Natural antisense transcript of natriuretic peptide precursor A (NPPA): structural organization and modulation of NPPA expression

<p>Abstract</p> <p>Background</p> <p>Mammalian transcriptome contains a large proportion of diverse and structurally complex noncoding RNAs. One class of such RNAs, natural antisense transcripts (NATs), are derived from the opposite strand of many protein-coding genes. Although the exact structure and functional relevance of most of the NATs is unknown, their emerging role as gene expression regulators raises the hypothesis that NATs might contribute to development of complex human disorders. The goal of our study was to investigate the involvement of NATs in regulation of candidate genes for blood pressure.</p> <p>Results</p> <p>First we analysed blood pressure candidate genes for the presence of natural antisense transcripts. <it>In silico </it>analysis revealed that seven genes (<it>ADD3</it>, <it>NPPA</it>, <it>ATP1A1</it>, <it>NPR2</it>, <it>CYP17A1</it>, <it>ACSM3</it>, <it>SLC14A2</it>) have an antisense partner transcribed from the opposite strand. We characterized <it>NPPA </it>and its antisense transcript (<it>NPPA-AS</it>) in more detail. We found that <it>NPPA-AS </it>is expressed in a number of human tissues as a collection of alternatively spliced isoforms and that <it>NPPA-AS </it>and <it>NPPA </it>can form RNA duplexes <it>in vivo</it>. We also demonstrated that a specific <it>NPPA-AS </it>isoform is capable of down-regulating the intron-retained <it>NPPA </it>mRNA variant. We studied the evolutionary conservation of <it>NPPA-AS </it>and were able to detect the presence of <it>Nppa-as </it>transcript in mouse.</p> <p>Conclusion</p> <p>Our results demonstrate functional interaction of <it>NPPA-AS </it>with <it>NPPA </it>at the RNA level and suggest that antisense transcription might be an important post-transcriptional mechanism modulating <it>NPPA </it>expression.</p

Allele frequencies of variants in Ultra Conserved Elements identify selective pressure on transcription factor binding

Ultra-conserved genes or elements (UCGs/UCEs) in the human genome are extreme examples of conservation. We characterized natural variations in 2884 UCEs and UCGs in two distinct populations ; Singaporean Chinese (n=280) and Italian (n=501) by using a pooled sample, targeted capture, sequencing approach. We identify, with high confidence, in these regions the abundance of rare SNVs (MAF&lt;0.5%) of which 75% is not present in dbSNP137. UCEs association studies for complex human traits can use this information to model expected background variation and thus necessary power for association studies. By combining our data with 1000 Genome Project data, we show in three independent datasets that prevalent UCE variants (MAF&gt;5%) are more often found in relatively less-conserved nucleotides within UCEs, compared to rare variants. Moreover, prevalent variants are less likely to overlap transcription factor binding site. Using SNPfold we found no significant influence of RNA secondary structure on UCE conservation. All together, these results suggest UCEs are not under selective pressure as a stretch of DNA but are under differential evolutionary pressure on the single nucleotide level

Lysimeter-based full fertilizer 15N balances corroborate direct dinitrogen emission measurements using the 15N gas flow method

