New insights into the regulation of bile acids synthesis during the early stages of liver regeneration: A human and experimental study.

BACKGROUND AND AIMS: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol. METHODS: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot. RESULTS: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in sham-operated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet. CONCLUSIONS: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.

MCPIP1 Inhibits Hepatic Stellate Cell Activation in Autocrine and Paracrine Manners, Preventing Liver Fibrosis.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by enhanced deposition of extracellular matrix (ECM), which results from the wound healing response to chronic, repeated injury of any etiology. Upon injury, hepatic stellate cells (HSCs) activate and secrete ECM proteins, forming scar tissue, which leads to liver dysfunction. Monocyte-chemoattractant protein-induced protein 1 (MCPIP1) possesses anti-inflammatory activity, and its overexpression reduces liver injury in septic mice. In addition, mice with liver-specific deletion of Zc3h12a develop features of primary biliary cholangitis. In this study, we investigated the role of MCPIP1 in liver fibrosis and HSC activation. METHODS: We analyzed MCPIP1 levels in patients' fibrotic livers and hepatic cells isolated from fibrotic murine livers. In vitro experiments were conducted on primary HSCs, cholangiocytes, hepatocytes, and LX-2 cells with MCPIP1 overexpression or silencing. RESULTS: MCPIP1 levels are induced in patients' fibrotic livers compared with their nonfibrotic counterparts. Murine models of fibrosis revealed that its level is increased in HSCs and hepatocytes. Moreover, hepatocytes with Mcpip1 deletion trigger HSC activation via the release of connective tissue growth factor. Overexpression of MCPIP1 in LX-2 cells inhibits their activation through the regulation of TGFB1 expression, and this phenotype is reversed upon MCPIP1 silencing. CONCLUSIONS: We demonstrated that MCPIP1 is induced in human fibrotic livers and regulates the activation of HSCs in both autocrine and paracrine manners. Our results indicate that MCPIP1 could have a potential role in the development of liver fibrosis.

Plant Production Protocols from Seeds of Threatened Atropa baetica and Widespread A. belladonna, Both Rich in Alkaloids.

Members of the genus Atropa contain various tropane alkaloids, including atropine ((±)-hyoscyamine) and scopolamine, which possess medicinal properties. Preserving the diverse genetic background of wild populations via optimal plant production from seeds could be essential for avoiding the loss of potential uses. We analyzed the germination ecology of two Atropa species comprising the threatened A. baetica and widespread A. belladonna to determine the: (1) influence of temperature, light, and seed age on germination patterns; (2) effects of cold stratification and gibberellic acid (GA3); (3) phenology of seedling emergence in outdoor conditions; (4) phenology of dormancy break and loss of viability in buried seeds; and (5) ability to form persistent soil seed banks. Freshly matured seeds exhibited conditional physiological dormancy, with germination at high temperatures (32/18 °C) but not at low and cold ones (5, 15/4, 20/7 °C). The germination ability increased with time of dry storage and with GA3, thereby suggesting nondeep physiological dormancy. Under outdoor conditions, no seedlings emerged during the first post-sown autumn, but emergence peaks occurred in late winter-early spring. Both species could form small persistent soil seed banks with short durations (3-5 years). A plant production protocol from seeds was established for both taxa.

Comprehensive analysis of epigenetic and epitranscriptomic genes’ expression in human NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition with a complex etiology. Its incidence is increasing globally in parallel with the obesity epidemic, and it is now considered the most common liver disease in Western countries. The precise mechanisms underlying the development and progression of NAFLD are complex and still poorly understood. The dysregulation of epigenetic and epitranscriptomic mechanisms is increasingly recognized to play pathogenic roles in multiple conditions, including chronic liver diseases. Here, we have performed a comprehensive analysis of the expression of epigenetic and epitranscriptomic genes in a total of 903 liver tissue samples corresponding to patients with normal liver, obese patients, and patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), advancing stages in NAFLD progression. We integrated ten transcriptomic datasets in an unbiased manner, enabling their robust analysis and comparison. We describe the complete landscape of epigenetic and epitranscriptomic genes’ expression along the course of the disease. We identify signatures of genes significantly dysregulated in association with disease progression, particularly with liver fibrosis development. Most of these epigenetic and epitranscriptomic effectors have not been previously described in human NAFLD, and their altered expression may have pathogenic implications. We also performed a comprehensive analysis of the expression of enzymes involved in the metabolism of the substrates and cofactors of epigenetic and epitranscriptomic effectors. This study provides novel information on NAFLD pathogenesis and may also guide the identification of drug targets to treat this condition and its progression towards hepatocellular carcinoma. (AU)

