33 Thus, it is an apparent paradox that in DEN-exposed mice an increased as well as decreased β-catenin expression appears to enhance the susceptibility to HCC. A surprising and unexpected result of our study was that β-catenin mutations are comparatively late events and that chromosomal instability clearly precedes the occurrence this website of β-catenin mutations. In tumors at weeks 32-42 there was no clear correlation between β-catenin mutation status and chromosomal instability.

Thus, β-catenin mutations do not appear to be the driving force behind chromosomal instability in this mouse model. The apparent lack of correlation between β-catenin mutations and chromosomal instability may explain why, in contrast to previous reports,35, 36 our tumors with β-catenin mutations clearly show chromosomal instability. Importantly and also in contrast to previous

work, β-catenin-activating mutations are—at least in the DEN model—not involved in HCC initiation but rather in tumor progression, which may explain the differences reported in the two aforementioned studies.32, 33 In Idasanutlin solubility dmso summary, our study suggests that the majority of the current knowledge about genomic changes in HCC is based on advanced tumor lesions and that systematic analyses of the chronologic order including early lesions may reveal new, unexpected findings. Such findings include that at least in the DEN-induced HCC mouse model β-catenin mutations are not early events and that these mutations occur independently of chromosomal instability. Loss of the distal region of the mouse chromosome 4q, which is syntenic to the distal human 1p region, is likely an early driver mutation. The loss of the known tumor suppressor genes Runx3 and Nr0b2/Shp within this region N-acetylglucosamine-1-phosphate transferase may be critical in the initiation or first steps

in HCC development. We thank Prof. Michael Trauner and Dr. Johannes Haybaeck for critically reading the article and stimulating discussions. We thank Mag. Maria Langer-Winter for editing the article. We thank Dusan Babic for expert help in the preparation of the figures and Dr. Ivan Kanchev for help with pathologic samples. The help of Dr. Walter Spindelböck with the statistical analysis is highly appreciated. Additional Supporting Information may be found in the online version of this article. “
“Nonalcoholic fatty liver disease (NALFD) encompasses a wide spectrum of liver injury ranging from bland hepatic steatosis to nonalcoholic steatohepatitis (NASH). Bland steatosis follows a relatively benign clinical course, but NASH may progress to cirrhosis. NAFLD affects about one third of the adult US population and up to 10% of children and teenagers. The demographics of NAFLD in the general population mirrors that of the metabolic syndrome, which is characterized by obesity, diabetes, hypertension, and dyslipidemia.

33 Thus, it is an apparent paradox that in DEN-exposed mice an increased as well as decreased β-catenin expression appears to enhance the susceptibility to HCC. A surprising and unexpected result of our study was that β-catenin mutations are comparatively late events and that chromosomal instability clearly precedes the occurrence INCB018424 mouse of β-catenin mutations. In tumors at weeks 32-42 there was no clear correlation between β-catenin mutation status and chromosomal instability.

Thus, β-catenin mutations do not appear to be the driving force behind chromosomal instability in this mouse model. The apparent lack of correlation between β-catenin mutations and chromosomal instability may explain why, in contrast to previous reports,35, 36 our tumors with β-catenin mutations clearly show chromosomal instability. Importantly and also in contrast to previous

work, β-catenin-activating mutations are—at least in the DEN model—not involved in HCC initiation but rather in tumor progression, which may explain the differences reported in the two aforementioned studies.32, 33 In Dorsomorphin cost summary, our study suggests that the majority of the current knowledge about genomic changes in HCC is based on advanced tumor lesions and that systematic analyses of the chronologic order including early lesions may reveal new, unexpected findings. Such findings include that at least in the DEN-induced HCC mouse model β-catenin mutations are not early events and that these mutations occur independently of chromosomal instability. Loss of the distal region of the mouse chromosome 4q, which is syntenic to the distal human 1p region, is likely an early driver mutation. The loss of the known tumor suppressor genes Runx3 and Nr0b2/Shp within this region Mannose-binding protein-associated serine protease may be critical in the initiation or first steps

in HCC development. We thank Prof. Michael Trauner and Dr. Johannes Haybaeck for critically reading the article and stimulating discussions. We thank Mag. Maria Langer-Winter for editing the article. We thank Dusan Babic for expert help in the preparation of the figures and Dr. Ivan Kanchev for help with pathologic samples. The help of Dr. Walter Spindelböck with the statistical analysis is highly appreciated. Additional Supporting Information may be found in the online version of this article. “
“Nonalcoholic fatty liver disease (NALFD) encompasses a wide spectrum of liver injury ranging from bland hepatic steatosis to nonalcoholic steatohepatitis (NASH). Bland steatosis follows a relatively benign clinical course, but NASH may progress to cirrhosis. NAFLD affects about one third of the adult US population and up to 10% of children and teenagers. The demographics of NAFLD in the general population mirrors that of the metabolic syndrome, which is characterized by obesity, diabetes, hypertension, and dyslipidemia.