The $^{15}$N gas flux ($^{15}$NGF) method allows for direct in situ quantification of dinitrogen (N$_2$) emissions from soils, but a successful cross-comparison with another method is missing. The objectives of this study were to quantify N$_2$ emissions of a wheat rotation using the $^{15}$NGF method, to compare these N$_2$ emissions with those obtained from a lysimeter-based $^{15}$N fertilizer mass balance approach, and to contextualize N$_2$ emissions with $^{15}$N enrichment of N$_2$ in soil air. For four sampling periods, fertilizer-derived N$_2$ losses ($^{15}$NGF method) were similar to unaccounted fertilizer N fates as obtained from the $^{15}$N mass balance approach. Total N$_2$ emissions ($^{15}$NGF method) amounted to 21 ± 3 kg N ha− 1, with 13 ± 2 kg N ha− 1 (7.5% of applied fertilizer N) originating from fertilizer. In comparison, the $^{15}$N mass balance approach overall indicated fertilizer-derived N$_2$ emissions of 11%, equivalent to 18 ± 13 kg N ha− 1. Nitrous oxide (N$_2$O) emissions were small (0.15 ± 0.01 kg N ha− 1 or 0.1% of fertilizer N), resulting in a large mean N$_2$:(N$_2$O + N$_2$) ratio of 0.94 ± 0.06. Due to the applied drip fertigation, ammonia emissions accounted for < 1% of fertilizer-N, while N leaching was negligible. The temporal variability of N$_2$ emissions was well explained by the δ$^{15}$N$_2$ in soil air down to 50 cm depth. We conclude the $^{15}$NGF method provides realistic estimates of field N$_2$ emissions and should be more widely used to better understand soil N$_2$ losses. Moreover, combining soil air δ$^{15}$N$_2$ measurements with diffusion modeling might be an alternative approach for constraining soil N$_2$ emissions

Hypervariable intronic region in NCX1 is enriched in short insertion-deletion polymorphisms and showed association with cardiovascular traits

<p>Abstract</p> <p>Background</p> <p>Conserved non-coding regions (CNR) have been shown to harbor gene expression regulatory elements. Genetic variations in these regions may potentially contribute to complex disease susceptibility.</p> <p>Methods</p> <p>We targeted CNRs of cardiovascular disease (CVD) candidate gene, <it>Na(+)-Ca(2+) exchanger (NCX1) </it>with polymorphism screening among CVD patients (n = 46) using DHPLC technology. The flanking region (348 bp) of the 14 bp indel in intron 2 was further genotyped by DGGE assay in two Eastern-European CVD samples: essential hypertension (HYPEST; 470 cases, 652 controls) and coronary artery disease, CAD (CADCZ; 257 cases, controls 413). Genotype-phenotype associations were tested by regression analysis implemented in PLINK. Alignments of primate sequences were performed by ClustalW2.</p> <p>Results</p> <p>Nine of the identified <it>NCX1 </it>variants were either singletons or targeted by commercial platforms. The 14 bp intronic indel (rs11274804) was represented with substantial frequency in HYPEST (6.82%) and CADCZ (14.58%). Genotyping in Eastern-Europeans (n = 1792) revealed hypervariable nature of this locus, represented by seven alternative alleles. The alignments of human-chimpanzee-macaque sequences showed that the major human variant (allele frequency 90.45%) was actually a human-specific deletion compared to other primates. In humans, this deletion was surrounded by other short (5-43 bp) deletion variants and a duplication (40 bp) polymorphism possessing overlapping breakpoints. This indicates a potential indel hotspot, triggered by the initial deletion in human lineage. An association was detected between the carrier status of 14 bp indel ancestral allele and CAD (<it>P </it>= 0.0016, OR = 2.02; Bonferroni significance level alpha = 0.0045), but not with hypertension. The risk for the CAD development was even higher among the patients additionally diagnosed with metabolic syndrome (<it>P </it>= 0.0014, OR = 2.34). Consistent with the effect on metabolic processes, suggestive evidence for the association with heart rate, serum triglyceride and LDL levels was detected (<it>P </it>= 0.04).</p> <p>Conclusions</p> <p>Compared to SNPs targeted by large number of locus-specific and genome-wide assays, considerably less attention has been paid to short indel variants in the human genome. The data of genome dynamics, mutation rate and population genetics of short indels, as well as their impact on gene expressional profile and human disease susceptibility is limited. The characterization of <it>NCX1 </it>intronic hypervariable non-coding region enriched in human-specific indel variants contributes to this gap of knowledge.</p

Resequencing PNMT in European hypertensive and normotensive individuals: no common susceptibilily variants for hypertension and purifying selection on intron 1