Comprehensive analysis of epigenetic and epitranscriptomic genes' expression in human NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition with a complex etiology. Its incidence is increasing globally in parallel with the obesity epidemic, and it is now considered the most common liver disease in Western countries. The precise mechanisms underlying the development and progression of NAFLD are complex and still poorly understood. The dysregulation of epigenetic and epitranscriptomic mechanisms is increasingly recognized to play pathogenic roles in multiple conditions, including chronic liver diseases. Here, we have performed a comprehensive analysis of the expression of epigenetic and epitranscriptomic genes in a total of 903 liver tissue samples corresponding to patients with normal liver, obese patients, and patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), advancing stages in NAFLD progression. We integrated ten transcriptomic datasets in an unbiased manner, enabling their robust analysis and comparison. We describe the complete landscape of epigenetic and epitranscriptomic genes' expression along the course of the disease. We identify signatures of genes significantly dysregulated in association with disease progression, particularly with liver fibrosis development. Most of these epigenetic and epitranscriptomic effectors have not been previously described in human NAFLD, and their altered expression may have pathogenic implications. We also performed a comprehensive analysis of the expression of enzymes involved in the metabolism of the substrates and cofactors of epigenetic and epitranscriptomic effectors. This study provides novel information on NAFLD pathogenesis and may also guide the identification of drug targets to treat this condition and its progression towards hepatocellular carcinoma.

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds.

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species.

Impact of Liver Inflammation on Bile Acid Side Chain Shortening and Amidation.

Bile acid (BA) synthesis from cholesterol by hepatocytes is inhibited by inflammatory cytokines. Whether liver inflammation also affects BA side chain shortening and conjugation was investigated. In human liver cell lines (IHH, HepG2, and HepaRG), agonists of nuclear receptors including the farnesoid X receptor (FXR), liver X receptor (LXR), and peroxisome proliferator-activated receptors (PPARs) did not affect the expression of BA-related peroxisomal enzymes. In contrast, hepatocyte nuclear factor 4α (HNF4α) inhibition down-regulated acyl-CoA oxidase 2 (ACOX2). ACOX2 was repressed by fibroblast growth factor 19 (FGF19), which was prevented by extracellular signal-regulated kinase (ERK) pathway inhibition. These changes were paralleled by altered BA synthesis (HPLC-MS/MS). Cytokines able to down-regulate cholesterol-7α-hydroxylase (CYP7A1) had little effect on peroxisomal enzymes involved in BA synthesis except for ACOX2 and bile acid-CoA:amino acid N-acyltransferase (BAAT), which were down-regulated, mainly by oncostatin M (OSM). This effect was prevented by Janus kinase (JAK) inhibition, which restored BA side chain shortening and conjugation. The binding of OSM to the extracellular matrix accounted for a persistent effect after culture medium replacement. In silico analysis of four databases (n = 201) and a validation cohort (n = 90) revealed an inverse relationship between liver inflammation and ACOX2/BAAT expression which was associated with changes in HNF4α levels. In conclusion, BA side chain shortening and conjugation are inhibited by inflammatory effectors. However, other mechanisms involved in BA homeostasis counterbalance any significant impact on the serum BA profile.

Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis.

Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent studies have shown that defective function of hepatocyte nuclear factor 4 alpha (HNF4a) and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein, an integrative analysis of hepatic RNA sequencing and liquid chromatography-tandem mass spectrometry was performed to identify plasma protein signatures for patients with mild and severe AH. Alcohol-related liver disease cirrhosis, nonalcoholic fatty liver disease, and healthy subjects were used as comparator groups. Levels of identified proteins primarily involved in hepatocellular function were decreased in patients with AH, which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified, including thrombin, hepatocyte growth factor α, clusterin, human serum factor H-related protein, and kallistatin, which exhibited large abundance shifts between severe and nonsevere AH. The combination of thrombin and hepatocyte growth factor α discriminated between severe and nonsevere AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.

New molecular mechanisms in cholangiocarcinoma: signals triggering interleukin-6 production in tumor cells and KRAS co-opted epigenetic mediators driving metabolic reprogramming.

BACKGROUND: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. METHODS: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. RESULTS: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. CONCLUSIONS: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures.

OBJECTIVE: Despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). DESIGN: A prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. RESULTS: An initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. CONCLUSION: Implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Dual Pharmacological Targeting of HDACs and PDE5 Inhibits Liver Disease Progression in a Mouse Model of Biliary Inflammation and Fibrosis.

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated Mdr2-KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor ß (TGFß). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strategy.

Optimization of Plant Production by Seed Treatment in Two Wild Subspecies of Narcissus pseudonarcissus Rich in Alkaloids.

