Study of a novel evolutionarily conserved pattern of histone acetylation

Le génome eucaryote est empaqueté dans une structure hautement ordonnée appelée chromatine. Même si la structure de la chromatine est importante pour le maintien de l'intégrité génomique, elle constitue une barrière à de nombreux processus basés sur l'ADN tels que la réplication de l'ADN, la transcription et la réparation de l'ADN. Les histones contiennent une diversité déconcertante de modifications covalentes qui sont concentrées principalement, mais non exclusivement dans leurs queues amino-terminales. Les modifications des histones jouent un rôle central dans la régulation de la structure et de la fonction de la chromatine. Cependant, la détermination de la stoechiométrie des modifications à des sites spécifiques, l'identification des motifs de modifications et l'établissement de leurs rôles physiologiques restent des défis redoutables. Dans cette étude, nous avons utilisé la spectrométrie de masse pour déterminer la stoechiométrie de l'acétylation de résidus lysine spécifiques des histones. En général, les résidus lysine des histones dépourvus d'acétylation sont dérivatisés pour rendre les peptides résultants chimiquement équivalents à leurs homologues acétylés, mais pouvant être distingués par spectrométrie de masse. Cependant, cette méthode est insuffisante pour étudier les peptides contenant plus d'une lysine acétylable, tels que ceux dérivés de la queue N-terminale des histones H3 et H4. La digestion trypsique de tels peptides génère des «isomères de position», des isomères qui ont la même masse, mais qui portent des groupes acétyle à des positions différentes. La quantification précise de l'acétylation d'un site spécifique dans ces peptides est donc un défi analytique majeur. Dans le deuxième chapitre, nous décrivons une nouvelle méthode, pour quantifier l'acétylation à un site spécifique dans les peptides co-éluants isomériques et isobariques, qui combine des données LC-MS / MS à haute résolution avec un nouvel algorithme bioinformatique, Iso-PeptidAce. En utilisant des spectres de masse en tandem (MS/MS) de peptides synthétiques, les produits de fragmentation caractéristiques de chaque isomère de position ont été identifiés et utilisés pour déconvoluer des spectres provenant de mélanges d'isomères de position et quantifier l'abondance de chaque isomère. Nous avons ensuite testé l'applicabilité de l'Iso-PeptidAce pour quantifier les augmentations en fonction du temps de l'acétylation des histones des cellules d'érythroleucémie K562 traitées avec des inhibiteurs d’histone déacétylase (HDAC). En utilisant notre méthode, nous avons également trouvé que les histones H3 et H4 associées à CAF-1, un facteur d'assemblage de la chromatine, ont une stoechiométrie élevée d’acétylation sur plusieurs résidus de H3 et H4, par rapport aux histones totales. Dans le chapitre 3, nous avons appliqué Iso-PeptidAce pour déterminer la stoechiométrie de l'acétylation chez la levure de fission présentant un mutant d’histone désacétylase. Conformément aux études antérieures impliquant Clr3 et Sir2 dans la régulation de l’hétérochromatine, nous avons observé que les cellules dépourvues de ces HDAC présentaient une augmentation de l'acétylation H3-K14 uniquement sur les peptides coexistant avec H3-K9 di / tri méthylé, une marque caractéristique de l'hétérochromatine. Au chapitre 4, nous décrivons la découverte de très hauts niveaux d'acétylation sur deux résidus de lysine. Nous avons trouvé qu'une stoechiométrie élevée d’acétylation à H3-K14 et H3-K23 et une faible stoechiométrie d’acétylation à H3-K9 et H3-K18 est un profil global de H3 conservé sur le plan évolutif d’acétylation. En utilisant des souches de levures de fission (S. pombe) où la seule source de gènes d'histone porte des mutations H3-K14R et / ou H3-K23R qui empêchent l'acétylation, nous avons démontré que H3-K14 et H3-K23 ont des fonctions distinctes. De façon surprenante, nous avons trouvé que les phénotypes observés dans les cellules mutantes H3-K14R sont largement dus à la mutation du résidu lysine, plutôt qu'à la perte d'acétylation. En utilisant des souches de S. pombe dépourvues d'histone acétyltransférases (HAT), nous avons identifié les acétyltransférases qui contribuent à H3-K14ac et à H3-K23ac in vivo. Très peu d'études ont cherché à déterminer spécifiquement les stoechiométries d'acétylation des histones. Nos résultats suggèrent qu’en moyenne, sur l'ensemble du génome, chaque deuxième ou troisième