<p>Abstract</p> <p>Background</p> <p>Human linkage and animal QTL studies have indicated the contribution of genes on Chr17 into blood pressure regulation. One candidate gene is <it>PNMT</it>, coding for phenylethanolamine-N-methyltransferase, catalyzing the synthesis of epinephrine from norepinephrine.</p> <p>Methods</p> <p>Fine-scale variation of <it>PNMT </it>was screened by resequencing hypertensive (n = 50) and normotensive (n = 50) individuals from two European populations (Estonians and Czechs). The resulting polymorphism data were analyzed by statistical genetics methods using Genepop 3.4, PHASE 2.1 and DnaSP 4.0 software programs. <it>In silico </it>prediction of transcription factor binding sites for intron 1 was performed with MatInspector 2.2 software.</p> <p>Results</p> <p><it>PNMT </it>was characterized by minimum variation and excess of rare SNPs in both normo- and hypertensive individuals. None of the SNPs showed significant differences in allelic frequencies among population samples, as well as between screened hypertensives and normotensives. In the joint case-control analysis of the Estonian and the Czech samples, hypertension patients had a significant excess of heterozygotes for two promoter region polymorphisms (SNP-184; SNP-390). The identified variation pattern of <it>PNMT </it>reflects the effect of purifying selection consistent with an important role of PNMT-synthesized epinephrine in the regulation of cardiovascular and metabolic functions, and as a CNS neurotransmitter. A striking feature is the lack of intronic variation. <it>In silico </it>analysis of <it>PNMT </it>intron 1 confirmed the presence of a human-specific putative Glucocorticoid Responsive Element (GRE), inserted by <it>Alu</it>-mediated transfer. Further analysis of intron 1 supported the possible existence of a full Glucocorticoid Responsive Unit (GRU) predicted to consist of multiple gene regulatory elements known to cooperate with GRE in driving transcription. The role of these elements in regulating <it>PNMT </it>expression patterns and thus determining the dynamics of the synthesis of epinephrine is still to be studied.</p> <p>Conclusion</p> <p>We suggest that the differences in PNMT expression between normotensives and hypertensives are not determined by the polymorphisms in this gene, but rather by the interplay of gene expression regulators, which may vary among individuals. Understanding the determinants of PNMT expression may assist in developing PNMT inhibitors as potential novel therapeutics.</p

Practices of implementation of sustainable development goals in Estonian organizations

