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Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.The multi-subunit Elongator complex mediates the addition of a carboxymethyl group to wobble uridines in eukaryotic tRNAs. This tRNA modification is crucial to preserve the integrity of cellular proteomes and to protects us against severe neurodegenerative diseases. Elongator is organized in two distinct modules (i) the larger Elp123 subcomplex that binds and modifies the suitable tRNA substrate and (ii) the smaller Elp456 subcomplex that assists the release of the modified tRNA. The presented cryo-EM structures of Elongator show that the assemblies are very dynamic and undergo conformational rearrangements at consecutive steps of the process. Last but not least, the study provides a detailed reaction scheme and shows that the architecture of Elongator is highly conserved from yeast to mammals.

Species boundaries and geographic distribution of corticioid fungi (resupinate Basidiomycota) are often poorly known. Our recent study on Subulicystidium showed that species diversity in this genus is at least twice as high as previously recognized. This re-estimation of the species diversity was based on a study of only a part of the genus. The present study sheds light on molecular and morphological diversity of three more species. We generated 27 ITS and 24 28S nuclear ribosomal DNA sequences from 49 specimens labelled as Subulicystidium cochleum, S. longisporum and S. perlongisporum and collected in distant geographic localities. We assessed pairwise dissimilarities and phylogenetic relationships of DNA sequences with Bayesian and maximum likelihood methods. We correlated phylogenetic information with morphological data on spores and cystidia. We found that the three species are not closely related, despite their similarity in spore shape and size. In one of the species, S. perlongisporum, we detected the presence of two sympatric lineages. These lineages are not morphologically distinct, except for a small difference in the mean length of cystidia. Our study provides a further example of transoceanic species distribution in Agaricomycetes.

// Katarzyna Szarc vel Szic 1, 4 , Ken Declerck 1 , Rene A.J. Crans 1, 3 , Jolien Diddens 1 , David B. Scherf 2 , Clarissa Gerhauser 2 and Wim Vanden Berghe 1 1 Laboratory of Protein Chemistry, Proteomics and Epigenetic Signaling (PPES), Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium 2 Workgroup Cancer Chemoprevention and Epigenomics, Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Current address: Laboratory for GPCR Expression and Signal Transduction (L-GEST), Department of Biochemistry and Microbiology, University of Ghent, Ghent, Belgium 4 Current address: Division of Hematology, Oncology and Stem Cell Transplantation, Center for Translational Cell Research, The University Medical Center Freiburg, Freiburg, Germany Correspondence to: Wim Vanden Berghe, email: wim.vandenberghe@uantwerpen.be Keywords: triple negative breast cancer, luminal breast cancer, epigenetics, Withaferin A, DNA methylation Received: February 15, 2016 Accepted: March 30, 2017 Published: April 13, 2017 ABSTRACT Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. Withaferin A (WA) is a plant derived steroidal lactone which holds promise as a therapeutic agent for treatment of breast cancer (BC). We determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines, following 72h treatment to a sub-cytotoxic concentration of WA. In contrast to the DNA demethylating agent 5-aza-2’-deoxycytidine (DAC), WA treatment of MDA-MB-231 cells rather tackles an epigenetic cancer network through gene-specific DNA hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 ( ADAM8 ), urokinase-type plasminogen activator ( PLAU ), tumor necrosis factor (ligand) superfamily, member 12 ( TNFSF12 ), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1 ), or mitochondrial metabolism (malic enzyme 3, ME3 ). Gene expression and pathway enrichment analysis further reveals epigenetic suppression of multiple cancer hallmarks associated with cell cycle regulation, cell death, cancer cell metabolism, cell motility and metastasis. Remarkably, DNA hypermethylation of corresponding CpG sites in PLAU , ADAM8 , TNSF12 , GSTM1 and ME3 genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ESR)/progesterone receptor (PR) status in primary BC tumors. Moreover, upon comparing differentially methylated WA responsive target genes with DNA methylation changes in different clinical subtypes of breast cancer patients in the cancer genome atlas (TCGA), we found that WA silences HER2/PR/ESR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks, with an improved therapeutic sensitivity. In this respect, WA may represent a novel and attractive phyto-pharmaceutical for TNBC treatment.