However, such acceleration of clearance can give rise to a decline of only log (1/aV) = 0.75-0.86 log10 copies/mL (Fig. 2), because viral load during accelerated clearance will reach a new steady state at V0/a (Supporting Material, Equation 6). This is considerably less than the viral decline observed here, and thus accelerated clearance is a necessary but not sufficient explanation of the antiviral mechanism. The observed HBV

DNA kinetics during HepeX-B infusion can be explained by assuming a combination of two mechanisms of action: an accelerated CH5424802 HBV clearance from the circulation (aV > 1), mediated by the infused antibodies, together with partial blocking of virion release from infected cells (1 > εV > 0), as shown in Fig. 2. Using this assumption, we performed nonlinear fitting for all patients (Fig. 3 and Table 1A), assuming either a slow or a rapid intrinsic half-life of HBV

virions, thus giving, accordingly, low and high estimates of the effectiveness in blocking virion release of εV = 76.2%-96.1% (97%-99.5%). The analysis of HBsAg kinetics following HepeX-B infusion yields similar conclusions with higher acceleration of clearance and effectiveness in blocking release (Fig. 3). Analysis of HBsAg decline during treatment with lamivudine shows decline with a half-life of 38 days, which we use as a maximal estimate of HBsAg half-life, with a minimal estimate of 1 day (data not shown). Nonlinear fitting of HBsAg decline during HepeX-B gives a half-life of 0.09-0.19 Adriamycin hours, thus corresponding to a minimal (maximal) estimate of the acceleration of HBsAg clearance from circulation of aA = 126-282 (4,800-10,729) times an intrinsic half-life of 1 (38) days. As for the HBV DNA kinetics, accelerated clearance of HBsAg cannot by itself explain the total decline, and thus a maximal (minimal) estimate of effectiveness in

blocking release of HBsAg particles from infected cells is εA = 98.6-99.5% (46.2%-82.4%). Note, that because all patients reached undetectable levels of HBsAg, the decline is probably larger and thus the estimates of effectiveness of blocking HBsAg release are most probably underestimated next here. In addition to the three patients who received a single infusion of 40 mg HepeX-B, six patients received multiple HepeX-B doses of 40 and 80 mg (Fig. 1C-F), but those patients had less frequent sampling and the duration of infusion was only 4 hours. We have therefore tested in the first three patients with frequent samples whether the estimation of decline half-life and total decline from 0.5 to 4 hours is accurate enough. We found that although both values were slightly underestimated with less frequent samples, the estimate of effectiveness in blocking release with the 4-hour sample was still accurate within 90%-110% of the nonlinear fitting (Table 1B). In five of six patients who received multiple doses of HepeX-B, a rapid decline with a half-life 0.31-0.66 hours and magnitude of 1.6-3.

The promoter activities of RANTES and PRDII in transfected cells mock infected or infected with HCV for the indicated periods were determined using the luciferase reporter assay, as previously described.12, 14 Detailed methods selleck screening library have been presented elsewhere.7, 12, 15 The following polyclonal (pAb) and monoclonal (mAb) antibodies were used: anti-IκBα (C-21) and anti-p65 (C-20) pAbs and anti-Lamin A/C (636) mAb (Santa Cruz Biotechonology, Santa Cruz, CA); anti-HCV NS5A (9E10) mAb (a gift

from Charles Rice, Center for the Study of Hepatitis C, The Rockefeller University); anti-actin mAb (Sigma-Aldrich, St. Louis, MO); anti-ISG15 (a gift from Dr. Arthur Haas, Louisiana State University), and anti-ISG56 pAbs12; and peroxidase-conjugated secondary antirabbit and antimouse pAbs (Southern Biotech, Birmingham, AL). The protein bands were visualized by enhanced chemiluminescence (Millipore, Billerica, MA), followed by exposure to X-ray films. 7.5-TLR3 cells (∼5 × 106) were mock infected or infected with HCV (multiplicity of infection [MOI] = 0.5) for 48 hours, then were processed for chromatin immunoprecipitation (ChIP) assay, as described previously.14 The antibodies used for ChIP were