Magistritöö Keskkonnakorralduse ja -poliitika õppekavalTänapäeval, kui inimkond on muutumas rohkem teadlikumaks planeetide piiridest ja globaalsetest ühiskondlikest väljakutsetest nõutakse kõikidelt organisatsioonidel oma valdkonnas säästva arengu suunas panustamist. Ülemaailmsed ja ka kohalikud poliitilised institutsioonid pööravad üha rohkem tähelepanu kuidas läbi ÜRO säästva arengu eesmärkide panustada jätkusuutlikumasse looduskeskkonda arvestavasse tulevikku. Käesoleva töö eesmärgiks oli uurida säästva arengu eesmärkide praktilisi näiteid Eesti era- ja avaliku sektori organisatsioonides, kirjeldada takistusi ja väljakutseid rakendamise protsesse ning kirjeldada jätkusuutlikku ärimudeli kasutust. Uuringu eesmärgi täitmiseks viidi läbi seitse poolstruktureeritud süvaintervjuud (kokku 26 küsimust), neist kolm erasektori ettevõtete ja nelja avaliku sektori organisatsiooniga. Töö tulemusena selgus, et erasektor on säästvaarengu eesmärkidesse panustamisel oluliselt eespool kui avalik sektor. Säästvaarengu eesmärgid jõudsid erasektorisse peamiselt ülevalt alla(juhtkond) kaskaadimise tulemusena, aga ka valdkonna põhiselt alt üles. Tegevusi tehakse süsteemselt organisatsiooni kõiki tasandeid arvesse võttes. Kesksel kohal on jätkusuutlikkusega seotud mõtteviisi ja uute kestlike teenuste või toodete arendamine, mida toetavad pidev kommunikatsiooni, koolitused ja koostöö teiste sidusrühmadega. Võrreldes erasektoriga on avalik sektor säästva arengu eesmärkide rakendamisega seotud tegevusi alustanud hiljuti või on alles planeerimise ja kaasamise faasis, kus puudub selge juhtimine, kommunikatsioon ja sidusrühmade kaasamine. Piiravaks teguriks on sageli rahaliste vahendite ja inimressursi puudus. Mõlemad sektorid näevad säästva arengu eesmärkide vahel mitmeid koostöö võimalusi nagu õpe, teadustegevused, kommunikatsioon, koolitused ja parimate praktikate jagamine. Suur vajadus on koolitusprogrammide (nt mikrokraadid) järele, mis aitaksid säästva arengu teemasid laiemalt ühiskonda viia. Jätkusuutliku ärimudeli kasutuspraktikast selgus, et pikaajaline strateegiline tase (visioon) oli olemas mõlema sektori plaanides. Samas ei olnud selget vahet keskmise ja lühiajalise tasandi vahel ning nende tegevus pigem kattus. Eestis on sarnast uuringut viidud läbi Rootsi Instituudi projekti IntAG2030 raames, kus võrreldi Eesti, Läti ja Rootsi erinevate organisatsioonide säästva arengu eesmärke kaasavaid praktikaid. Projekti esialgsed tulemused ühtivad käesoleva uuringu tulemustega, kus säästva arengu eesmärkide elluviimise edu saavutati juhtkonna (sh rahalise) mandaadiga, millele lisandus selge suhtlus ja kaasamine nii organisatsiooni erinevate tasandite kui ka teiste huvirühmade vahel.Becoming more aware of our planetary boundaries and global societal challenges, all the organizations are expected to contribute towards sustainable development. Both global as well as local political institutions are paying more attention to the UN sustainable developmental goals, using it as a tool to build more sustainable future and reduce their impact on the environment. The aim of this research was to explore the practical examples of sustainable developmental goals in Estonian private and public sector organizations as well as describe the obstacles and challenges with the implementation process and describe the usage of sustainable business model. Seven in-depth, semi-structured interviews (in total 26 questions) were conducted, three with the private sector companies and four public sector organisations. The interviews revealed that the private sector is using sustainable developmental goals more systematically in their daily activities compared to the public sector. The goals reached to the private sector mainly by the cascading from the management as a top down process, with the few exceptions being bottom up. The work with the goals was systemic, taking into account all the levels of the organization where the sustainability-related mind-set played a crucial role in it. Supported by the continuous communication, training and cooperation with other stakeholders helped to reach out to the new sustainable services and products. Compared to the private sector, public sector either has recently started implementing sustainable development goals or is still in the phase of planning and involvement process with no clear management, communication and involvement of the stakeholders. The limiting factor behind that is often the lack of financial and human resources. The usage practice of the sustainable business model revealed that the long-term strategic level (vision) was represented in both sectors’ plans. At the same time, there was no clear distinction between the medium and short-term levels, and their activities rather overlap. Both sectors see that to reach the aims of the sustainable developmental goals more crosssectorial collaboration is crucial. There is need for educational programs, research activities and in depth communication as well as training and sharing of best practices. Specifically there is a great need for training programs (e.g. micro-degrees) that would help bring the info to the wider society and thereby helps to boost the development of the sustainable mind-sets and future. In Estonia, one similar study has recently conducted within the international project called IntAG2030, funded by the Swedish Institute. The initial results of the project coincide with the result of the current research, where the implementation success of sustainable developmental goals were gained with the mandate from the management (including financial) complementing with the clear communication and engagement of different organisational levels as well as other stakeholders

Südameveresoonkonna funktsioonis osalevad geenid: uued pärilikud DNA variandid Eesti ja Tšehhi populatsioonides

