrolides, azithromycin , fluoroqMixed hepatic injury is defined as R between 2 and 5 being clinical and laboratory abnormalities intermediate between the cholestatic and hepatocellular injury. The manifestations of drug allergy are more common. Almost all drugs that induce cholestatic hepatitis can also cause this type of liver injury .Chronic DILI (unresolved injury 6 months after onset) was much more frequent among cholestatic cases 31%) than either hepatocellular (13%) or mixed-injury cases (14%) in the DILIN analysis [% than eiThe definition of chronicity in DILI remains a matter of debate, The International Consensus Conference  it is re+) were more numerous in immunostains of liver biopsies from patients with idiopathic AIH as compared with those of drug-induced AIH [An increasingly reported phenotype is that of DILI with autoimmune features . This phuced AIH . The latuced AIH , and moruced AIH  and statuced AIH .Recently, immune check-point blockade by manipulating either cytotoxic-T lymphocyte A-4 (CTLA-4) or programed death-1 (PD-1) receptors has attracted attention in its relationship to idiosyncratic DILI pathogenesis indirectly supporting the importance of immune pathways. CTLA-4 mediated immune checkpoint is induced in T cells at the time of their initial response to antigen, dampening the amplitude of the initial response. In contrast, the major role of the PD1—an inhibitory molecule of lymphocyte activation-pathway is not at the initial T cell activation stage but rather to regulate inflammatory responses in tissues by effector T cells recognizing antigen in peripheral tissues. Thus, immune checkpoint blockade leads to vigorous immune responses and this concept is used to uncover potent antitumor immune responses that are depressed in cancer . Intereset al. [Steatosis is another clinicopathological pattern that can result from the toxic effect of drugs. Macrovesicular steatosis is characterized by variable degrees of accumulation of large fat droplets with peripheral displacement of the nucleus without significant inflammation or cholestasis. Tetracyclines, steroids, gold, 5-fluorouracil, methotrexate or tamoxifen are examples of drugs capable of inducing this pattern of injury ,23. The et al.   is secondary to endothelial cell injury to small hepatic venules that manifests as endothelial swelling and thrombosis. This pattern is related to cytotoxic drugs, myeloablation before stem cell transplantation and bone marrow transplantation . GeneticThe idiosyncratic DILI is basically thought to be due to the interaction of three factors: a drug that has the potential to be harmful to the liver, a genetically susceptible subject and the intervention of other host and environmental factors . The rarTherefore, only in carriers of the HLA risk alleles would occurs the presentation of the haptenized reactive metabolite by antigen-presenting cells to the adaptive immune system (“hapten hypothesis”) ,52.Often, it is necessary a co-stimulus initiated by releasing DAMPS (Damage Associated Molecular Patterns), which are released by the reactive metabolites that damage liver cells and starts a sterile inflammation, which stimulates the innate immune system through the “toll-like receptors” of antigen-presenting cells. The production of proinflammatory cytokines that follows stimulate the adaptive immune system, which induces a direct cytotoxic response or mediated by antibodies against hepatocytes . For many drugs in which the risk of DILI is associated with certain HLA alleles, toxicity will occur only in carriers of the alleles of interest, but people without this genetic profile would be free of risk . In contDILI is generally divided into intrinsic and idiosyncratic reactions based on its dose dependency and predictability. Intrinsic hepatotoxicity is dose dependent and can be predicted in animal models. Idiosyncratic DILI is not related to the pharmacological properties of a drug and, hence, unpredictable. However, it is now becoming apparent that idiosyncratic DILI is not entirely a dose independent event. In fact, drugs given at a low daily dose are rarely associated with a high incidence of idiosyncratic ADRs, because even for allergic reactions a threshold dose (≥10 mg) is usually required . FurtherLipophilicity is an important pharmacological property, correlating with drug uptake and metabolism. Analysis of large sets of compounds revealed that the lipophilic compounds are more likely to produce toxic events ,60.P > 3), which is indeed, simply a surrogate for extensive biotransformation and hepatic exposure to a parent drug or reactive metabolite has been called the “rule of 2” and was present among a majority of drugs with proven liver toxicity in two independent databases of the Food and Drug Administration (FDA)-approved drugs labeled for the presence or absence of liver injury [et al. [The combination of both factors in a given drug (daily dose > 100 mg/day) and high lipophilicity  can directly damage DNA, proteins, enzymes, and lipids in cells and tissues and induce immune-mediated liver damage . In an iu et al.  used conIn a recent study  in rat lThe crucial role of mitochondria as cellular energy supplier can explain its importance in the pathogenesis of DILI. Mitochondrial damage can trigger apoptosis and/or hepatic necrosis, leading to the activation of a signaling pathway of cell death when a threshold of mitochondrial damage is exceeded . It has SOD2 Ala) and glutathione peroxidase (GPX1 Leu) genes, which are involved in mitochondrial oxidative stress, are more prone to develop cholestatic DILI particularly from drugs forming electrophilic metabolites or that are mitochondrial hazardous [A recent study  on isolaazardous .An interesting hypothesis gives support to the antibiotics risk of inducing mitochondrial impairment. Being of bacterial ancestry, the mitochondria share many genetic and structural similarities with bacteria that could render mitochondria more vulnerable towards antibiotics. This may explain, at least in part, (other explanation being their effects on microbioma) why antibiotics top the list of causative agents among DILI cases identified to date .In vitro testing of 200 benchmark compounds by the use of human BSEP inverted vesicles demonstrated that most tested compounds found to have potent inhibitory effect on BSEP (IC50 ≤ 25 µM) for example troglitazone, ketoconazole, nefazodone and lapatinib, have been associated with human liver liabilities [in vitro testing the median potency of BSEP inhibition was higher among drugs that caused cholestatic/mixed DILI than among drugs that caused hepatocellular or no DILI [et al. [Inhibition of the hepatic bile salts efflux transporter bile salt export pump (BSEP) leads to accumulation of toxic bile salts in hepatocytes, which could lead to cell damage. Consequently, drugs that exhibit inhibitory effects on BSEP can have hepatotoxic potential . In vitrbilities . In anot no DILI . However no DILI . Interes no DILI . Thus, d [et al.  found thin vitro models focusing on a single transporter do not reflect the true activity in vivo. In cholestatic conditions, the multidrug resistance-associated protein 3 and 4  transporters eliminate bile acid from the hepatocyte into the blood in order to control intracellular bile acid accumulation. Moreover, the multidrug resistance-associated protein 2 (MRP2) can eliminate divalent bile acids from the hepatocyte [However, patocyte . Therefopatocyte ,84.Aging is known to cause pharmacokinetic changes due to decreased renal function, hepatic mass, blood flow and cytochrome-mediated hepatic metabolism. Furthermore, age-related lean body mass reductions can affect the volume of distribution . Despiten = 133), 77% of cases were women [For some drugs like diclofenac, tetracyclines and nitrofurantoin, liver damage occurs more frecuently in females ,90; but re women .Ethnical differences in DILI incidence rates among countries can be a reflection of genetic differential background as well as variations in many other factors including life-style, consumption of herbals, regional medication policies and prescription habits. In a multiracial country such as USA, the DILIN group was unable to find an ethnic difference in DILI frequency. However, Asian race was an independent risk factor for transplantation and Afroamerican race was a major determinant for chronic evolution in a 6-months prospective follow-up of the DILIN Registry .Pre-existing liver disease is not associated with increased risk of idiosyncratic hepatotoxicity for the bulk of medications, an exception being patients with alcoholic liver disease treated with methotrexate, and patients with chronic B and C hepatitis, especially if they are co-infected with HIV . For exaversus 5.2%) in a recent update of the DILIN database [Contradicting results with regards to the role of chronic viral co-infections as risk factors in anti-TBC hepatotoxicity have been reported with studies being unable to find any association ,96 and odatabase .An increased risk of toxicity of methotrexate in patients with psoriasis was observed compared to patients with rheumatoid arthritis, although there are confounding factors that limit these observations  ,102. DiaDrugs and other xenobiotics may theoretically modulate the hepatotoxic potential of other drugs by inducing/inhibiting its metabolism through cytochrome P-450 (CYP) or competing at the level of membrane transporters . The besA retrospective study from the General Practice Research Database (GPRD)  found inIn an analysis of patients with statin hepatotoxicity reported to the Swedish Adverse Drug Reactions Advisory Committee, no clinically significant interaction was found with concomitant medication .In summary, Clinical Networks in Drug-Induced Liver Injury have offered an opportunity for advancing Safety Science and translational research in DILI, providing new insights in clinical phenotypes and severity, and allowing performing pharmacogenetic and mechanistic studies. The complex and multilayered aspects of DILI will require in the near future an integrative approach taking into consideration the combined role of genetic and drug-host-enviromental interactions. Systems biology applied to this field is promising and offers the opportunity to identify clinical risk modifiers and improve our comprehension of the multiple factors involved in idiosyncratic DILI and their interaction. Hopefully, this will enable personalized therapy and safer treatment strategies . In the