Genome-wide association studies have highlighted three major lung cancer susceptibility regions at 15q25.1, 5p15.33 and 6p21.33. To gain insight into the possible mechanistic relevance of the genes in these regions, we investigated the regulation of candidate susceptibility gene expression by epigenetic alterations in healthy and lung tumor tissues. For genes up or downregulated in lung tumors, the influence of genetic variants on DNA methylation was investigated and in vitro studies were performed. We analyzed 394 CpG units within 19 CpG islands in the susceptibility regions in a screening set of 34 patients. Significant findings were validated in an independent patient set (n=50) with available DNA and RNA. The most consistent overall DNA methylation difference between tumor and adjacent normal tissue on 15q25 was tumor hypomethylation in the promoter region of CHRNB4 with a median difference of 8% (P

Genetic case-control association studies often include data on clinical covariates, such as body mass index (BMI), smoking status, or age, that may modify the underlying genetic risk of case or control samples. For example, in type 2 diabetes, odds ratios for established variants estimated from low–BMI cases are larger than those estimated from high–BMI cases. An unanswered question is how to use this information to maximize statistical power in case-control studies that ascertain individuals on the basis of phenotype (case-control ascertainment) or phenotype and clinical covariates (case-control-covariate ascertainment). While current approaches improve power in studies with random ascertainment, they often lose power under case-control ascertainment and fail to capture available power increases under case-control-covariate ascertainment. We show that an informed conditioning approach, based on the liability threshold model with parameters informed by external epidemiological information, fully accounts for disease prevalence and non-random ascertainment of phenotype as well as covariates and provides a substantial increase in power while maintaining a properly controlled false-positive rate. Our method outperforms standard case-control association tests with or without covariates, tests of gene x covariate interaction, and previously proposed tests for dealing with covariates in ascertained data, with especially large improvements in the case of case-control-covariate ascertainment. We investigate empirical case-control studies of type 2 diabetes, prostate cancer, lung cancer, breast cancer, rheumatoid arthritis, age-related macular degeneration, and end-stage kidney disease over a total of 89,726 samples. In these datasets, informed conditioning outperforms logistic regression for 115 of the 157 known associated variants investigated (P-value = 1×10−9). The improvement varied across diseases with a 16% median increase in χ2 test statistics and a commensurate increase in power. This suggests that applying our method to existing and future association studies of these diseases may identify novel disease loci., Author Summary This work describes a new methodology for analyzing genome-wide case-control association studies of diseases with strong correlations to clinical covariates, such as age in prostate cancer and body mass index in type 2 diabetes. Currently, researchers either ignore these clinical covariates or apply approaches that ignore the disease's prevalence and the study's ascertainment strategy. We take an alternative approach, leveraging external prevalence information from the epidemiological literature and constructing a statistic based on the classic liability threshold model of disease. Our approach not only improves the power of studies that ascertain individuals randomly or based on the disease phenotype, but also improves the power of studies that ascertain individuals based on both the disease phenotype and clinical covariates. We apply our statistic to seven datasets over six different diseases and a variety of clinical covariates. We found that there was a substantial improvement in test statistics relative to current approaches at known associated variants. This suggests that novel loci may be identified by applying our method to existing and future association studies of these diseases.