nucléosome contient une molécule H3 avec une acétylation K14 et / ou K23. Cela nous amène à penser que l'acétylation de l'histone H3 à l'échelle du génome peut jouer un rôle important dans la fonction chromosomique. Il est impératif de comprendre la signification fonctionnelle de ce modèle d'acétylation étant donné que la thérapie épigénétique est activement étudiée comme stratégie pour traiter de nombreuses maladies.The eukaryotic genome is packaged into a highly ordered chromatin structure. Even though chromatin structure is important for maintaining genomic integrity, it is a barrier to numerous DNA-based processes such as DNA replication, transcription and DNA repair. Histones contain a bewildering diversity of covalent modifications that are mostly but not exclusively concentrated within their amino-terminal tails. Histone modifications play a central role in regulating chromatin structure and function. However, determining the stoichiometry of site-specific modifications, identifying patterns of modifications and establishing their physiological roles remain formidable challenges. In this study, we exploited mass spectrometry to determine the stoichiometry of acetylation at specific histone lysine residues. In general, histone lysine residues lacking acetylation are derivatized to render the resulting peptides chemically equivalent but distinguishable by mass from their acetylated counterparts. However, this method is insufficient to study peptides that contain more than one acetylatable lysine, such as those derived from the N-terminal tail of histones H3 and H4. Tryptic digestion of such peptides generates ‘positional isomers’, isomers that have the same mass but bearing acetyl groups located at different positions. Accurate quantification of site-specific acetylation in those peptides is, therefore, a major analytical challenge. In the second chapter, we describe a novel method for quantifying site-specific acetylation of co-eluting isomeric and isobaric peptides that combines high-resolution LC-MS/MS data with a novel bioinformatics algorithm, Iso-PeptidAce. Using tandem mass spectra (MS/MS) of synthetic peptides, fragmentation products diagnostic of each positional isomer were identified and were used to deconvolute spectra that arise from mixtures of positional isomers and quantify the abundance of each isomer. We then tested the applicability of Iso-PeptidAce to quantify time-dependent increases in histone acetylation of K562 erythroleukaemia cells treated with histone deacetylase (HDAC) inhibitors. Using our method, we also found that histones H3 and H4 associated with CAF-1, a chromatin assembly factor, have a high stoichiometry of acetylation on multiple residues of H3 and H4, compared with total histones. In Chapter 3, we applied Iso-PeptidAce to determine the stoichiometry of acetylation in fission yeast histone deacetylase mutants. Consistent with previous reports implicating Clr3 and Sir2 in heterochromatin function, we observed that cells lacking these HDACs showed an increase in H3-K14 acetylation only on those peptides where it co-exists with di/trimethylated H3-K9, a mark of heterochromatin. In chapter 4, we describe the discovery of very high levels of acetylation on two lysine residues. We found that a high stoichiometry of acetylation at H3-K14 and H3-K23, and low stoichiometry of acetylation at H3-K9 and H3-K18, is an evolutionarily conserved global pattern of H3 acetylation. Using fission yeast (S. pombe) strains harboring histone mutations H3-K14R and/or H3-K23R that prevent acetylation, we demonstrate that H3-K14 and H3-K23 have separable functions. Surprisingly, we found that the phenotypes observed in H3-K14R mutant cells are largely due to mutation of the lysine residue, rather than loss of acetylation. Using S. pombe strains that lack histone acetyltransferases (HATs) we identified the acetyltransferases that contribute to H3-K14ac and H3-K23ac in vivo. Very few studies have aimed at specifically determining the acetylation stoichiometries of histones. Our results suggest that, on average, over the entire genome, every second or third nucleosome contains an H3 molecule with K14 and/or K23 acetylation. This leads us to surmise that genome-wide acetylation of histone H3 may have an important role in chromosome function. It is imperative to understand the functional significance of this acetylation pattern given that epigenetic therapy is actively pursued as a strategy to treat many diseases