Insulin is required for maintenance of glucose homeostasis. Despite the importance of insulin sensitivity to metabolic health, the mechanisms that induce insulin resistance remain unclear. Branched-chain amino acids (BCAAs) belong to the essential amino acids, which are both direct and indirect nutrient signals. Even though BCAAs have been reported to improve metabolic health, an increased BCAA plasma level is associated with a high risk of metabolic disorder and future insulin resistance, or type 2 diabetes mellitus (T2DM). The activation of mammalian target of rapamycin complex 1 (mTORC1) by BCAAs has been suggested to cause insulin resistance. In addition, defective BCAA oxidative metabolism might occur in obesity, leading to a further accumulation of BCAAs and toxic intermediates. This review provides the current understanding of the mechanism of BCAA-induced mTORC1 activation, as well as the effect of mTOR activation on metabolic health in terms of insulin sensitivity. Furthermore, the effects of impaired BCAA metabolism will be discussed in detail. Branched-chain amino acids (BCAAs) are critical nutrient signals that affect metabolism, either directly or indirectly. The BCAAs comprise leucine, isoleucine, and valine, which are essential amino acids . BCAAs aEven though the association between a high level of BCAAs and IR has been shown in numerous studies in humans and in rodent models, elevating the level of BCAAs leads to positive effects that improve metabolic parameters such as body composition, glycaemia levels, and satiety.Hypothalamic leucine is a potential nutrient signal that may reduce food intake by activating mammalian target of rapamycin (mTOR) . mTOR isBCAAs control hormone release in both the gastrointestinal tract and in fat deposits. Treatment with leucine for six weeks increased adiponectin and decreased cholesterol in the plasma of previously obese mice, without changing body weight or fat mass . BCAAs aw/v) doubled leucine level in the plasma of high fat diet fed mice, leading to improvement of glucose tolerance and insulin sensitivity, as well as decrease in hepatic steatosis and in inflammation of adipose tissue without affecting food intake and weight gain [Together with insulin, BCAAs also function as anabolic signals to alter the growth of energy-consuming tissues such as skeletal muscle and adipose tissue. Amino acids are required for protein synthesis. Amino acids, BCAAs in particular, also stimulate mTORC1 signaling pathways to regulate mRNA translation . Leucineght gain .Taken together, both BCAA-rich diets and BCAA supplementation have positive roles in metabolism, supporting the dietary recommendation for protein to increase BCAA levels.Recent metabolomics studies and comprehensive metabolic profiling studies have consistently showed a disturbance of normal amino acid metabolism and an increase of specific amino acids, often BCAAs, in some rodent models of obesity or T2DM, and in patients with obesity or T2DM ,19,20,21Newgard et al. showed that BCAAs, aromatic amino acids, and BCAA byproducts are most strongly correlated with insulin sensitivity and the homeostasis model assessment-insulin resistance index (HOMA-IR) than many other lipid species . They meIt has been suggested that BCAAs influence brain function by competing with the uptake of the amino acid precursors of dopamine and 5-hydroxytryptamine in the brain . Large nDespite the positive effects of BCAAs on metabolism, the strong association of BCAA levels with insulin resistance and metabolic syndrome suggests that increased levels of BCAAs may cause insulin resistance and T2DM, although this remains a speculation for now. The mechanism underlying that correlation is not yet fully understood. The possible mechanism by which BCAAs contribute to IR will be discussed in the following section.Even though it is not still clear whether BCAAs are causative factors in the development of IR, or whether they are biomarkers of impaired insulin action, the involvement of nutrient signaling in insulin resistance has emerged. High levels of BCAAs persistently activate mTORC1 (mTOR complex 1), resulting in insulin resistance through the phosphorylation of insulin receptor substrate 1 (IRS-1).mTOR is a serine/threonine kinase belonging to the phosphatidylinositol (PI) kinase-related protein kinase family . The mTOTuberous sclerosis complex 1/2 (TSC1/2) transduces the upstream signal to mTORC1 . TSC is Compared to other mTOR upstream signals, amino acids do not impinge on the TSC-Rheb axis. The Rag GTPase-Regulator axis mediates amino acid signal to mTORC1 . Four meA recent report identified GATOR complexes as negative regulators of Rag proteins. GATOR1 interacts directly with Rag proteins, functioning as a GAP toward Rag A and Rag B, leading to inhibition of mTORC1 activation . GATOR2 2+-calmodulin during Vps34 activation has been suggested, the upstream regulators that activate Vps34 remain unknown.The identification of amino acid mediators on mTORC1 translocation to the lysosome surface notwithstanding, the Vps34-phospholipase D1 (PLD1) axis was also suggested to activate mTORC1 on the lysosome . PhosphamTOR is a central signaling mediator of crosstalk between amino acids and insulin. Insulin activates mTOR, followed by activation of ribosomal S6K1, which regulates initiation of translation and elongation by phosphorylating S6 . Either Insulin triggers autophosphorylation of the insulin receptor, leading to the recruitment and phosphorylation of IRS1/2 at multiple tyrosine residues . These sDespite several lines of experimental evidence supporting the view that the effect of BCAAs on insulin resistance occurs through mTORC1 activation, some observations are controversial.Increasing the level of BCAAs, by supplementation or genetic modification, correlates with an improvement in metabolism, in spite of activated mTORC1 signaling. Deletion of mitochondrial branched-chain aminotransferase (BCATm) increased energy expenditure and improved insulin sensitivity . BCATm cIn addition, it is not clear whether the small changes in the BCAA level could increase IRS-1/2 serine phosphorylation under physiological conditions. Since many studies administrated larger doses of leucine compared to the BCAA levels in diabetic patients, it has not been shown that physiological levels of BCAAs can induce mTOR activation and the subsequent serine phosphorylation of IRS1 and IRS2. In line with this, Weickert et al. observed the only transient high-protein diets affect insulin sensitivity . Under iIn order to understand the potential role of BCAAs on metabolism, it would be beneficial to understand the processes that promote high blood levels of BCAAs. Considering that BCAAs are essential amino acids that cannot be synthesized de novo in organisms, the level of circulating BCAAs could be contributed to by dietary intake and by degradation of protein in tissue. This idea is supported by the fact that BCAAs regulate protein degradation by diminishing autophagy and proteasomal activity. BCAAs impair autophagy by activating mTORC1, a negative regulator of the initiation of autophagy. In agreement with this view, leucine restriction promotes protein breakdown in muscle cells by inhibiting mTOR, leading to an induction of autophagy . DietaryProtein turnover is controlled by insulin, in addition to amino acids. Although insulin activates protein synthesis in newborn piglets , the enhIn addition, gut microbiota influence BCAAs level in the plasma . Gut micAlong with persistent mTORC1 activation, impairments in BCAA metabolism are linked to insulin resistance and T2DM by the accumulation of possibly toxic intermediates and BCAA levels in plasma.BCAT2) catalyzes the first step in the metabolism of BCAAs in most peripheral tissues. BCAA metabolites are diminished in the peripheral tissues of BCAT2-/-mice [BCAT2-/-mice do not have insulin resistance, which would be expected. Instead, BCAT2-/-mice exhibit ameliorated glycemic control and insulin sensitivity with high energy expenditure, probably due to the loss of gluconeogenic precursors, indicating that muscle transaminases play a critical role in the generation of gluconeogenic substrates for the liver.The mitochondrial isoform of branched-chain aminotransferase  . BCKDC oIn the adipose tissue of patients with obesity and T2DM with insulin resistance, the expression of genes encoding the enzymes of BCAA metabolism is significantly decreased through an undefined mechanism—at least those genes encoding enzymes catalyzing the first two steps—leading to an increased plasma level of BCAAs ,79. ConsAltered gene expression, caused by either mutations or epigenetic regulation, affects all the enzymatic activities of BCAA metabolism. Tiffin et al. identified BCKDH4, the gene encoding the regulatory subunit of BCKDC, as one of two primary susceptibility genes that affect the risk of developing both T2DM and obesity via computational disease prioritization methods . FurtherThe association between the level of circulating BCAAs, insulin resistant obesity, and T2DM prompted consideration of BCAA levels as a predictor for future insulin resistance or T2DM, in spite of the beneficial effects of BCAA supplementation and a BCAA-rich diet. Elevated BCAAs stimulate mTORC1, a nutrient sensing complex, and following IRS-1 serine phosphorylation, result in insulin resistance and other metabolic disorders. However, recent works sparked questions on whether mTOR activation by physiological changes in the level of BCAAs is sufficient or necessary to cause insulin resistance, and subsequent metabolic disorders. The correlation between BCAAs level and insulin resistance was matched to HOMA-IR before diet intervention, but not after . Recent In summary, recent studies propose a close association between BCAAs and insulin resistance. Mechanisms whereby increased BCAAs induce insulin resistance have been proposed. The new findings relating to the BCAA signaling pathway, and to BCAA metabolism, broaden our understanding of insulin resistance. However, whether BCAAs are simply markers of insulin resistance, or whether they are direct contributors to insulin resistance remains uncertain, and this issue is attracting increased research interest. Furthermore, the signaling pathways and metabolism of BCAAs could be therapeutic targets for the treatment of insulin resistance and T2DM.