Epigenetic changes occur in all types of cancers and include alterations in deoxyribonucleic acid (DNA) methylation, histone modification, or expression changes of non-coding ribonucleic acid (RNA). There is now evidence that epigenetic alterations influence cancer risk and could be developed into useful biomarkers. While it is clear that epigenetic mechanisms can play a key role in mediating environmental influences including dietary folate intake, smoking, other environmental stressors, and age, many of the mechanisms involved remain obscure. There are numerous mechanisms by which genetic and epigenetic factors interact and thus the field of epigenetic epidemiology of cancer faces considerable challenges in tearing apart gene-epigene-environment interactions and in determining how they modulate individual cancer risk. An important question for epigenetic epidemiology of cancer is whether epigenetic changes can be identified in surrogate tissues; e.g., DNA isolated from peripheral lymphocytes, plasma samples, urine, or buccal swabs. The current state of the art of cancer epigenetic epidemiology studies as relating to epigenetic cancer risk factors and markers of diagnostic and prognostic significance is reviewed. In the past, epigenetic work has been carried out in comparative studies of primary human normal/tumor tissue pairs, but recent advances in technologies have made large scale analyses on surrogate tissues within defined cohorts possible. Interindividual variations exist in epigenetic markers, but their origin and relevance needs to be determined. In future studies it will be important to characterize the interaction between environmental, dietary, genetic, and epigenetic factors. Echoing the development in the field of genetics, candidate gene approaches employed thus far will be replaced by genome-wide epigenome analyses in the future. Approaches integrating genetic, epigenetic, and well-documented epidemiological and clinical data are most promising and may improve the currently available risk models for cancer in the future.

SummaryThe subepicardial injection of 0.1 cc of digitalis or of a strophanthin preparation, as well as of other substances, over the sinus area leads to the immediate but transient appearance of sinoauricular block. This phenomenon is attributed to a reflex inhibition of the sinus node centers caused by the mechanical stimulus and not to a specific effect of the injected substances. In some instances the centers of the auriculo-ventricular node are also inhibited.

The prognosis for lung cancer patients treated with chemotherapy is poor. Single nucleotide polymorphisms (SNPs) in matrix metalloproteinase (MMP) genes could influence treatment outcome by altering apoptotic pathways. Eight SNPs with known or suspected phenotypic effect in six genes (MMP1, MMP2, MMP3, MMP7, MMP9 and MMP12) were investigated. For 349 Caucasian patients with primary lung cancer, receiving first-line chemotherapy, three different endpoints were analysed: response after the second cycle, progression free survival (PFS) and overall survival (OS). The prognostic value of the SNPs was analysed using multiple logistic regression for all patients and histology-, stage- and treatment-specific subgroups. Hazard ratio estimates for PFS and OS were calculated using Cox regression methods. None of the investigated polymorphisms modified response significantly in the whole patient population. However, tumour stage IIIB variant allele carriers of MMP2 C-735T showed a significantly worse response. PFS was significantly prolonged in MMP1 G-1607GG variant allele carriers and OS in small cell lung cancer patients carrying the MMP12 A-82G variant allele. In conclusion, this study identified SNPs in MMP1, MMP2, MMP7 and MMP12 for further investigation as possible predictors of chemotherapy outcome in lung cancer patients.

The inhibition of ectopic impulse formation by carbon dioxide has gained practical importance with the advent of refrigeration and hypothermia anesthesia. Ventricular fibrillation is one of the commoner hazards of this procedure and its seriousness cannot be overemphasized. It has been stated recently that the danger of experimental ventricular fibrillation could be markedly decreased when the cooled animals inhaled a mixture of 5 to 10 per cent carbon dioxide with oxygen. Similar results have been obtained clinically. 4,9,10 The study reported in this paper is concerned with the effect of inhalation of carbon dioxide on atrial fibrillation caused by aconitine and on ventricular fibrillation elicited by focal cooling of a rapid heart. Previous experiments have shown that focal cooling of the rapidly beating ventricles leads to ventricular fibrillation. 14

SummaryThe intravenous administration of 0.5 mg of dihydroergocornine to 24 patients with left ventricular hypertrophy caused no electrocardiographic changes indicative of coronary vasoconstriction. Although 6 of the 24 patients suffered from anginal pain and many more certainly had coronary disease, no angina pectoris followed the injection.This particular alkaloid of ergot seems to be a safe drug in patients with coronary artery disease.

Extrasystoles with constant coupling are elicited in the dog by topical application of veratrine and the inhalation of 20 per cent of carbon dioxide with 80 per cent oxygen. Warming the focus of origin of the extrasystoles shortens, cooling lengthens, the duration of the coupling. After the extrasystoles disappear, they are brought out again in the form of bigeminal rhythm by warming the area on which veratrine has been applied. In the opinion of the authors, these results are best explained if one assumes that extrasystoles are caused by the phenomenon of after-discharge in an altered cell.