Tuberculosis and metastatic carcinoma coexistence in axillary lymph node: A case report

BACKGROUND: Coexistence of cancer and tuberculosis in axillary lymph nodes is rare. Only seven cases have been reported in the literature. CASE REPORT: We report here a case of infiltrating ductal carcinoma breast metastasizing to the axillary lymph node along with tubercular granuloma in the same lymph node without primary mammary or pulmonary tuberculosis. CONCLUSION: Primary tuberculosis coexisting with carcinoma is of rare occurrence. A possibility should always be borne in mind especially in patients from endemic areas

Optical Properties of High-Frequency Radio Sources from the Australia Telescope 20 GHz (AT20G) Survey

Our current understanding of radio-loud AGN comes predominantly from studies at frequencies of 5 GHz and below. With the recent completion of the Australia Telescope 20 GHz (AT20G) survey, we can now gain insight into the high-frequency radio properties of AGN. This paper presents supplementary information on the AT20G sources in the form of optical counterparts and redshifts. Optical counterparts were identified using the SuperCOSMOS database and redshifts were found from either the 6dF Galaxy survey or the literature. We also report 144 new redshifts. For AT20G sources outside the Galactic plane, 78.5% have optical identifications and 30.9% have redshift information. The optical identification rate also increases with increasing flux density. Targets which had optical spectra available were examined to obtain a spectral classification. There appear to be two distinct AT20G populations; the high luminosity quasars that are generally associated with point-source optical counterparts and exhibit strong emission lines in the optical spectrum, and the lower luminosity radio galaxies that are generally associated with passive galaxies in both the optical images and spectroscopic properties. It is suggested that these different populations can be associated with different accretion modes (cold-mode or hot-mode). We find that the cold-mode sources have a steeper spectral index and produce more luminous radio lobes, but generally reside in smaller host galaxies than their hot-mode counterparts. This can be attributed to the fact that they are accreting material more efficiently. Lastly, we compare the AT20G survey with the S-cubed semi-empirical (S3-SEX) models and conclude that the S3-SEX models need refining to correctly model the compact cores of AGN. The AT20G survey provides the ideal sample to do this.Comment: Accepted for publication in MNRA

In Situ Recrystallization of Co-Evaporated Cu(In,Ga)Se\u3csub\u3e2\u3c/sub\u3e Thin Films by Copper Chloride Vapor Treatment Towards Solar Cell Applications

Cu(In,Ga)Se2 (or CIGS) thin films and devices were fabricated using a modified three-stage process. Using high deposition rates and a low temperature during the process, a copper chloride vapor treatment was introduced in between the second and third stages to enhance the films properties. X-ray diffraction and scanning electron microscopy demonstrate that drastic changes occur after this recrystallization process, yielding films with much larger grains. Secondary ion mass spectrometry shows that the depth profile of many elements is not modified (such as Cu, In and Se) while others change dramatically (such as Ga and Na). Because of the competing effects of these changes, not all parameters of the solar cells are enhanced, yielding an increase of 15% in the device efficiency at the most

Learning without contingencies induces higher order asynchrony in brain networks in schizophrenia

Schizophrenia (SCZ) is characterized by both cognitive and reward impairments. A recent study suggests that SCZ is associated with a loss of synchrony between learning and reward circuits (Robison et al., 2019) and higher levels of dis-organization of functional brain networks may underpin failures in learning that characterize SCZ (Hütt et al., 2014). Therefore, here we examined inter-group (HC ≠ SCZ) 4th order differences in statistical regularity across a connectome of cognition and reward brain circuits. The analyses were conducted on fMRI time series data from a previous learning paradigm (Stanley et al., 2017) with periods of Encoding and Retrieval. 75 participants (46 SCZ, 18 Age 50) consented for fMRI. Time Series were extracted from eight bilateral a priori nodes (across learning and reward sub-networks). 2nd order undirectional functional connectivity was characterized across all nodal pairs during Encoding, Retrieval, and their subsequent rest periods. From this, a 4th order cross-correlation matrix was produced within each group and condition. Significant 4th order differences were projected to chords in the 4th order connectomic rings for Encoding and Retrieval (Figure 1). Two effects are evident: 1) SCZ are characterized by a massive loss of 4th order synchrony during both Encoding and Retrieval and 2) Retrieval evokes a greater loss in 4th order statistical synchrony than does Encoding. These results appear to validate the idea the SCZ is characterized by a loss of synergy between cognition and reward circuits, and that this loss of synergy is evident at higher order scales

The Australia Telescope 20GHz (AT20G) Survey: analysis of the extragalactic source sample