Südameveresoonkonna haigusi (SVH) peetakse tänapäeva lääne ühiskonna üheks sagedasemaks enneaegse surma põhjustajaks. Vastuvõtlikust SV haiguste suhtes on sõltuv pärilikkusest kuid ka elustiilist ja keskkonnast tulenevatest teguritest. Olulist rolli mängivad see juures ülekaalulisus, vähene füüsiline aktiivsus, liigne soola- ja alkoholitarbimine ning suitsetamine. Perekondadel põhinevad ja kaksikute uuringud on näidanud, et SVH pärilik komponent võib olla väga varieeruv. Lisaks keskkonnast tingitud riskiteguritele on geneetilised uuringute tulemusena tuvastanud, et erinevate SVHte vormide kujunemiseni võivad viia mutatsioon(id), mis esinevad ühes geenis (üksiku geeni ehk monogeenne vorm) kui ka mitmed mutatsioonid erinevates geenides (multigeenne vorm) korraga. Levinumad on neist multigeensed vormid, mis teevad haiguse kujunemisel osalevate mutatsioonide leidmise protsessi keeruliseks ja aeganõudvaks tegevuseks. Inimeste haigustega seotud geene ning nendes esinevate mutatsioonide leidmiseks on läbi aegade kasutatud genoomis esinevaid kindlaid geneetilisi piirkondi ehk markereid. Neist kõige levinumad on ühe nukleotiidsed polümorfismid (ÜNPd). ÜNPdel põhinevaid tänapäeval laialdast kasutust leidnud üle kogu genoomi analüüsid, mille tulemusena on kaardistatud mitmeid sh. SVHte kujunemisel osalevaid DNA lookusi. Kuid lisaks ÜNPdele, esineb genoomis ka veel teisi pikemaid geneetilisi markereid nagu insertsioonid ja deletsioonid (indelid) ning mille rolli inimese haiguste kujunemisel on oluliselt vähem uuritud. Kirjeldades geneetilist varieeruvust mitmetes inimese SVHte kandidaatgeenides selgus, et DNA järjestuse varieeruvus geenide regulatoorsetes piirkondades, nagu seda on promootorid ja evolutsiooniliselt konserveerunud mittekodeerivad piirkonnad, on suhteliselt madal. Vähene varieeruvus geenide funktsiooni mõjutavates alades annab alust arvata, et uuritud geenide roll SVH kujunemisel ei sõltu mitte ainult nendes leiduvate polümorfismide poolt, vaid ilmselt hoopis keerulisemas koostöös individuaalsete ekspressiooni reguleerivate üksustega. Uuringute käigus tuvastasin ühe konserveerunud indiviiditi väga varieeruva piirkonna, inimese SVH kandiaatgeenis. Leitud piirkonnas esines mitmeid eri pikkusega seni varasemalt kirjeldamata indeleid. Šimpansi, reesusmakaagi ja inimese vastavate genoomipiirkondade võrdlemisel selgus veel, et tegemist on inimese spetsiifilise deletsiooniga. Läbi viidud assotsiatsioonianalüüs kahes Ida-Euroopa populatsioonis näitas seost uuritud geeni sagedasema variandi, 14bp indeli, kandjastaatuse ja suurenenud CAD haigestumise riski vahel tšehhi valimis. Risk oli veelgi kõrgem metaboolse sündroomiga koronaartõvega patsientidel. Lisaks esines seos ka 14bp indel variandi ja triglütseriidide tasemete vahel tšehhidel ning 14bp indel variandi ja HR ja LDL tasemete vahel mõlema valimi (tsehhid ja eestlased) tervetel kontrollidel. Nii käesolev töö kui ka mõned varasemad tööd on näidanud, et seni vähe uuritud geneetilised variandid, nagu indelid võivad omada palju olulisemat rolli inimeste komplekshaiguste (nagu SVHd) kujunemisel kui seni arvatud.Cardiovascular diseases (CVD) are known as one of the main causes of premature deaths and disability in Western societies. CVDs are complex diseases influenced by the interplay of multiple genes as well as controllable (diet, exercise, stress) and uncontrollable (age, sex, family history) including environmental determinants. To date there are many genetic loci already known to have an impact on the etiology of CVD causing both monogenic and complex forms of the disease. In case of monogenic forms a single alteration in the gene may lead to the disease manifestation whereas in complex forms more than one gene has an impact to the disease phenotype. Genetic studies focused on families and twin cohorts have been previously shown that the approximate inheritance of CVD may range from 17% up to 66%. Despite of the great knowledge of the loci leading to the susceptibility to CVD, still many loci are remained to be described. To find these genomic regions a wide range of genetic markers are widely used. The most common markers in the genome are single nucleotide polymorphisms (SNPs). SNPs located in the regulatory or protein coding region may have an impact on gene function and thereby lead to the disease development. But there are also other genetic markers in the genome, like short insertions and deletions, which may have even higher impact on diseases development than previously expected. Studying variation pattern of CVD candidate genes in two population sample of European descent showed low genetic variation along regulatory regions (for example promoters, conserved non-coding regions) of the studied genes. This phenomenon indicates that the function of these genes is not determined only by the variations in these regions but with the complex interplay with other transcription regulators. Interestingly, during the variation detection one hypervariable genomic fragment in human CVD candidate gene was found. This region included multiple indel variations with the range of 5bp up to 43bp in size. Comparison of this region against chimpanzee and rhesus macaque revealed that the most common variant, 14p indel was human-specific deletion located in intronic CNR of NCX1 gene. In association study of two CVD phenotypes (coronary artery disease (CAD) and essential hypertension (EH)) this indel variant showed significant association with coronary artery disease and was even higher between the indel variant and among patients additionally diagnosed with metabolic syndrome in Czech population. A suggestive evidence of association with indel variant and serum triglyceride levels was observed in Czechs as well as with indel variant and heart rate and LDL levels in healthy control individuals among both (Czechs and Estonians) cohorts. Current research, as well as other recent studies have shown that non-SNP variations are a substantial source of polymorphism in humans and may have even larger role in complex disease (like CVD) than previously thought