Resume of previously reported experimental studies upon conduction in the A-V bundle and bundle branches which show that: 1. 1. In nodal rhythm, with the P-waves buried in the ventricular complexes (pacemaker in the lower part of the node) a ventricular or auricular extrasystole, under certain conditions, will cause a positive P-R interval in the first post-extrasystolic A-V complex, the P-wave gradually moving back to its original position in the following complexes. 2. 2. In nodal rhythm with positive P-R intervals (pacemaker in upper part of node) a ventricular or auricular extrasystole, under certain conditions, will cause the P-wave of the first post-extrasystole A-V beat to be buried in the ventricular part of that complex and gradually to move back to its original position in the following complexes. 3. 3. The cause of these phenomena, we believe, is an altered conductivity of the specific tissue lying below the point of origin of the impulse, caused by a lengthened or shortened rest and recovery time of that tissue. We therefore bring evidence to support the Engelmann and Wenckebach conception of the cause of the pathological prolongation of the P-R interval. 4. 4. Neither “shifting of the pacemaker” nor “sinus escape” can explain these findings. 5. 5. In our studies on conduction in the bundle branches, when one branch was cut and the other one pressed upon with the dull side of a small knife, we obtained curves showing all forms and degrees of complete and partial A-V block. 6. 6. We can have conduction disturbances in the bundle branches giving us the above curves, without the A-V node or the ventricular musculature coming at all into consideration. 7. 7. Curves of Type I of partial block (gradual lengthening P-R interval with dropped beats) were produced by anatomical lesions. This fact rather argues against the impression of Mobitz that this type of block is due only to functional disturbances. Cardiograms presented, expamples of many obtained in the course of other experiments, are analyzed in the sense of our studies. They also tend to show: 1. 1. In animal experiments, ano-esophageal lead, positive or diphasic P-waves can be seen in undoubted nodal rhythm. 2. 2. Ventricular extrasystoles are conducted backward in the bundle and bundle branches for a greater or lesser distance, causing a greater demand upon that tissue, though they do not provoke an auricular contraction.

1. Der sichere Nachweis, das Flattern und Flimmern auf eine Kreisbewegung zuruckzufuhren sind, steht noch aus.

SummaryIn cats and dogs Viscum album produces a characteristic high take-off. If this is distinctly developed, one can no longer recognize a disturbance of intraventricular conduction (bundle-branch block) and stimulation of the accelerans does not alter the shape of the terminal deflection.

Alternating tachycardias with regular or varying cycle lengths may result from stimulus formation in one or two centers. This was established by both experimental and clinical observations. Experimentally rapid impulse formation may evoke an alternating tachycardia from two ventricular centers as in a double parasystole or from a single focus in the A-V node. Clinically alternation could be abolished by carotid sinus pressure with or without slowing; be simulated by interpolated ventricular extrasystoles; be recorded with an increase in A-V nodal rate in patients with bigeminy, and be caused by extrasystoles with different couplings and different forms.

SummaryStretching of the auricular muscle fibers during a tachycardia induced by aconitine leads to an increase of rate and to transient auricular fibrillation. Transient auricular fibrillation (or flutter) can be elicited by stretching during a period of 15-20 minutes after the aconitine arrhythmia has subsided and sinus rhythm prevails.Because there is much evidence that stretching changes impulse formation and not impulse conduction these results are believed to bring further evidence against the circus movement hypothesis.The experiments offer an explanation for the appearance of paroxysmal fibrillation induced by physical exertion.

In two series of cases we studied the effect on the heart of the intravenous injection of hypertonic glucose solution. In Group I it was observed that in patients with effort angina and coronary arteriosclerosis the injection of hypertonic glucose solution produced marked electrocardiographic changes and frequently caused pain. In Group II it was observed that patients with effort angina and pathologic alterations of the electrocardiogram after exercise developed the same, and even more marked, alterations of the electrocardiogram when hypertonic glucose solution was given shortly before the performance of the exercise test. The various theories concerning the effect of hypertonic glucose solution on the blood and coronary arteries were discussed, and the possible causes for the production of cardiac pain after the injection of hypertonic glucose solution were reviewed.