The Australia Telescope 20 GHz (AT20G) survey is a blind survey of the whole Southern sky at 20 GHz with follow-up observations at 4.8, 8.6, and 20 GHz carried out with the Australia Telescope Compact Array (ATCA). In this paper we present an analysis of radio spectral properties in total intensity and polarisation, sizes, optical identifications, and redshifts of the sample of the 5808 extragalactic sources in the survey catalogue of confirmed sources over the whole Southern sky excluding the strip at Galactic latitude |b|<1.5deg. The sample has a flux density limit of 40 mJy. Completeness has been measured as a function of scan region and flux density. Averaging over the whole survey area the follow-up survey is 78% complete above 50mJy and 93% complete above 100mJy. 3332 sources with declination <-15deg have good quality almost simultaneous observations at 4.8, 8.6, and 20GHz. The spectral analysis shows that the sample is dominated by flat-spectrum sources. The fraction of flat-spectrum sources decreases from 81% for 20GHz flux densities S>500mJy, to 60% for S<100mJy. There is also a clear spectral steepening at higher frequencies with the median spectral index decreasing from -0.16 between 4.8 and 8.6GHz to -0.28 between 8.6 and 20GHz. Simultaneous observations in polarisation are available for all the sources at all the frequencies. 768 sources have a good quality detection of polarised flux density at 20GHz; 467 of them were also detected in polarisation at 4.8 and/or at 8.6GHz so that it has been possible to compare the spectral behaviour in total intensity and polarisation. We have found that the polarised fraction increases slightly with frequency and decreases with flux density. Cross matches and comparisons have been made with other catalogues at lower radio frequencies, and in the optical, X-ray and gamma-ray bands. Redshift estimates are available for 825 sources.Comment: 15 pages, 16 figures, accepted for publication in MNRA

An Erythropoietin-Independent Mechanism of Erythrocytic Precursor Proliferation Underlies Hypoxia Tolerance in Sea Nomads

The Bajau Sea Nomads were recently demonstrated to have evolved larger spleens as an adaptation to millennia of a marine foraging lifestyle. The large-spleen phenotype appears to derive from increases in thyroid hormone (TH) production as a result of reduced expression of phosphodiesterase 10A (PDE10A), though the exact mechanism remains unknown. Through pharmacological inhibition of PDE10A using the selective inhibitor MP-10 in mice, we were able to mimic the Bajau adaptation and show that treated mice had significantly larger spleens than control animals. This difference appears connected to an excess of early stage erythrocytes and an apparent increase in red blood cell (RBC) precursor proliferation in response to increased TH. However, we determined that the stimulation of RBC production in the mouse model via TH is Erythropoietin (EPO)-independent, unlike in the altitude (chronic hypoxemia) response. We confirmed this using human GWAS data; although the Bajau PDE10A variants are significantly associated with increased TH levels and RBC count, they are not associated with EPO levels, nor are other strongly thyroid-associated SNPs. We therefore suggest that an EPO-independent mechanism of stimulating RBC precursor proliferation via TH upregulation underlies the increase in spleen size observed in Sea Nomad populations

Exosomal microRNAs in breast cancer: towards theranostic applications

Breast cancer is one of the top two reproductive cancers responsible for high rates of morbidity and mortality among women globally. Despite the advancements in the treatment of breast cancer, its early diagnosis remains a challenge. Recent evidence indicates that despite the adroit use of numerous strategies to facilitate rapid and precision-oriented screening of breast cancer at the community level through the use of mammograms, Fine-needle aspiration cytology (FNAC) and biomarker tracking, no strategy has been unequivocally accepted as a gold standard for facilitating rapid screening for disease. This necessitates the need to identify novel strategies for the detection and triage of breast cancer lesions at higher rates of specificity, and sensitivity, whilst taking into account the epidemiologic and social-demographic features of the patients. Recent shreds of evidence indicate that exosomes could be a robust source of biomaterial for the rapid screening of breast cancer due to their high stability and their presence in body fluids. Increasing evidence indicates that the Exosomal microRNAs- play a significant role in modifying the tumour microenvironment of breast cancers, thereby potentially aiding in the proliferation, invasion and metastasis of breast cancer. In this review, we summarize the role of ExomiRs in the tumour microenvironment in breast cancer. These ExomiRs can also be used as candidate biomarkers for facilitating rapid screening and triaging of breast cancer patients for clinical intervention