Anaphylaxis due to Hymenoptera stings is one of the most severe consequences of IgE-mediated hypersensitivity reactions. Although allergic reactions to Hymenoptera stings are often considered as a general model for the underlying principles of allergic disease, diagnostic tests are still hampered by a lack of specificity and venom immunotherapy by severe side effects and incomplete protection. In recent years, the knowledge about the molecular composition of Hymenoptera venoms has significantly increased and more and more recombinant venom allergens with advanced characteristics have become available for diagnostic measurement of specific IgE in venom-allergic patients. These recombinant venom allergens offer several promising possibilities for an improved diagnostic algorithm. Reviewed here are the current status, recent developments, and future perspectives of molecular diagnostics of venom allergy. Already to date, it is foreseeable that component-resolution already has now or will in the future have the potential to discriminate between clinically significant and irrelevant sensitization, to increase the specificity and sensitivity of diagnostics, to monitor immunotherapeutic intervention, and to contribute to the understanding of the immunological mechanisms elicited by insect venoms. Allergy to the venom of Hymenoptera species is a classical IgE-mediated allergic disease caused by the crosslinking of receptor-bound IgE antibodies on the surface of mast cells and basophils. Hymenoptera venom allergy is one of the most severe hypersensitivity reactions with regard to the high risk of anaphylactic reactions with potentially fatal outcome.Although venom allergy is one of the most frequent triggers of anaphylactic reactions in adults –3, the tApocrita which consists of the superfamilies Apoidea (Apinae and Bombinae subfamilies) and Vespoidea . In western and central Europe, the predominant elicitors of venom allergy are stings of honeybees (Apis mellifera) and yellow jackets (Vespula vulgaris). In southern Europe and the United States (US), additionally allergic reactions to paper wasps (Polistinae) are common [Solenopsis invicta) [Myrmecia pilosula) [Globally, all allergy-eliciting Hymenoptera belong to the suborder e common . In Euroinvicta)  and Austilosula) .For patients with anaphylactic reactions to Hymenoptera venom, the only causative treatment which is effective in reducing the risk of subsequent systemic reactions is venom immunotherapy (VIT). Particularly in Hymenoptera, venom-allergic patients specific immunotherapy is very effective in inducing tolerance with a protection rate ranging from 75 to 98 % . PrerequThe diagnosis of Hymenoptera venom allergy comprises the past medical history of a systemic sting reaction, a positive skin test response, and the detection of venom-specific IgE antibodies. Especially when the patient was not able to definitely identify the culprit insect, in clinical practice, the correct diagnosis is not always straightforward due to inherent problems and limitations of both tests. On the one hand, there are patients with a convincing history of anaphylaxis but negative diagnostic tests and, on the other hand, up to 50 % of patients show positive tests with more than one venom. Moreover, to date, no molecular tools are available which allow the prediction of the success of venom immunotherapy. Several limitations of diagnostic tests are based on the use of whole venom preparations for diagnosis. Venom preparations just like many other allergen extracts such as pollen or mite extracts , 20 ofteIn recent years, significant progress has been made in the identification of novel Hymenoptera venom allergens, the detailed characterization of established allergens, and the development of suitable strategies for the recombinant production of venom allergens. To date, analyses on a molecular level are able to overcome at least some of the problems of diagnostic approaches and to contribute to an improved therapeutic intervention right now , 24•.Hymenoptera venoms are complex cocktails of low molecular weight substances such as biogenic amines, basic peptides, toxins, and of higher molecular weight proteins, many of them with enzymatic activity, together with a variety of other components, all of which may contribute to sensitization, allergic symptoms, and success of immunotherapy.Apis mellifera, which surely is due to the outstanding importance of beekeeping and thus of the honeybee as elicitor of venom allergy all over the world and moreover, to the availability of detailed proteomic data of pure venom [Vespula vulgaris. Among the best characterized honeybee venom (HBV), allergens are phospholipase A2 (Api m 1), hyaluronidase, (Api m 2) and melittin (Api m 4), all constituting medium to higher abundance proteins [The certainly best characterized venom is that of the honeybee re venom  and genore venom . Additioproteins . Promineproteins , 29. Recproteins , 31.Mainly by proteomic approaches in the last years, much progress has been made in identifying important allergens of low abundance. The gene of the well-known acid phosphatase (Api m 3) of HBV was identified and recombinantly produced , 32, andMyrmecia venom are small peptides (pilosulins) which partially form homo- or heterodimers [Bumblebee venom closely resembles honeybee venom and has two allergens of known sequence, phospholipase A2, and a protease. The honeybee and bumblebee venom phospholipases A2 show extensive sequence identity with each other, while no sequence identity is given with vespid phospholipase A1 , which drodimers , but addOnly few allergens are present in substantial amounts in the venom. For honeybee venom, for example, Api m 1 and Api m 4 are predominant with amounts of dry weight of 12 and 50 %, respectively. Since low abundance allergens are difficult to isolate in substantial amounts, their recombinant availability can be considered a prerequisite for their detailed characterization and their use for diagnostic applications. However, even the purification of allergens of higher abundance from allergen extracts and their subsequent use for diagnosis has several disadvantages such as the danger of remaining impurities with other allergens and the presence of cross-reactive carbohydrate determinants (CCDs), both of which might severely impair and falsify analyses on a molecular diagnostic level.All these problems can be bypassed by the application of recombinant technologies. Some allergens have been traditionally produced in bacteria, but for several venom allergens produced in this way, conformational IgE epitopes are affected due to the lack of proper posttranslational modifications and correct folding. Although, the bacterial system impresses with its production rates and cost-effectiveness, this recombinant approach is only feasible for structurally less complex allergens. So far, only the HBV allergen Api m 10 could be easily produced with authentic IgE reactivity in bacteria ••. OtherIn the last decade, insect cell lines have developed to one of the most appropriate systems for the production of correctly folded authentic venom allergens , 51–53, Polistes dominula venom are available for routine molecular diagnostics. Ves v 1 and Ves v 5 in different patient populations allow the identification of 92 to 96 % of patients with confirmed YJV allergy [So far, only the major allergens Api m 1 (phospholipase A2) of HBV, Ves v 1 (phospholipase A1) and Ves v 5 (antigen 5) of YJV, and Pol d 5 (antigen 5) of  allergy , 60••. I allergy •, 63, 64 allergy ••. The sIn addition to the added value of recombinant insect venom allergens in serological IgE diagnosis, also other diagnostic tests such as the basophil activation test could be improved by the additional use of recombinant venom allergens, thus leading to the development of more reliable and efficient in vitro tests for molecular allergy diagnostics .Positive results in skin testing or sIgE testing to conventional Hymenoptera venom extracts do not always reflect a clinically relevant sensitization . In clinPolistinae) which show no immunologically detectable CCD-reactivity [On the one hand, cross-reactivity may be based on the recognition of common protein epitopes of homologous allergens, present in both venoms as described for hyaluronidases (Api m 2 and Ves v 2), dipeptidyl peptidases (Api m 5 and Ves v 3) and vitellogenins (Api m 12 and Ves v 6) , 34. On activity .Fig. 1MoSpodoptera frugiperda (Sf9) insect cells allows the recombinant production of properly glycosylated and thus correctly folded but CCD-free allergens due to a lack of immunologically detectable alpha-1,3-core fucosylation [Nowadays, molecular diagnostics using recombinant allergens has remarkable potential to discriminate between protein- and CCD-based IgE reactivity Table . The usesylation , 43. Thesylation •. Both sTaken together, in most cases where the extract-based diagnostics does not allow the identification of the culprit venom due to clinically irrelevant cross-reactivity, the analysis on a molecular level applying species-specific venom allergens, devoid of CCDs, enables the detailed characterization of sensitization profiles and the identification of the venom causing clinical symptoms Fig.  59, 61,, 77••.Polistes dominula, increasingly spread all over Europe as well as in the US from the warmer to the more moderate climate zones. Cross-reactivity between Polistinae and Vespinae  venoms is frequently observed [Polistes venoms, only a very limited number of allergens is described, all of which are highly homologous to Vespinae allergens. Although it was proposed that Polistes and Vespula venom allergy should be discriminated by measurement of sIgE to antigens 5 (Pol d 5 and Ves v 5) and phospholipases (Pol d 1 and Ves v 1) [However, in the coming years, the field of molecular IgE diagnostics for the elucidation of multiple sensitization to Hymenoptera venoms will face additional challenges that need better solutions. In addition to their established importance in North America and Mediterranean regions of Europe, paper wasps, especially observed , 78 indeobserved . For PolVes v 1) , the knoAnother diagnostic problem arises in patients with a convincing history of a systemic sting reaction but undetectable specific IgE in classical extract-based diagnostic approaches. A prominent example is a study comprising 308 patients with a systemic reaction to a yellow jacket sting ••. Only If a similar phenomenon also holds true for other venom allergens is not known so far. Furthermore, the example of the predominance of particular allergens such as Api m 1 and Api m 4 in HBV extract creates very likely a diagnostic information bias by favoring the detection of high abundant allergens in natural venom extracts. Recently, an underrepresentation or complete lack of the major allergens Api m 3 and Api m 10 was demonstrated for several therapeutic venom preparations which are licensed in European countries for routine venom immunotherapy ••. On thAlthough controversially discussed –82, moleApart from a well-documented field sting without a systemic reaction, the only recommended diagnostic method for the prediction of success of venom immunotherapy is the sting challenge with a living insect . Most paAlready today, molecular sIgE diagnostics of Hymenoptera venom allergy represent more than just an advanced diagnostic strategy. It has created added clinical value over the last decade. A component-resolution based on recombinant CCD-free species-specific allergens enables the differentiation between true sensitization and cross-reactivity and thus, in many patients improves the selection of the appropriate venom immunotherapy or the unnecessary therapy with multiple venoms instead of a single venom. Additionally, the availability of recombinant allergens facilitates to bypass the inherent limitations of venom extracts caused by heterogeneity and underrepresentation of particular important allergens. Moreover, for the future, component-resolved analyses possess the potential to improve the longitudinal monitoring of patients in the course of VIT on the level of sIgE and/or sIgG1/4 to molecular allergens. Further clinical studies will have to demonstrate, whether these new diagnostic tools will indeed provide superior diagnostic information to the clinician in charge of the venom-allergic patient, thus enabling the a priori identification of patients, at risk to inadequately respond to conventional extract-based venom immunotherapy.