from Santa Cruz (anti-p65) and Active Motif (control immunoglobulin G). The ChIP-enriched samples were subjected to qPCR quantification of various chemokine and Trefoil family factor 1 (TFF1) (negative control) promoter sequences using BMN 673 nmr specific primers presented online in Supporting Table 3. To investigate whether TLR3 plays a role in hepatoceullar proinflammatory response to HCV

infection, we determined the production of cytokines/chemokines in HCV-infected 7.5-TLR3 cells, which were derived from Huh7.5 cells by stably reconstituting the expression of human TLR3. As controls, we studied Huh7.5 cells expressing the control vector (Vect) and mutant TLR3s defective for signaling as a result of incapability of dsRNA binding (H539E and N541A). All these cells are RIG-I defective, and the effects observed were the result of activation of the TLR3 pathway in the absence of RIG-I. We have previously utilized these cell lines to demonstrate that TLR3 senses HCV infection and triggers a weak ISG response, thereby moderately reducing HCV propagation DOK2 when cells were infected at low MOIs.12 We infected cells with JFH1 virus at an MOI of 0.2 for 72 hours, then harvested the culture supernatants to measure cytokine/chemokine production by a Bio-Plex Multiplex Cytokine Assay. At this MOI, HCV did not replicate differentially among these Huh7.5 derivatives,12 and all the cells were infected at 72 hours, as determined by immunostaining of NS5A (data not shown). Compared to mock-infected cells, six chemokines/cytokines were strongly induced by HCV infection in 7.5-TLR3 cells, including RANTES (37-fold), MIP-1β (25-fold), MIP-1α (17-fold), IL-6 (13-fold), IP-10 (10-fold), and tumor necrosis factor alpha (TNF-α) (10-fold) (Fig. 1).

Late apoptosis was determined by TUNEL (DNAse-I) staining. Proliferation was assessed by Ki-67 and PCNA staining. HIF-1 alpha induction induces significantly higher PCNA/Ki-67 expression paralleled by bax/bcl-2 ratio < 1 favorable for anti-apoptotic conditions. By downregulation of CUX1 after HIF-1-alpha induction early and late apoptosis markers caspase 3/7 and their cleavage products and DNASe-I were highly significantly induced followed PLX4032 by significantly loss of PCNA and Ki-67 positive cells. CUX1 and its role in hcc has to be further investigated a potential new therapeutic target. Disclosures:

The following people have nothing to disclose: Carmen Rothmund, Pietro Di Fazio, Heidi Griesmann, Benjamin Kuehnemuth, Thomas Gress, Patrick Michl, Thaddaeus T. Wissniowski Background The role of alpha-feto protein (AFP) in the diagnosis of hepatocellular carcinoma (HCC) is getting smaller due to the advances of imaging modalities. However, consecutive increment of AFP level in liver cirrhosis patients is

presumed to be associated with the higher risk of developing HCC in clinical settings. Such a notion instigated us to analyze serial AFP levels of HCC patients in a retrospective manner. Methods From January 2002 to December 2012, 1931 patients were diagnosed with HCC in Seoul St. Mary’s hospital. Among them 133 patients were found to have a serial record of AFP measurements for over one year. We assessed AFP levels at the time the diagnosis of HCC was made and compared them with that of patients Tigecycline at 3,6 and 12 months prior to the diagnosis. Results At the time the diagnosis was made, the patients’ baseline characteristics were as follows; mean age was 58.24 years (32-87), median tumor size was 2.2cm (0.9-26.3), median AFP level was 45.53 ng/mL (1.4-32134). Median AFP level of 12 months, 6 months and 3 months before the diagnosis of HCC was 6.19 ng/mL (1.12-513), 7.53 ng/mL (0.96-1287.86), 11.94 ng/mL (0.91-1461), 45.53 ng/mL (1.4-23134), respectively. Consecutive increment of AFP level was statistically significant in time dependent

manner (p<0.000) with linear relationship (P=0.001). these We divided patients by two groups; one was AFP over 45 ng/mL at the time of diagnosis of HCC, and the other one was not. In elevated AFP group (n=67), median AFP level of 12 months, 6 months and 3 months before the diagnosis of HCC and at the time of the diagnosis of HCC was 11.76 ng/mL (1.3513), 26.82 ng/mL (1.4-1287.86), 76.92 ng/mL (3-1461), 476 ng/mL (45.53-23134), respectively. In non-elevated AFP group (n=66), median AFP level was 5.37 ng/mL (1.1274.78), 6.09 ng/mL (0.96-91), 5.51 ng/mL (0.91-30.01), 5.63 ng/mL (1.4-40.1), respectively. In elevated AFP group, consecutive increment of AFP level was statistically significant in time dependent manner (p≤0.000). However, there was no significant change of consecutive AFP level In non-elevated AFP group.