The daffodil Narcissus pseudonarcissus L. contains alkaloids of pharmaceutical interest. Wild daffodil populations have diverse genetic backgrounds and various genetic traits of possible importance. Developing protocols for plant production from seeds may ensure the availability of a large reservoir of individuals as well as being important for species with bulbs that are difficult to acquire. The closely related Narcissus pseudonarcissus subsp. munozii-garmendiae and subsp. nevadensis were investigated in this study because the alkaloids isolated from both are of high pharmacological interest. At the dispersal time, the seeds of both were dormant with underdeveloped embryos, i.e., morphophysiological dormancy (MPD). Experiments were conducted outdoors and under controlled laboratory conditions. Embryo growth and the percentages of radicle and seedling emergence were calculated under different temperature-light stratifications. In N. munozii-garmendiae, embryo growth occurred during warm stratification (28/14 °C or 25/10 °C) and the radicle then emerged when the temperature decreased, but the shoot was dormant. In N. nevadensis, the seeds germinated when cold stratified (5 °C) and then incubated at cool temperatures. Thus, N. munozii-garmendiae and N. nevadensis exhibit different levels of MPD, i.e., deep simple epicotyl and intermediate complex, respectively. Plant production protocols from seeds were established for both taxa in this study.

Pilot Multi-Omic Analysis of Human Bile from Benign and Malignant Biliary Strictures: A Machine-Learning Approach.

Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign (n = 36) and malignant conditions, CCA (n = 36) or PDAC (n = 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease and identifying biomarkers for the differential diagnosis of biliary strictures. Comprehensive analyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids (n = 10) and proteins (n = 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy.

Seed germination in Narcissus yepesii (Amaryllidaceae): clinal variation in the morphophysiological dormancy levels.

Seed dormancy classes determine both population and species-level processes which can be crucial in the life cycle of many plants. However, there are no studies of a dormancy cline between levels of morphophysiological dormancy (MPD). We aimed to determine the class of seed dormancy of Narcissus yepesii exhibits in order to explore links between different dormancy levels, previously characterized in two closely related phylogenetic congeners, N. alcaracensis and N. longispathus. Experiments were carried out under both near-natural temperature and controlled laboratory conditions. The parameters calculated were mean embryo length, radicle and shoot emergence percentages. The effects of different periods of storage; and different periods with or without GA3 of warm, cold or warm plus cold were analysed. The Narcissus populations from the Baetic System of mountain ranges in south-eastern Spain show clinal variation in a northeast-southwest gradient from intermediate to non-deep complex MPD, through the coexistence of intermediate and non-deep complex MPD in N. yepesii (21 % and 74 %, respectively). In addition, 54 % of stored seeds were able to show both levels of MPD. Narcissus yepesii occupies an intermediate position between N. alcaracensis and N. longispathus in the geographical distribution and in the clinal germination ranges. It strongly suggests an evolutionary gradient, which connects the intermediate complex MPD with the non-deep complex MPD in southern Iberian daffodils. This is the first study showing a gradient in the evolution between levels of MPD. Our results demonstrate a cline in these levels in response to both an environmental gradient and genetic differences.

Physiology, morphology and phenology of seed dormancy break and germination in the endemic Iberian species Narcissus hispanicus (Amaryllidaceae).

BACKGROUND AND AIMS: Only very few studies have been carried out on seed dormancy/germination in the large monocot genus Narcissus. A primary aim of this study was to determine the kind of seed dormancy in Narcissus hispanicus and relate the dormancy breaking and germination requirements to the field situation. METHODS: Embryo growth, radicle emergence and shoot growth were studied by subjecting seeds with and without an emerged radicle to different periods of warm, cold or warm plus cold in natural temperatures outdoors and under controlled laboratory conditions. KEY RESULTS: Mean embryo length in fresh seeds was approx. 1·31 mm, and embryos had to grow to 2·21 mm before radicle emergence. Embryos grew to full size and seeds germinated (radicles emerged) when they were warm stratified for 90 d and then incubated at cool temperatures for 30 d. However, the embryos grew only a little and no seeds germinated when they were incubated at 9/5, 10 or 15/4 °C for 30 d following a moist cold pre-treatment at 5, 9/5 or 10 °C. In the natural habitat of N. hispanicus, seeds are dispersed in late May, the embryo elongates in autumn and radicles emerge (seeds germinate) in early November; however, if the seeds are exposed to low temperatures before embryo growth is completed, they re-enter dormancy (secondary dormancy). The shoot does not emerge until March, after germinated seeds are cold stratified in winter. CONCLUSION: Seeds of N. hispanicus have deep simple epicotyl morphophysiological dormancy (MPD), with the dormancy formula C(1b)B(root) - C(3)(epicotyl). This is the first study on seeds with simple MPD to show that embryos in advanced stages of growth can re-enter dormancy (secondary dormancy).