It is interesting that a decrease in NKp46, a representative activating receptor, is common between NK cells in PBMC culture with asbestos and those of mesothelioma patients. Moreover, it was observed that CD8+ lymphocytes may be stimulated by some kind of “nonself” cells in plaque-positive individuals and in mesothelioma patients, whereas CTL in mesothelioma is impaired by poststimulation maintenance of cytotoxicity. These findings suggest that analysis of immunological parameters might contribute to the evaluation of health conditions of asbestos-exposed individuals and to a greater understanding of the pathology of malignant mesothelioma.Malignant mesothelioma is caused by exposure to asbestos, which is known to have carcinogenic effects. However, the development of mesothelioma takes a long period and results from a low or intermediate dose of exposure. These findings have motivated us to investigate the immunological effects of asbestos exposure and analyze immune functions of patients with mesothelioma and pleural plaque, a sign of exposure to asbestos. Here, we review our knowledge concerning natural killer (NK) cells and cytotoxic T lymphocytes (CTL). NK cells showed impaired cytotoxicity with altered expression of activating receptors upon exposure to asbestos, while induction of granzyme Inhalation of naturally occurring particles and fibers causes not only pulmonary fibrosis following an inflammatory response, but also tumor and autoimmune diseases. To date, we have focused on and examined the effect of asbestos exposure on the functions of various kinds of immune competent cells. These studies confirmed that functional decreases in T helper (Th) cells, natural killer (NK) cells, and cytotoxic T lymphocytes (CTLs) were caused by exposure to asbestos, decreases that were also partly observed in patients with malignant mesothelioma, following examination of cell lines and primary cells cultured with asbestos and analyzing cells prepared from the peripheral blood of patients  1–8]. R–8. R1–8]Asbestos is a kind of naturally occurring mineral fiber that has valuable physical and chemical characteristics including flexibility, as well as fire and heat resistance, which has resulted in the enormous use of asbestos globally. However, in the latter part of the 20th century many reports established that inhalation of asbestos causes malignant mesothelioma, which marked asbestos as one of the representative carcinogenic materials –16. Maliγ→ c-Jun N-terminal kinase 1 (JNK1) pathway. Perforin and granzymes released by degranulation cause target cells to undergo apoptosis  asbestos at 5 μg/mL for 7 days and assayed for the expression of activating receptors on the cell surface of NK cells. As shown by NK cells of mesothelioma patients, NK cells showed a decrease in NKp46 in the culture upon CB exposure, whereas NKG2D and 2B4 did not differ from the control culture  prepared from peripheral blood were assayed for cytotoxicity against K562 cells and the expression level of activating receptors on the cell surface of NK cells, and results were compared between healthy and mesothelioma individuals. To evaluate the lytic activity of a given cell number, the cytotoxicity per 5000 NK cells was calculated from the percentage of NK cells in PBMCs. Mesothelioma patients showed lower cytotoxicity of NK cells than healthy individuals, and also exhibited alteration in expression of activating receptors in their NK cells, which differed from YT-CB5. The NK cells of mesothelioma patients exhibited a characteristic decrease in expression of NKp46, whereas NKG2D and 2B4 showed normal expression  [5]. PBM culture . In addiTo examine the relationship between low levels of cytotoxicity and activating receptors, peripheral blood NK cells were isolated from the PBMCs of healthy individuals and assayed regarding cytotoxicity for K562 cells, cell surface expression of activating receptors, and phosphorylation of ERK1/2 after stimulation with antibodies to receptors, and results were compared between individuals. When individuals were put in descending order of cytotoxicity, an individual with high cytotoxicity showed high expression of NKp46 and NKG2D, whereas another with low cytotoxicity showed the opposite trend . In acco+ T lymphocytes play a more crucial role in cytotoxicity against target cells in an antigen-specific manner, together with the natural cytotoxicity of NK cells [+ T lymphocytes have to be selected clonally and proliferate and differentiate into functional CTLs in order to exert matured cytotoxicity for targets [+ T lymphocytes then migrate into the nodes and communicate with APC, and those having specificity for the antigen presented are chosen [+ T lymphocytes capable of recognizing the same antigen also have to migrate into the nodes and communicate with APC to lead to differentiation of functional CTLs [In antitumor immunity, CD8NK cells . CTLs alNK cells . However targets . The dif targets . Naïve Ce chosen . Moreovenal CTLs . Thus, t in vitro induction of CTL function from naïve CD8+ T cells. PBMCs were cultured with a stimulator of allogeneic PBMCs upon exposure to CB asbestos, and examined for the characteristics and allogeneic cytotoxicity of CD8+ T cells [+ lymphocytes, which when sorted from the culture showed a decrease in cytotoxicity against allogeneic targets compared with those from the control culture. CD8+ lymphocytes from the CB-exposed culture also showed low percentages of intracellular granzyme B and IFN-γ and cell surface CD25 and CD45RO, and a high percentage of CD45RA. In addition, suppressed cell proliferation of CD8+ lymphocytes upon exposure to CB was also confirmed by the CFSE labeling method. In contrast, those lymphocytes did not differ in apoptosis from those of the control group. Moreover, the productions of TNF-α and IFN-γ in the supernatant from the CB-exposed culture were low, whereas IL-2 did not differ in production. These findings therefore indicate that the induction of CTL function in MLR was suppressed by asbestos, and it was found that asbestos exposure has the potential to exert a suppressive effect on CTL induction following antigen stimulation.We thus attempted to investigate the effect of asbestos on acquired immunity in the antitumor response using the mixed lymphocyte reaction (MLR), an experimental method to induce cell-mediated acquired immunity using an allogeneic set of PBMCs or lymphoid cells, and an easy tool to mimic T cells . CB expo+ lymphocytes in individuals positive for pleural plaque and patients with malignant mesothelioma revealed the similarities and differences between these groups. Individuals in the pleural plaque and malignant mesothelioma groups showed higher percentages of perforin+ cells and CD45RA− cells in fresh CD8+ lymphocytes than healthy individuals. However, patients in the mesothelioma group showed a decrease in perforin+ cells following stimulation with PMA and ionomycin, whereas most of the healthy and plaque-positive individuals retained those cells after stimulation  CTL function is impaired in patients with malignant mesothelioma in comparison to plaque-positive individuals. Following these studies, we have continued to examine the effect of asbestos exposure on immune function and analyze specimens from asbestos-exposed individuals and mesothelioma patients. We hope that our studies will contribute to a greater understanding of asbestos exposure-related health disorders, including malignant mesothelioma, in order to improve the cure rate of those diseases.Our overall findings highlight the following points. (1) Exposure to asbestos has the potential to suppress the function of NK cells and CTLs. (2) It is possible that analysis of immunological parameters, such as NKp46 expression, might contribute to the evaluation of health conditions in asbestos-exposed individuals. (3) CD8


S-transferase A1 (GSTA1) and glutathione peroxidase 1 (GPX1) targets pattern was found to be a special characteristic of toxic PAs with stepwise multiple linear regressions; Furthermore, the molecular mechanism underlying the interactions within toxic PAs and these two targets was demonstrated with the ligand-protein interaction analysis; Finally, GSTA1 and GPX1 were proved to be significant nodes in GSH metabolism. Overall, toxic PAs could be identified by GSTA1 and GPX1 targets pattern, which suggests their common hepatotoxicity mechanism: the interfering of detoxication in GSH metabolism. In addition, all the strategies developed here could be extended to studies on toxicity mechanism of other toxins.Pyrrolizidine Alkaloids (PAs) are currently one of the most important botanical hepatotoxic ingredients. Glutathion (GSH) metabolism is the most reported pathway involved in hepatotoxicity mechanism of PAs. We speculate that, for different PAs, there should be a common mechanism underlying their hepatotoxicity in GSH metabolism. Computational methods were adopted to test our hypothesis in consideration of the limitations of current experimental approaches. Firstly, the potential targets of 22 PAs (from three major PA types) in GSH metabolism were identified by reverse docking; Secondly, glutathione  Pyrrolizidine Alkaloids (PAs) are well known as a type of the most hepatotoxic toxins in nature. Currently, more than 660 kinds of isolated PAs and their nitrogen oxide derivatives have been identified in over 6000 plants . PAs maiIn vitro experiment was regarded as an important approach in studying metabolic toxicity, and cell models including Primary cultured hepatocytes  (IDH2).The targets number distribution of the toxic PAs and non-toxic compounds are different b. The poTo find some commonalities of targets to distinguish toxic PAs from non-toxic compounds, specific targets of toxic PAs were enriched with four stepwise multiple linear regression models. There are four targets enriched in the best model , accordiR-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine) were used to dock GSTA1 and GPX1 respectively   network to confirm their importance in GSH metabolism. A PPI network with 91 proteins and 194 interactions was produced by linking each pair of 44 proteases in GSH metabolism with the shortest path, and the importance of these proteins was measured by betweenness, degree and pagerank a. Under Our study identified the potential targets of PAs in GSH metabolism, and demonstrated the GSTA1 and GPX1 targets pattern can distinguish toxic PAs from non-toxic compounds with the accuracy over 94%. This pattern suggests toxic PAs probably bind to both these two proteins and influence their function. Furthermore, we have detected potential molecular interactions between secondary metabolites of toxic PAs and GSTA1, GPX1 respectively, which helps understand how PAs affect these two important enzymes.In GSH metabolism , as an iIntracellular GSH could serve as the electron donor in the reduction of peroxides catalyzed by GPX . As a glBesides, under the catalysis of GST, GSH can conjugate the metabolites of exogenous toxins . GSTs arTaken together, toxic PAs could bind with GSTA1 and GPX1 in liver and interfere two important detoxication pathways in GSH metabolism by decreasing the activities of these two major cellular antioxidant enzymes. This would disrupt the balance of free radicals in the body and hinders the excretion of toxic compounds out of the cell, therefore indirectly resulting in the toxicity on hepatocytes.Reverse docking is an important method to calculate and predict the targets of small molecules in