Typically, serum levels of adiponectin, a potent anti-inflammatory cytokine, are reduced in NASH. “Immuno-metabolism” is an emerging field of research that investigates the co-regulation of metabolic and inflammatory pathways within immune cells. Chronic exposure of the immune system to particular nutrients can lead to metabolically triggered inflammation (meta-inflammation). In this context tissue resident macrophages play a pivotal role

in monitoring tissue function and restoring metabolic homeostasis. We explored how the intrinsic metabolic state of liver resident macrophages (Kupffer cells) change in NASH. Materials and Methods: Male C57BL/6 adiponectin knockout (ADN) and wild type mice were fed either normal chow (NC) or a high cholesterol CP-690550 cost (HC) diet for 12 weeks. At the end of this Paclitaxel mw period, serum and hepatic cholesterol were measured. Liver tissues were examined by haematoxylin and eosin (H&E), and by CD68 immunostaining. For in vitro analyses of immuno-metabolism, rat Kupffer cells were isolated and cultured in various concentrations of glucose and treated with LPS +/− adiponectin, and palmitate. qPCR and western blots were performed

to determine mRNA expression and to study changes in signaling molecules in liver tissue and kupffer cells. Results: When fed the high cholesterol diet, both genotypes had similar increases in average liver/total body weight ratio and reduced fat pad weight. Biochemistry and histology showed that KO mice fed the HC (ADN-HC) diet had significantly increased ALT levels, and elevated hepatic total (148 vs. 101 mg/dl, p < 0.05), crotamiton and free cholesterol (114 vs. 80 mg/dl, p < 0.05). ADN-HC livers had increased gene expression for TNFα (11-fold, p < 0.05) CD68 (6-fold, p < 0.05), IL-1β (1.4-fold, p < 0.05), CCL19 (6.0-fold, p < 0.01), liver pyruvate kinase (L-PK), (WT-HC 6.7-fold, ADN-HC 5-fold, p < 0.05), and 1.8-fold increase in expression of RelB by

western blot. In preliminary in vitro experiments, we find that treatment of Kupffer cells with LPS leads to increased glucose uptake. We detected incremental increases in LPS mediated activation of the NFkB pathway in macrophages cultured without glucose, low glucose (5 mM) and high glucose (25 mM) media. Adiponectin increased glucose uptake and adding adiponectin to the media increased NFkB activation, whereas palmitate reduced the uptake of glucose and suppressed activation of NFkB pathways. Conclusion: In vivo, adiponectin KO mice fed the high cholesterol diet had severe inflammation, with glycolytic pathway activation. Likewise, exposure to glucose modified the inflammatory status of Kupffer cells. These data suggest that Kupffer cell driven meta-inflammation plays an important role in hepatic immunometabolism and NASH pathogenesis.

Triptans are not commonly used during pregnancy mainly because of the fact that conclusive evidence on their safety profiles is still lacking with only a few studies having been conducted so far.8-11 In one study, the sumatriptan

exposed group was found to be at an increased risk of preterm delivery compared with migraine controls (OR = 6.3; 95% CI: 1.2-32.0) and all pregnant migraineurs who delivered at term were found to be at an increased risk of low birth weight compared with nonmigraine controls (OR = 3.0; 95% CI: 1.3-7.0).9 Pharmaceutical selleck inhibitor company registry studies12,13 have only published a few data on birth defect rates. In these studies, the frequencies of major congenital malformations were reported to be 4.7% for sumatriptan used in 599 pregnancies12 and 3.1% for rizatriptan used in 51 pregnancies;13 these percentages all lie within the normal expected range of birth defect rates for the general population. However, studies based on pharmaceutical company registries are often limited because of a lack of control groups and recall

bias because Compound Library of retrospective reporting and data collection. In general, congenital malformation rates amount to 3.8% in Norway14 2.2% in Europe,14 3.0% in the United States of America15 and 2.8% in Latin America.16 It should, however, be noted Akt inhibitor that these data are not directly comparable, as the inclusion criteria vary between countries. While it is necessary to exercise caution when using pharmacotherapy during pregnancy, untreated or inadequately managed severe migraine may also pose a risk to both the mother and child. Some studies have found a significant association between migraine