Identification of putative transciption regulating elements within human intron 1

<p><b>Copyright information:</b></p><p>Taken from "Resequencing in European hypertensive and normotensive individuals: no common susceptibilily variants for hypertension and purifying selection on intron 1"</p><p>http://www.biomedcentral.com/1471-2350/8/47</p><p>BMC Medical Genetics 2007;8():47-47.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1947951.</p><p></p> Putative transcription factors binding sites (TFBS) predicted by MatInspector 2.2 software and regulatory elements identified by manual inspection are depicted upon the sequence of intron 1 (951 bp). The Glucocorticoid Responsive Element (GRE; consensus GGTACAnnnTGTTCT), a core for a potential Glucocorticoid Responsive Unit (GRU), is given in bold. The human-specific element is underlined. The two-directional arrows indicate the predicted binding sites for regulatory factors: IRE – Insulin Responsive Element (consensus T(G/A)TTT(T/G)(G/T)); ERE – Estrogen Responsive Element (consensus GGTCAnnnTGACC); NFκB – Nuclear Factor kappa B; Sp1/2 – Specificity protein 1/2; Egr1 – Early Growth Response 1; MAZ – Myc-Associated Zinc finger protein; ZBP-89 – Zinc finger Binding Protein 148, ZNF148; HMGI/Y – High Mobility Group protein isoform I and Y, HMGA1; RORA (RORα) – Retinoic acid receptor-related Orphan Receptor α; E4BP4 – mammalian transcription factor E4 Binding Protein 4

Upper white bar depicts the positions of identified SNPs (Table 2) relative to the location of gene ATG (A denoted as 1)

Singleton polymorphisms were excluded from calculations.<p><b>Copyright information:</b></p><p>Taken from ", a positional candidate for blood pressure and renal regulation: resequencing, association and study"</p><p>http://www.biomedcentral.com/1471-2350/9/25</p><p>BMC Medical Genetics 2008;9():25-25.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2330028.</p><p></p