a large scale as well as in a non-laboratory environment. However, it yet has two main limitations. On the one hand, it may generate false positives from computing models and docking procedures. On the other hand, there are still no available 3D structures for some receptors, which is the foundation of docking study. Here, only receptors with 3D structures were selected for molecular docking, which may inevitably generate biased results. Besides, there is a need to further optimize computation models and statistical models to decrease false positives. Despite all this, our study could still lay a foundation to future PAs’ toxicity mechanism research, especially to the researches on the detoxication mechanisms regarding the GSTA1 and GPX1.The positive and negative datasets of PAs  are retrThe 3D structures of 34 proteins involved in the Glutathione metabolism in KEGG are acquired from protein data bank (PDB) .http://www.rcsb.org/) [INVDOCK  was usedsb.org/) ,53,54. Psb.org/) . The firIn this study, only when a protein was identified as the target of all the three states of a same PA, could it be considered as the potential target of this PA. As a result, 14 out of 34 proteins were hit by PAs.Stepwise multiple linear regression analysis was applied to seek the specific targets to distinguish toxic PAs (positive dataset) and non-toxic compounds (negative dataset). Four linear regression models were established based on the targets information, and the differences among them lie in the targets set of positive dataset, shown as follows:Model 1: Targets set only contains the targets of the PAs prototype.Model 2: Targets set only contains the targets of DHP.Model 3: Targets set contains the targets of all small molecules, whether they are PAs prototype, DHP or DHR.Model 4: Targets set contains the targets which were hit by all the three states of certain PA at the same time.Key targets filtered out from each of the four models were further combined to predict whether a molecule is a toxic PA. The information of other three models and prediction precisions of different combinations of features are shown in The binding sites and possible molecular interactions within toxic PAs and GSTA1, GPX1 were identified by Simulations module in the commercial software Molecular Operating Environment (MOE).In order to investigate the connection of proteases related to the GSH metabolism on the human protein-protein interaction network links (PPI), in particular the importance of GSTA1 and GPx1 pattern, we constructed a subnetwork for GSH metabolism.Firstly, we constructed a human PPI which composed of 55,363 edges and 10,523 nodes based on 6 PPI databases, including HPRD (Human Protein Reference Database) , MINT (tSecondly, we mapped 44 proteases in GSH metabolism on the human PPI and made them interconnected by remaining some background nodes. Under this condition, a GSH metabolism network with 91 nodes and 194 edges was constructed a. FinallOverall, our study suggests that toxic PAs and their metabolites are most likely to be identified by GSTA1 and GPX1 targets pattern. After binding with GSTA1 and GPX1, toxic PAs could further exert toxicity by breaking the oxidant-antioxidant balance and intercepting detoxication of the exogenous toxins. This pattern could provide some support in the future studies on PAs’ hepatotoxicity mechanism. Moreover, the computational workflow employed in this study may throw light upon the toxic mechanisms studies on other toxins.


Metastatic melanoma represents the most deadly form of skin cancer. The poor response to chemotherapy and the brief response to the anti-BRAF vemurafenib in selected population of patients, make the identification of new therapeutic approaches an urgent need. Our goal is the evaluation of the efficacy of barasertib, an aurora B kinase inhibitor impairing cytokinesis, in both mutated and non-mutated melanoma cell lines.Panel of melanoma cells: BRAFV600E mutated cells (MBA72 and Hmel1), the same cell in which the resistance to vemurafenib was induced by chronic exposure to it (MBA72R and Hmel1R) and BRAF wt (HBL and LND1).Cells were characterized for vemurafenib and barasertib effectiveness on cell growth by MTT assay after 3 and 6 days of continuous exposure.Cell cycle was determined by flowcytometry and migration was evaluated by wound-healing assay.Cells with BRAFV600E mutation are sensitive to vemurafenib conversely, those with BRAF wt and the resistant ones showed an IC50 of at least 10 folds higher.3days-barasertib exposure strongly reduced cell growth  in all cell lines; when the drug was given together with vemurafenib, no gain in effectiveness was evident. Prolonged exposure to barasertib (6 days) showed a progressive increase of effectiveness particularly in cells BRAF wt.The analysis of cell death mechanisms involved in determining the effectiveness of barasertib and vemurafenib showed that the first drug induced both apoptosis and necrosis conversely, the latter mainly apoptosis.Cell cycle analysis demonstrated that barasertib induced an increase in cell size and in polyploidia, suggesting also the mitotic catastrophe as a further cell death mechanism.Moreover, the anti-metastatic behaviour of this agent has been evaluated in function of drug concentration and time exposure. Preliminary results showed a strong reduction of cell migration after drug exposure.The sensitivity of melanoma cells to barasertib is irrespective to BRAF mutational status; however, cells BRAF wt show an higher nuclear modification. In conclusion, our results suggest barasertib as a novel therapeutic approach in melanoma treatment irrespective of BRAFV600E mutation.


Mitochondrial dysfunction is one of the earliest and most prominent features in the brains of Alzheimer’s disease (AD) patients. Recent studies suggest that mitochondrial dysfunction plays a pivotal role in the pathogenesis of AD. Neurons are metabolically active cells, causing them to be particularly dependent on mitochondrial function for survival and maintenance. As highly dynamic organelles, mitochondria are characterized by a balance of fusion and fission, transport, and mitophagy, all of which are essential for maintaining mitochondrial integrity and function. Mitochondrial dynamics and mitophagy can therefore be identified as key pathways in mitochondrial quality control. Tremendous progress has been made in studying changes in these key aspects of mitochondrial biology in the vulnerable neurons of AD brains and mouse models, and the potential underlying mechanisms of such changes. This review highlights recent findings on alterations in the mitochondrial dynamics and mitophagy in AD and discusses how these abnormalities impact mitochondrial quality control and thus contribute to mitochondrial dysfunction in AD. At synaptic terminals, mitochondria take up excess intracellular Ca2+ and release Ca2+ to prolong residual levels, maintaining Ca2+ homeostasis , and oxidative phosphorylation components within a mitochondrial network. While damaged mitochondria can be repaired through fusion with healthy mitochondria for mixture of contents, mitochondrial elongation upon nutrient deprivation maximizes energy production and promotes cell survival by preventing autophagy-mediated degradation of mitochondria  is the major motor that drives mitochondrial transport  that function as the motor domain for force production, and intermediate (DIC), light intermediate (DLIC), and light chains (DLC) that function in cargo attachment and motility regulation. Dynein motors associate with in vitro studies, their involvement in this pathway has recently been supported by proteomic analyses in Drosophila under normal physiological conditions -containing protein NIP3-like X , was shown to play an important role in the elimination of mitochondria in erythrocytes  cellular analysis of depolarization of mitochondrial membrane potential with Parkin translocation, co-localization with autophagy markers (mitophagosome formation), and lysosomal sequestration of defective mitochondria; (2) ultrastructural analysis by electron microscopy based on alterations in mitochondrial structure and morphological features of autophagic vacuoles (AVs) containing mitochondrial profiles; and (3) western blot analysis of increased association of mitophagy machinery with mitochondria and reduced levels of mitochondrial proteins upon mitophagy induction. These assays need to be combined with the application of mitochondrial membrane potential dissipating agents and a flux inhibitor to trap newly formed autophagosomes.Mitochondrial quality control involves surveillance and protective strategies at multiple levels in order to limit mitochondrial damage and ensure mitochondrial integrity. The quality control occurs at the molecular, organellar, and cellular levels. At the molecular level, a proteolytic system, involving molecular chaperones and ATP-dependent proteases in the matrix and the inner membrane (IM) of mitochondria, degrades damaged and misfolded proteins and/or dissolves protein aggregates for proteolysis  or oligomeric Aβ, as well as in primary neurons cultured from AβPP mice  was shown to degrade Aβ within mitochondria and thus reduce its toxic effects on mitochondrial function —decreased fusion and fission rates due to reduced levels of OPA-1 and Drp-1. In contrast, overexpressing wild-type tau was shown to have protective effects on both mitochondrial dynamics and function, including the enhancement of complex I activity  responsible for stabilizing axonal MTs. It has also been shown to regulate axonal transport of membranous organelles, including mitochondria (Stamer et al., While mutant tau expression results in mitochondrial dysfunction, deregulation of mitochondrial dynamics, and impaired mitochondrial transport, one recent study reported that pathogenic tau truncation might also contribute to abnormal mitophagy in AD. A 20–22 kDa NH2-tau fragment detectable in cellular and animal AD models and human AD subjects was shown to be stably associated with Parkin and UCHL-1. As a result, it led to aberrant recruitment of Parkin and UCHL-1 to mitochondria and excessive Parkin-dependent mitochondrial turnover (Corsetti et al., Mitochondrial dysfunction is a prominent feature in AD, but it remains unclear whether the cellular mechanisms maintaining mitochondrial quality are defective and further augments mitochondrial pathology. Mitochondrial quality control involves multiple levels of surveillance and protective strategies to limit mitochondrial damage and efficiently eliminate defective mitochondria for the maintenance of mitochondrial homeostasis. Mitochondrial fusion and fission control mitochondrial shape and function, while mitochondrial transport plays a critical role in regulating mitochondrial distribution and removing aged and damaged mitochondrial from distal axons and synapses for lysosomal degradation in the soma. These key features of mitochondria operate strongly in conjunction with mitophagy as the key pathways of mitochondrial quality control. Studies reviewed here suggest that a complex network of dynamic and reciprocal interactions among mitochondrial fusion, fission, transport, and mitophagy governs mitochondrial integrity and function. Accumulating evidence notes that perturbed mitochondrial dynamics and abnormal mitophagy exist in AD brains (Chen and Chan, QC conceived ideas and wrote the manuscript. PT contributed to some sections related with AD.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.