and preeclampsia,17-21 and preeclampsia is known to be associated with intrauterine growth retardation and prematurity. An association between migraine during pregnancy and ischemic stroke in the mother has also been found.22 However, the impact of migraine on pregnancy outcome remains uncertain as direct associations between a disease as such and adverse pregnancy outcomes are often difficult to determine. No previous studies on the safety of triptans during pregnancy have taken the possible effect of the underlying disorder into consideration. The main aim of this study was to provide more information on the safety of triptan therapy during pregnancy. More specifically, associations between triptan therapy and congenital malformations, other adverse pregnancy outcomes (including miscarriage/stillbirth, death of the newborn or infant, prematurity, low birth weight and low Apgar scores), and perinatal complications (including atonic uterus, prolonged labor, and extensive maternal blood loss at delivery) were the focus of this study.

In order to better assess the quality of the liver, in this review we focus on some liver-specific donor risk indices. LIVER STEATOSIS IS strongly associated with poor graft function after LT. The impact of severe steatosis on allograft survival appears greater than other donor factors, including MG-132 mw the calculated DRI.[1] Moreover, clinicopathological studies have demonstrated an inverse correlation between steatosis and graft survival.[9] Steatosis is typically characterized qualitatively and quantitatively. Fatty infiltration is separated into two categories, macrosteatosis and microsteatosis. Macrosteatosis is characterized by a single, bulky fat vacuole

in hepatocytes, displacing the

nucleus to the edge of the cell. In microsteatosis, the cytoplasm of the hepatocytes contains tiny lipid vesicles without nuclear dislocation. Microsteatosis seems SB203580 molecular weight to have a low impact on the postoperative liver function. Macrosteatosis and microsteatosis most often present simultaneously at different degrees in the liver.[10] The quantitative evaluation is traditionally based on percentages of visualized hepatocytes containing fat vacuoles within the cytoplasm, classified as mild (<30%), moderate (30–60%) or severe (>60%).[11-13] Livers with more than 40–50% macrosteatosis should not be used.[14] In all such Rolziracetam cases, the procurement surgeon has to make the definitive decision. A percutaneous liver biopsy performed at the bedside, before organ procurement, may help prevent unnecessary donor laparotomy. In addition, livers with severe steatosis from donation after

cardiac death donors, combined with a prolonged cold ischemic time have a high risk for developing early allograft dysfunction which is correlated with shorter graft survival. Therefore, severe steatosis livers should only be considered for LT in selected recipients without the presence of additional risk factors.[15] Although hepatic steatosis is a widely accepted risk factor for postoperative complications after LT, studies have been inconsistent regarding the relevant amount of fat or type of fat. All those observations lead to controversies in the field. Some studies showed that liver grafts containing moderate degrees of microsteatosis significantly increase the rate of organ failure after LT,[16] while other groups recommended the use of microsteatotic grafts, regardless of the total amount, to safely expand the donor pool.[17] The main reason for the inconsistent outcome is the estimation of steatosis using frozen section liver biopsy which is both difficult and subjective.[18, 19] Quantification of hepatic steatosis in histological sections is strongly observer-dependent, not reproducible. El-Badry et al.

After densitometric analysis corrected for GAPDH expression, the expression of MMP-12 showed no significant changes across all groups (data not shown). Given the tightly regulated activity of MMPs,9, 10 it was important to detect whether active MMP-12 was present. One of the main factors in determining MMP activity is the ratio with their tissue inhibitors, especially TIMP-1. TIMP-1 mRNA and protein (Fig. 3B) were increased after 8 and 12 weeks injury. In order to establish the degree of inhibition of MMP-12

by TIMP-1 in our model system, we coimmunoprecipitated the two proteins and analyzed the samples by zymography. After immunoprecipitation of MMP-12, casein zymography (Fig. 3C) showed a similar pattern to the samples used for western blot, indicating even efficiency of precipitation. selleck products Additionally, when we immunoprecipitated TIMP-1 and performed casein zymography (Fig. 3C) the signal increased through 4 to 8 and 12 weeks (Fig. 3C), indicating that there is increasing amounts of TIMP-1 bound to MMP-12 in increasingly fibrotic liver. Thus, MMP-12