Ventricular ectopic tachycardias were created by the focal injection of 0.05 c.c. of a 30 per cent solution of sodium chloride, or a 3.8 per cent solution of sodium citrate or oxalate into the subepicardial layers of the ventricular myocardium. These tachycardias were often followed by a parasystolic rhythm. The experiments show that a rapidly firing center is protected from the normal impulses which spread over the heart, and, thus, the hypothesis of entrance (protection) block is not necessary. These parasystoles provide an opportunity for study of the distance between ectopic beats and preceding normal beats (coupling). It was found that this distance varies little if normal and ectopic rhythms are rapid and have an almost equal rate. Marked variations in the coupling occur if the rates differ. Little variation in the coupling may occur if the impulses of a rapid parasystolic center become effective only shortly after a conducted beat.

Four instances of parasystole with the ectopic center in the upper portion of the A-V node are described, and several similar observations were collected from the literature. The astonishing fact is stressed that the automaticity of the ectopic center is slow in most of these observations. Unlike ventricular parasystole this form seems to appear in persons with healthy hearts.

Ventricular tachycardia provoked by focal application of Hypertonic solutions of sodium chloride on the exposed heart of the dog is little influenced by the intravenous injection of 0.01 to 0.02 cc. per kg. of the commercial solution of epinephrine. Only a moderate acceleration of the rate is seen when epinephrine is injected in the same dosage after a ventricular tachycardia was obtained by focal application of sodium oxalate. In no experiment did epinephrine elicit ventricular fibrillation. These results and the experience that extrasystoles and tachycardias caused by administration of aconitine are abolished by administration of epinephrine or sympathetic stimulation encourage the use of the less stormily acting pressor amines in patients with ectopic arrhythmias and shock. Under the influence of nicotine or epinephrine applied focally, one mechanical impulse causes a long after-discharge in the form of chains of ectopic beats. The clinical implications of this finding are discussed.

Changes in the form of the T waves in patients with auricular flutter and varying atrioventricular block, as well as in auricular fibrillation, depend mainly on variations of rate. Other additional factors seem also responsible. These alterations involve only the terminal phase of the T wave, the RS-T segment remaining unchanged. They are seen mostly in Leads V 3 , V 4 and V 5 (particularly V 4 ) and are less pronounced or even absent in the other leads. The T-wave changes of the first postextrasystolic beat and the "isolated T negativity" of Grant appear in the same chest leads. These variations may be present only temporarily, as illustrated in patients with acute myocardial infarctions. Deep inspiration may modify these changes in a manner which cannot be explained as yet. Carotid sinus pressure usually increases the negativity of the T wave following a prolonged diastole; however, modification in the opposite direction may be seen. The fact that only the later portion of the T wave is involved may lead to bizarre forms of the electrocardiogram. The changes are attributed to variations of the rate of repolarization in abnormal parts of the myocardium. These may be located even in the subendocardial layers.

Faradic stimulation of the right or left sympathetic nerves of the heart of the dog in situ leads to a distinct and often marked acceleration of auricular flutter caused by topical application of aconitine. Flutter is often transformed into fibrillation by this measure. Administration of atropine regularly changes auricular fibrillation into flutter or slows the rate of auricular flutter. Faradic stimulation of the sympathetic nerves or the injection of epinephrine, after atropinization, causes only a slight acceleration of the existing flutter rate; occasionally these measures remain without effect on the auricular rate despite the appearance of marked and typical changes of the RS-T segments. It is concluded that the more marked effect of sympathetic stimulation before atropinization is due to reflex increase in vagus tonus.

MANY common arrhythmias observed in both atria and ventricles are similar although the atrial types have a few important distinguishing features. Thus, so far as therapy is concerned, the atrial arrhythmias are less responsive to procaine amide than the ventricular ones. On the other hand, quinidine exerts a stronger action on the atria.1 In general, atrial tachycardias do not, if present without complications, endanger life. Atrial paralysis from the failure of impulse formation in the sinus node or a failure of conduction of the impulse from its point of origin is usually compensated immediately by the escape of the deeper . . .