Introduction: Genetic skeletal diseases (GSDs) are a diverse and complex group of rare genetic conditions that affect the development and homeostasis of the skeleton. Although individually rare, as a group of related diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. There are currently very few specific therapeutic interventions to prevent, halt or modify skeletal disease progression and therefore the generation of new and effective treatments requires novel and innovative research that can identify tractable therapeutic targets and biomarkers of these diseases.Areas covered: Remarkable progress has been made in identifying the genetic basis of the majority of GSDs and in developing relevant model systems that have delivered new knowledge on disease mechanisms and are now starting to identify novel therapeutic targets. This review will provide an overview of disease mechanisms that are shared amongst groups of different GSDs and describe potential therapeutic approaches that are under investigation.Expert opinion: The extensive clinical variability and genetic heterogeneity of GSDs renders this broad group of rare diseases a bench to bedside challenge. However, the evolving hypothesis that clinically different diseases might share common disease mechanisms is a powerful concept that will generate critical mass for the identification and validation of novel therapeutic targets and biomarkers. Genetic skeletal diseases (GSDs) are an extremely diverse and complex group of rare genetic conditions that primarily affect the development and homeostasis of the osseous skeleton There are more than 450 unique and well-characterized phenotypes that range in severity from relatively mild to severe and lethal forms and are described in detail in the 2011 Nosology and Classifications of the GSDs GSDs are difficult human diseases to treat, particularly when the pathological process begins before birth and can affect the entire skeletal system. Furthermore, since it is now known that the skeleton has close physiological relationships with many other tissue systems in the body, and mutant genes may have pleiotropic effects, patients affected by GSDs may also have serious complications with other organs, including the peripheral nervous system, brain, bone marrow, immune system, pancreas, kidney, heart, muscle and tendon.The translation of state-of-the-art technology into quantifiable patient benefits such as the development of new treatments or effective biomarkers has been extremely limited for GSDs. The few notable exceptions include Biomarin’s drug candidate for achondroplasia (ACH), a C-type natriuretic peptide (CNP) analogue PG-CNP37 (BMN-111) 2. in vivo. Several recent reviews have highlighted both strengths and weaknesses of various modelling approaches in vivo models to investigate skeletal pathobiology. These will also act as pre-clinical models when new therapeutic targets are identified and validated.Over the last 20 years, the generation and in-depth analysis of transgenic, knock-out, knock-in and ENU-derived mice models of GSDs have generated valuable knowledge of disease mechanisms 3. The extracellular matrix (ECM) of cartilage is a highly organized composite material comprising numerous structural macromolecules such as collagens , proteoglycans (aggrecan) and glycoproteins (matrilin-3 and cartilage oligomeric matrix protein [COMP]). Mutations have now been identified in all the genes encoding the major structural components of the cartilage ECM and result in a diverse group of both dominant and recessive GSDs. These assorted mutations fall into two broad classes: qualitative mutations, such as those that have dominant-negative (antimorphic) effects, and quantitative mutations that result in haploinsufficiency and/or a complete loss of protein function. This section will focus specifically on dominant-negative (antimorphic) mutations, which affect conserved residues that are structurally and functionally important for normal protein folding and function .The endoplasmic reticulum (ER) is a distinct organelle of eukaryotic cells and plays the major role in the synthesis, folding and trafficking of proteins entering the secretory pathway. The ER has a highly sophisticated quality control mechanism for ensuring that misfolded mutant proteins do not accumulate in, or enter the secretory pathway. Eukaryotic cells have a homeostatic mechanism for maintaining the protein-folding equilibrium of the ER, which is known as the unfolded protein response (UPR) N-oxide (TMAO) An extensive allelic series of glycine and non-glycine substitutions in type II collagen have been introduced into mice to model a diverse range of type II collagenopathies MATN3 (V194D) COMP  COL10A1 (N617K) The role of ER stress in GSDs might best be exemplified by mutations in COMP, matrilin-3 and type X collagen resulting in pseudoachondroplasia (PSACH), multiple epiphyseal dysplasia (MED) Matn3 (V194D) and Col10a1 (N617K) mutations cause misfolding and retention of the relevant mutant protein, inducing ER stress and a classical UPR, primarily characterized by the up-regulation of ER chaperones BiP, Grp94 and a range of protein disulphide isomerases (PDIA) Col10a1N617K and Matn3V194D mutant mice and also noted that two novel ER stress-related genes, Armet and Creld2, were also significantly upregulated in these two models Armet and Creld2 has resulted in mice with growth plate dysplasia, thus confirming their important role in skeletal development (our unpublished observations). Moreover, the recent cartilage-specific knock-out of PDIA3  caused ER stress resulting in reduced proliferation and accelerated apoptotic cell death of chondrocytes in the growth plate Interestingly, both In contrast, the accumulation of mutant COMP has been demonstrated to result in the induction of novel stress pathways, which are characterized by changes in the expression of groups of genes implicated in oxidative stress (ER dependent), cell cycle regulation and apoptosis N-oxide (TMAO) In summary, these recent studies using a complimentary group of genetically relevant mouse models and cartilage-specific knock outs have demonstrated the key role that ER stress plays in the initiation and progression of growth plate dysplasia and reduced bone growth . Moreove4. Missense mutations in the discordin domain receptor 2 (DDR2) have been shown to cause the rare autosomal recessive spondyo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) due to either protein trafficking defects or loss of ligand binding TRAPPC2 cause X-linked spondyloepiphyseal dysplasia Tarda (SEDT) SEC23A, sec23a and sec24d, which transport secretory proteins from the ER to Golgi, result in cranio-lenticulo-sutural dysplasia (CLSD) crusherbulldogTRIP11) causes a lethal skeletal dysplasia in both mice and humans (achondrogenesis type 1A) Similarly, the ER export of procollagen is also controlled in part by the ubiquitously expressed Sedlin (TrappC2), and antimorphic or loss of function mutations in In summary, these various genetic studies consistently demonstrate that ‘professionally secreting cells’ such as chondrocytes are highly susceptible to perturbations in ER homeostasis and defects in protein trafficking and secretion. This premise is supported by the observation that deletion or mutation of ubiquitously expressed components of the secretory pathway specifically causes chondrocyte disruption and cartilage defects . Modulat5. The retention of mutant protein in the ER of cells appears to be a major pathomolecular mechanism underpinning the disease aetiology in a range of GSDs . TherefoThe potential effectiveness of such treatment has been demonstrated for type IV collagen mutations causing hemorrhagic stroke. In a study described by Murray and colleagues However, it is important to note that the incorporation of mutant proteins into the ECM may also have deleterious effects and lead to a skeletal condition, as highlighted by the PSACH and MED-causing mutations in the C-terminal domain (CTD) of COMP An in-depth analysis of the structural proteins in the cartilage ECM, in particular the proteins that have a bridging/adaptor function in the ECM, using transgenic mouse models has shown that some of these molecules are actually redundant in cartilage. For example, the genetic deletion of COMP Mutations that result from aberrant splicing often lead to nonsense-mediated decay of mRNA and associated cell stress or the production of truncated molecules, which may have downstream dramatic effects. This is the case in some cases of Schwartz-Jampel syndrome (SJS) and in dyssegmental dysplasia, Silverman-Handmaker type (DDSH) where an exon skipping mutation leads to a truncated perlecan molecule 6. Primary cilium is an organelle existing on almost every cell type in the human body Disruptions to the columnar organization of proliferative chondrocytes have been described in several mouse models of rare skeletal conditions, in particular those where the ECM structure was affected Mechanosensing is important for cartilage development and homeostasis and mutations involving primary cilia molecules have been discovered in several chondrodysplasias to date, including the Verma-Naumoff syndrome, Majewski syndrome, Jeune syndrome, Ellis-van Creveld syndrome, the Sensenbrenner syndrome and Weyers acrofacial dysostosis 7. ECM molecules are important for the sequestration and diffusion of endocrine, paracrine and autocrine molecules in the dense cartilage tissue PTHR1 have also been found in certain forms of Jansen dysplasia Mutations in the genes encoding molecules important in cartilage signalling have also been implicated in GSDs. Eiken syndrome is a recessive skeletal condition resulting from a truncating mutation in the parathyroid hormone-related peptide type 1 receptor (PTHR1) and mutations in the FGFR1, FGFR2, FGFR2, FGFR1 