buy LBH589 is present in the liver but held in check by noncovalent binding to TIMP-1 with increasing duration of liver injury. Taken together, these data strongly suggest that the elastin content in scars is regulated by MMP-12-mediated degradation, with active MMP-12 being inhibited by increased interaction with TIMP-1 with worsening fibrosis in vivo. Previous work by Yoshiji et al.10, 28 using a TIMP-1 overexpression, however, suggests that the Timp-1 inhibition may not be maximal and MMP-mediated Branched chain aminotransferase degradation still occurs

in remodeling during progressive fibrosis. MMP-12 has been reported to be expressed by macrophages.29 We confirmed this by immunocytochemistry on human monocyte-derived macrophages stained for both MMP-12 and the macrophage marker CD-68 (Fig. 4A1-2); 100% of the cells were positive for both proteins. To define which cells express MMP-12 in vivo, we stained serial sections of rat tissue for MMP-12 and CD-68 (Fig. 4A3-4). We found that the cells positive for MMP-12 were macrophages but that only a proportion of the CD-68-positive macrophages were also positive for MMP-12. To confirm the macrophage origin of MMP-12, we used the transgenic mouse CD11b-DTR in which macrophages can be selectively depleted as described.22 These mice show a 50% decrease in macrophage populations and increased accumulation of elastin compared with WT mice after CCl4 administration. Staining of liver following macrophage depletion showed a significant decrease in MMP-12-positive cells (Fig. 4B1-3). qPCR analysis of these tissues (Fig. 4B4) showed no significant changes in the expression of either tropoelastin or neutrophil elastase, whereas MMP-12 expression was significantly decreased. To further confirm that macrophages are the major hepatic source of MMP-12, we costained mouse liver after CCl4 injury for MMP-12 and key liver cell markers.

In this strain, all four 16S rRNA genes were identical, which is in agreement with a study of Engene and Gerwick (2011). Two operons contained nearly identical 16S-23S ITS regions with both tRNA genes, and two operons contained nearly identical 16S-23S ITS regions lacking tRNA genes. This result SCH772984 manufacturer was consistent with what we found in all other Cylindrospermum strains, and we assume that all strains had this pattern even when one of the ITS operons was not recovered. The 16S-23S ITS regions were highly similar within the Cylindrospermum taxa in clades X and Y, permitting alignment of both operon 1 and operon 2. Phylogenies of these ITS regions had high bootstrap support,

and are superior to the 16S rRNA phylogenies for elucidating relationships among species (Fig. 1, b and c). These trees were in fairly close agreement with each other and with the 16S phylogeny. The ITS phylogenies were not in disagreement with our species assignments, i.e. no species was clearly not monophyletic. Secondary structure of the conserved domains of the ITS revealed a number of species-specific patterns. The D1-D1′ helices were identical for C. muscicola SAG 44.79, C. licheniforme CCALA 995, C. badium CCALA 1000, and the five strains of

C. catenatum, although sequence differences existed (note ambiguous bases W and R, group I; Fig. 2a). Cylindrospermum pellucidum CCALA 989/CCALA 992, C. stagnale PCC 7417, and C. moravicum CCALA selleck chemical 993 possessed helices structurally similar to the D1-D1′ helices of the C. catenatum group, but differed in minor

ways (Fig. 2, b and c). Cronbergia also had a D1-D1′ helix similar to all of the above (Fig. 2d). Cylindrospermum stagnale PCC 7417 had a single nucleotide in the terminal loop that differed from the above group (Fig. 2, a–d), such that the terminal loop was divided into a small terminal loop and a subterminal bilateral bulge (Fig. 2h). The C. catenatum group could close the large terminal loop in this position as well, but this structure would be less thermodynamically stable (GC….GU binds, but more weakly than the AC….GU in C. stagnale PCC 7417). A major break in structure occurred between the group including C. catenatum and the group of species Chlormezanone in the clade containing C. marchicum CCALA 1001, C. alatosporum CCALA 988/CCALA 994, and C. maius CCALA 998 (Fig. 2, e–g). The Hawaiian Cylindrospermum CCALA 1002 had different helices in the two different operons, both of which showed significant divergence from the helices of other species (Fig. 2, i and j). Aulosira bohemensis was also very distinct (Fig. 2k). The V2 helix is located between the tRNAIle and tRNAAla genes, and is thus only present in those operons, which contain the tRNA genes. This helix varied considerably among strains. C. catenatum (CCALA 990, 991, 996) and C.