and FGFR2, FGFR2, FGFR3, FGFR3, Conversely, numerous GSDs result from activating mutations affecting the signalling pathways in the cartilage PTH1R) GNAS, TRPV4) ; however, downstream signalling occurs through several different guanine-nucleotide binding proteins (G-proteins) GNAS encodes the α subunit of one of these G-proteins, Gsα. Constitutive activation of both PTH/PTHrP and Gsα leads to the excessive synthesis of cyclic AMP, disrupted chondrocyte signalling and bone pathology TRPV4  encodes a calcium permeable cation channel and disease-causing mutations constitutively activate this channel resulting in an uncontrolled influx of calcium into chondrocytes, activation of follistatin Other GSDs share this common disease basis with activating mutations found in different genes causing diverse disorders such as Jansen’s metaphyseal chondrodysplasia   [82]. SiFGFR1 construct in utero prevented suture fusion within the skull Existing therapies for GSDs caused by constitutively activating mutations remain limited, although insights into their molecular pathogeneses have yielded some exciting therapeutic targets . A murinFGFR3 that cause thanatophoric dysplasia, SADDAN and achondrogenesis were treated in vitro with meclozine and this ameliorated abnormally suppressed cell proliferation. Comparison with the effect of CNP on the same cell models revealed that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signalling. Subsequent studies validated this result in a mouse model of ACH, showing that the bone lengths of both wild-type and mutant mice treated with meclozine were significantly longer than in untreated mice A significant development in efforts to treat ACH was the discovery that CNP overexpression rescues the phenotype in an ACH mouse model by inhibiting FGFR3 downstream signalling through the MAPK pathway et al. have demonstrated that statins, previously used in the treatment of tumours and of disorders in the cardiovascular, nervous and immune systems, were able to upregulate the expression of sox9, type II collagen and aggrecan in cell and animal models of ACH and TD1 Ach mouse model of ACH. Interestingly, the decrease in the protein level did not correlate with a decrease in mRNA level for FRFR3, which was actually slightly increased upon the statin treatment. Treatment of chondrocytes derived from the Fgfr3Ach mice with lovastatin in the presence of proteasomal inhibitors revealed that the statin partially upregulated the proteosomal degradation of FGFR3 in the treated cells and animals The use of statins to treat FGFR3-related disorders, ACH and tanatophoric dysplasia type I (TD1) has recently been proposed Several approaches have been proposed as possible therapeutic interventions for TRPV4-related disease Perhaps the most effective clinically approved treatment for a GSD caused by a constitutively activating mutation is the intravenous administration of bisphosphonates to treat fibrous dysplasia. This class of drugs possesses a common basic structure similar to pyrophosphate that inhibits bone resorption and has been used with success for decades. In addition to a decreased turnover of bone, the radiological aspect of existing bone lesions is improved, as is the experience of pain reported by patients 8. Comp, Matn3 and Col2a1) Slc26a2) Several studies have recently demonstrated that reduced chondrocyte proliferation, increased and/or dysregulated apoptosis in the growth plates of mouse models is a major pathological component of various GSDs, including those resulting from mutations in genes encoding cartilage structural proteins , which provided the first opportunity for both clinical and research experts in GSDs to interact in a Pan-European environment and this ultimately fostered important collaborations and ground-breaking ideas that would lead to future GSD networks.The FP5-funded European Skeletal Dysplasia Network (ESDN) for research and diagnosis (2002 – 2006) was one of the first networks of expertise in the field of rare diseases to use information and communications technology tools for the purposes of tele-expertise and medical diagnosis. Since September 2003, ESDN has received over 2000 referrals through an on-line Case Manager and 450 users have accessed ESDN from 45 different countries worldwide. During this initial funding period, research activities within ESDN developed several relevant mouse models of GSDs and identified the first disease mechanisms, which would later be defined as hallmark features of disease pathology and be tentatively proposed as potential therapeutic targets.EuroGrow (2007 – 2010) was an FP6 project that focused on the phenotyping of mouse models of a select group of GSDs in order to develop and validate experimental approaches for deep-phenotyping including omics-based analysis. This project successfully identified a range of common disease mechanisms between phenotypically different GSDs and laid the foundation for future large-scale projects.http://www.sybil-fp7.eu) is a large-scale collaborative project that aims to functionally validate genetic determinants of common and rare skeletal diseases to gain a mechanistic understanding of the disease processes and age-related changes and to deliver new and validated therapeutic targets. The outcomes of this project will include the generation and deep phenotyping of a diverse range of cell and animal models of GSDs that will generate new knowledge on disease mechanisms. The major strength of this coordinated and multidisciplinary approach is the use of System Biology to underpin the extensive ‘omics’-based analysis that will identify common disease mechanisms and potential therapeutic targets in an unbiased and iterative process.The FP7-funded SYBIL project , SKELNET (Germany) and Skeldys.org (Switzerland) have provided a combination of both formal and informal discussion and diagnostic forums, whilst the establishment within several EU countries of National Centers of Excellence for specific GSDs has generated critical mass for delivering clinical best practice and translational research.In summary, trans-European networks of clinical and research excellence in GSDs have a proven track record in delivering an efficient world class diagnostic service and generating new knowledge on disease mechanisms that will translate into novel therapeutic targets that might even be shared amongst groups of different disease (i.e. Common amongst the Rare). Moreover, interactions with patient self-support groups, the International Rare Diseases Research Consortium (IRDiRC) and the establishment of a European Reference Network in the GSD domain will all help to accelerate and deliver translational research through broad international collaborations and the sharing of best practice.12. The development of potential therapeutic targets to the point at which clinical trials can be performed and pharmacological agents ultimately brought to market is a notoriously challenging process with high attrition rates Recent discussions with support groups around the clinical trials currently being carried out for the BioMarin product BMN-111 have emphasized the importance of these issues. Concerns have been expressed that undue focus on stature is inappropriate in the context of the symptoms people with ACH have on a daily basis, whilst a reduction of the neurological impairment associated with neural axis compression would be extremely welcome.The rarity of the GSDs will pose significant challenges not only to those developing therapeutic agents but also to regulators and healthcare commissioners and providers. The relatively small market available for drugs targeted at specific molecular targets or even specific genetic pathways limits the financial viability of such agents and is part of the wider debate around the development of treatments for rare diseases 13. The extensive clinical variability and genetic heterogeneity of GSDs, coupled with complex disease mechanisms, renders this extensive group of rare diseases a bench to bedside challenge. Indeed, this large number of different and highly complex phenotypes makes the identification, validation and development of potential therapies almost impossible for anything other than the most common GSDs. As an alternative approach, we might consider identifying genotype- and/or phenotype-independent ‘core disease mechanisms’ that are shared amongst families of clinically unrelated GSDs. This approach would allow the focusing of resources into several areas of concerted investigation that have the potential to identify and validate therapeutic targets with a broad application to GSDs, inherited connective tissues as a whole and rare genetic disease in general. Indeed, Jürgen Spranger first suggested the idea of ‘bone dysplasia families’ in 1985 In this context, ER stress has been associated with a diverse range of genetic diseases and chronic conditions such as skeletal dysplasia (as discussed in this review), myopathy in vitro and in vivo models  resulting from dominant-negative mutations in cartilage structural proteins.Disruption to protein trafficking in chondrocytes leads to a variety of chondrodysplasia phenotypes, which highlights that ‘professionally secreting cells’ such as chondrocytes are highly susceptible to perturbations in ER homeostasis and defects in protein trafficking and secretion.The incorporation of mutant proteins into the extracellular matrix leads to changes in the composition and properties of cartilage.Reduced chondrocyte proliferation, increased and/or dysregulated apoptosis are common downstream effects for a range of different GDS and are robust readouts for pre-clinical studies.Numerous GSDs result from mutations affecting signalling pathways in cartilage and these are the targets of new therapeutic interventions.This box summarizes key points contained in the article.


Saturation indices calculated with respect to calcite, aragonite, and dolomite inside the mats showed extremely high values across the mat profile. Such high values were caused by high pH and high carbonate alkalinity generated within the mats in conjunction with increased concentrations of calcium and magnesium that were presumably stored in sheaths and extracellular polymer substances (EPS) of the living cyanobacteria and liberated during their post-mortem degradation. The generated CaCO3 morphs were highly similar to morphs reported from heterotrophic bacterial cultures, and from bacterially decomposed cyanobacterial biomass emplaced in Ca-rich media. They are also similar to CaCO3 morphs precipitated from purely inorganic solutions. No metabolically  controlled formation of particular CaCO3 morphs by heterotrophic bacteria was observed in the studied mats. The apparent alternation of in vivo and post-mortem generated calcareous layers in the studied cyanobacterial mats may explain the alternation of fine-grained (micritic) and coarse-grained (sparitic) laminae observed in modern and fossil calcareous cyanobacterial microbialites as the result of a probably similar multilayered mat organization.Marine cyanobacterial mats were cultured on coastal sediments  for over three years in a closed system. Carbonate particles formed in two different modes in the mat: (i) through precipitation of submicrometer-sized grains of Mg calcite within the mucilage near the base of living cyanobacterial layers, and (ii) through precipitation of a variety of mixed Mg calcite/aragonite morphs in layers of degraded cyanobacteria dominated by purple sulfur bacteria. The δ Howeverhe light . In additotrophs .3 precipitates occurring in particular microbial zones showed that the morphologies and modes of distribution of carbonate grains were not random but tied to well-defined zones labeled from 1 to 4 in The present study is based on observations made on cryomicrotome sections of approximately three-year-old mats , the strmicrite and sparite layers Micrite: <1 µm-sized globular grains of Mg calcite  precipitated in the mucilage of the living cyanobacterial layers, particularly dense at their basal parts.(ii)Sparite: a variety of >1 µm-sized mixed Mg calcite/aragonite particles  precipitated in the zones of cyanobacterial necromass colonized predominantly by phototrophic purple bacteria with varying admixtures of green sulfur bacteria and heterotrophic bacteria.As shown schematically in micrite. The surface layer 1 was composed largely of mucilage embedding trichomous cyanobacteria identified as Calothrix and Pseudanabaena mixed with heterotrophic bacteria. At the base of this living mucilage-rich cyanobacterial layer, an almost continuous layer of spherical submicrometer-sized CaCO3 grains occurred (3)  b and, asmicritic) calcium carbonate precipitates occurring in the living filamentous cyanobacterial layers, the layers of variously degraded cyanobacterial necromass were dominated by purple sulfur bacteria and heterotrophic bacteria . The coarse-grained (sparite) calcium carbonate precipitated in these layers included larger (2‒4 µm) anhedral and subhedral grains of Mg calcite with an admixture of aragonite, some showing crystal faces (sparite) carbonate particles. Here the main CaCO3 morphs were 50‒120 µm large globular and subglobular grains composed of dagger-like crystals .The calcite, aragonite, and dolomite saturation values for the pore waters of the studied mats showed very high supersaturation values in the uppermost 2‒3 mm of the mat . These hThe distribution of different carbonate precipitates in the studied mats indicates that two processes were involved in their formation:2 and/or HCO3− by cyanobacteria, causing a significant increase in the carbonate ion and thus in the CaCO3 saturation level  high-Mg calcite grains associated with living cyanobacterial layers. The nucleation in such layers was most probably a spontaneous process resulting from the high pH values induced by photo assimilatory uptake of COon level . In many3 precipitation took place in zones occupied predominantly by purple bacteria and heterotrophic bacteria, with the latter degrading the cyanobacterial necromass. This probably less viscous precipitation environment favored the formation of larger carbonate particles of mixed Mg calcite/aragonite mineralogy. As in the living cyanobacterial layers, the nanometer-sized precursor particles were transformed much faster into large superstructured grains composed of ordered nanograins and nanocrystals (3 particles (ACC) are unstable and rapidly transform into macrocrystals [The second process of CaCOcrystals g. It is crystals ,60,61,623 layers  as compared with layers generated in zones of decaying cyanobacterial biomass (due to input of lighter respiratory carbon).The presence of two different microenvironments of calcium carbonate production within the mats is supported by the results of stable carbon isotope analyses of the CaCO3 layers . They shThe saturation indices measured in the uppermost 2 mm of the studied mats showed very high supersaturation . It has 3 ions. The Ca concentration in Nivå Bay was, at a salinity of 22 ppt, lower (by a factor of 22/35) than in average seawater of 35 ppt. On the other hand, the alkalinity of >8 meq L−1 in Nivå Bay was much higher than in average seawater . This is most probably a consequence of sulfate reduction in the sediments of the shallow bay. High alkalinity, however, is not a direct measurement of carbonate concentration, but the sum of the negative charges of the bicarbonate and carbonate ions. The partitioning between the two ions is governed by CO2 pressure, which in turn determines pH. In the present paper we have not considered the potential contribution of nitrate, ammonium and orthophosphate in alkalinity measurements. In a closed system the mats were cultured, any excessive influx of the anions was barely possible for a longer time. Thus, measuring pH, temperature, and alkalinity and calculating the other main ion concentrations, among them calcium, from the measured salinity enables the calculation of the SI. High alkalinity per se does not lead to a high SI because the pH is—for average surface seawater—relatively low, possibly because of ongoing respiration in the sediments (2 (and therefore high pH) and high Ca concentration, maintains an SI below 0.8 because of its relatively low alkalinity. Thus, carbonate precipitation can normally only be triggered if seawater is concentrated by substantial evaporation such as that occurring in highly evaporative  lagoons or intertidal flats and embayments [in vivo and/or early post-mortem calcified benthic cyanobacterial mats in ancient seas  [In Nivå Bay , from which the environmental samples originated, no carbonate precipitation was observed. The appearance of a spectrum of calcium carbonate morphs in the cultured mats was thus rather unexpected. Calcification is a function of a supersaturation. Supersaturation depends in turn on the concentrations of free Ca and COediments . Even avbayments ,13,22,27estones) ,75,76.i.e., living) cell configuration. Identification of the primary morphology of cyanobacteria in calcified mats is much easier in case of filamentous forms and difficult, or often impossible, in the case of coccoid cells that are usually much smaller. In these cases even slight re-crystallization of the primary fine-grained CaCO3 is a destructive factor , creating mineral coatings on cells or groups of cells. Apparently such mineral covers must be formed before bacteriolysis destroys the original . We con3 morphs in the studied mats seem to be transient in terms of their preservational potential. From the known microstructural and textural characteristics of ancient carbonate stromatolites and thrombolites studied thus far (for review: [micrite), whereas the larger aragonite/Mg calcite morphs precipitated in the purple bacterial zones recrystallized into a more or less uniform mosaic of Mg or low-Mg calcite crystallites (sparite).The majority of the primary CaCO review: ,73,74,77micrite and sparite layers are subfossil (micro) stromatolites from the quasi-marine Satonda Crater Lake  formed by colonial coccoid  cyanobacteria [in vivo precipitation of micritic aragonite in the living cyanobacterial layer due to high CaCO3 saturation in the mat ambience during dry season, and (ii) the post-mortem precipitation of sparitic aragonite in the decaying layers of coccoid cyanobacteria growing uncalcified or weakly calcified during the wet season when CaCO3 saturation in the mat ambience was lower. Stable carbon isotope (δ13C) signatures from neighboring individual laminae of the Satonda stromatolites, obtained with laser ablation sampler for stable isotope extraction (LASSIE)  b, show v(LASSIE) . In bothee e.g., ,80,81,823 polymorphs both from the studied artificial mats and those from the subfossil Satonda Crater Lake stromatolites, it can be supposed, by comparison with diagenetic processes recognized in other calcium carbonate sediments [Concerning the mineralogical stability of the CaCOediments  that, wimicritic) and coarse-grained (sparitic) layers to periodic environmental changes in the mat ambience. Much more laboratory and field studies are still needed to fully clarify which factor(s) are responsible for the origin of the micrite/sparite lamination in multilayered microbial mats like those presented herewith.The studied artificial mats were cultured under constant environmental lab conditions in terms of temperature, illumination, basic hydrochemistry and absence of macrobiota. Therefore, their biological and mineralogical zonation cannot be explained with the mode of origin suggested for the Satonda Crater Lake stromatolites and for many fossil calcareous stromatolites ,85,86,872, H2S, So, or certain organic compounds as electron donors. Depending on the character of the donor, the amount of CO2 released during photosynthesis may be higher than that assimilated. Due to uptake of the CO2 excess from the purple bacteria zones by the photo-assimilating cyanobacteria and diatoms, increased pH and alkalinity (carbonate ion abundance) is generated in the upper 2‒3 mm of the mat. Altogether, this causes high carbonate supersaturation resulting in precipitation of a variety of CaCO3 particles, some with characteristic morphologies . The much larger dimensions of these particles, as compared with the sub-micrometer grains from the living cyanobacterial layers, probably result from higher nucleation kinetics in the less viscous medium as compared to the viscous EPS of the living cyanobacterial layers. High-nucleation kinetics media are favorable for faster transformation of the nanoprecursor grains into super-structured morphs built of ordered monocrystals  occurring in the studied multilayered mat is as follows: The growing surficial cyanobacterial layer is from time to time interrupted by a community of photosynthetic purple bacteria that rapidly create their own microenvironment, while the associated heterotrophic bacteria start to decompose the dead cyanobacterial biomass. The purple bacteria and associated heterotrophic bacteria divide the cyanobacterial layer in two (or even more) layers that still continue to grow. Weakly calcified, multilayered microbial mats described from modern natural settings may show similar dynamics ,28,88,89s e.g., ,94,95). ,95. 2, Hs . This s3 precipitates obtained from cyanobacterial mats decaying in calcium-rich solutions [3 particles from these experiments are strikingly similar, if not identical, to the spectrum of morphs generated in our mats. Spheres, hemispheres, dumbbells, and peanut-shaped particles have also been produced in cultures of heterotrophic bacteria growing in CaCO3-supersaturated solutions with and without addition of Mg  of the CaCO3 particles ,1103 prepolymers ,120,121 articles .3 morphs [in vivo EPS-bound (complexed) Ca2+ and Mg2+ cations to the precipitation medium and significantly increasing its CaCO3 saturation level  high-Mg calcite grains (micrite layers), whereas those precipitated in layers with dead (biodegraded) cyanobacteria settled by purple and green sulfur bacteria in association with heterotrophic bacteria are represented by a great variety of >1 μm-sized particles often of mixed high-Mg calcite/aragonite composition (sparite layers).Our study showed that precipitation of CaCOin vivo and post-mortem generated calcareous layers clearly visible in the studied mats may help to explain the alternation of fine- and coarse-grained (micritic and sparitic) laminae observed in many modern, subfossil and fossil calcareous cyanobacterial microbialites, as the result of a probably similar multilayered biological organization of the precursor mats.The alternation of 3 precipitation in the artificial mats is enhanced and controlled by seawater with higher calcium carbonate saturation levels comparing with average seawater. This may suggest that the past epicontinental seas sustaining often in vivo calcifying cyanobacterial microbialites (stromatolites), and cyanobacteria-associated fine-grained and peloidal limestones were characterized by significantly higher calcium carbonate saturation level than present-day seawater.The process of CaCO3 morphs identified in the artificial mat are comparable with CaCO3 morphs precipitated not only in other natural and artificial microbial mats, but also with morphs originating in laboratory experiments in calcium carbonate oversaturated solutions both with and without organic additives. Notably, no metabolically controlled activity of heterotrophic bacteria in the formation of CaCO3 morphs was observed in the studied mats.The CaCO