Human mitochondrial DNA is extensively methylated in a non-CpG context

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.Institut National du Cancer (INCa, France); European Commission (EC) Seventh Framework Programme (FP7) Translational Cancer Research (TRANSCAN) Framework; Foundation ARC pour la Recherche sur le Cancer (France); Plan Cancer-Eva-Inserm research grant (to Z.H.); Postdoctoral Fellowship from International Agency for Research on Cancer (to V.P.), partially supported by the EC FP7 Marie Curie Actions -People -Co-funding of regional, national and international programmes (COFUND)'. Funding for open access charge: Institut National du Cancer (INCa, France); European Commission (EC) Seventh Framework Programme (FP7) Translational Cancer Research (TRANSCAN) Framework; Foundation ARC pour la Recherche sur le Cancer (France); Plan Cancer-Eva-Inserm research grant (to Z.H.)

BREEDING SYSTEMATIZATION OF AUTOCHTHONOUS CATTLE BREED BUSHA

Buša je jedna od tri hrvatske izvorne pasmine goveda i prema veličini populacije druga je izvorna pasmina goveda u Republici Hrvatskoj. Za vjerodostojno i uspješno plansko vođenje uzgoja i provedbu uzgojnog programa, nužno je učiniti sistematizaciju uzgoja temeljem rodovničkih podataka, a plansko vođenje uzgoja (planski pripust) organizirati prema linijama bikova i rodovima krava. Iz tog razloga provedena je sistematizacija uzgoja buše (izgradnja rodovnika), a rezultat sistematizacije je definiranje 24 linije bikova i 154 rodova krava. Sistematiziran uzgoj prema linijama i rodovima omogućava plansko vođenje uzgoja u cilju sprečavanja reduciranja genetske varijabilnosti, vjerodostojnije utvrđivanje demografskih parametara, procjenu kompletne genetske varijabilnosti pasmine, efikasniju provedbu molekularnih istraživanja uz znatno manje troškove, smanjuje troškove pohrane genetskog materijala u banku gena te omogućava kontrolu uzgoja obzirom na izlučenje grla iz uzgoja (klanje).Busha is one of three Croatian autochthonous breeds of cattle and by population size is the second autochthonous cattle bree in the Republic of Croatia. For reliable and successful planned managing of breeding and implementation of breeding program, it is necessary to systemize breeding on the basis of pedigree data, and organize planned mating by sire lines and dam lines. For this reason, systematization of Busha breed has been conducted (pedigree building), and the result of systematization is defining 24 sire lines and 154 dam lines. Systemized breed by sire lines and dam lines allows planned leading of breeding with goal of avoiding reduction of genetic variability, more reliable determination of demographic parameters, evaluation of complete genetic variability of the breed, more efficient application of molecular research with significantly decreased costs, storage of genetic material in the gene bank, and provides control of breeding regarding to exclusion of heads out of the breeding (slaughtering)

EFFECTIVE POPULATION SIZE OF AUTOCHTHONOUS CATTLE BREED BUSHA

Svrha istraživanja je procijeniti efektivnu veličinu populacije (Ne) izvorne pasmine goveda buša pomoću rodovničkih podataka. Brojno stanje populacije buše kontinuirano se povećava i krajem 2011. godine (HPA, 2012) broj rasplodnih grla iznosi 341, odnosno 33 bika i 308 krava. Prema međunarodnoj klasifikaciji ugroženosti (FAO), hrvatska populacija buša kategorizira se u 2011. godini kao "ugrožena – kategorija K3". U cilju vjerodostojnije procjene Ne korišteno je više metodologija i različitih tipova rodovnika. Aktualna genetska varijabilnost nije rezultat uspješnog konzervacijskog programa, nego slabe informativnosti (dužine) i kompletnosti rodovnika te malog broja poznatih generacija predaka što potvrđuje niska vrijednost ekvivalenta kompletnih generacija (1,41). Rezultati istraživanja Ne buše pomoću analize rodovnika daju korisne informacije u monitoringu genetske raznolikosti, ali potvrđuju i činjenicu da rodovnički podaci mogu vrlo često biti manjkavi u procjeni Ne, odnosno genetske varijabilnosti. U takvim slučajevima metode molekularne genetike jedine omogućavaju vjerodostojniju informaciju o genetskoj varijabilnosti na razini strukture DNA, pa tako i u slučaju izvorne pasmine goveda buša.Purpose of research is to estimate effective population size (Ne) of autochthonous cattle breed busha with pedigree data. Numerical strength of busha population is continuosly increasing and at the end of 2011 (CAA, 2012) the number of breeding heads is 341, i.e. 33 bulls and 308 cows. According to international classification of endangerness (FAO), Croatian population of busha breed is categorized in 2011 as „endangered – category K3“. With goal of more reliable estimation Ne, several methodologies and different types of pedigrees were used. Actual genetic variability is not result of successful conservation program, but poor informativity (length) and completiveness of pedigree and small number of known ancestor generation which confirm low value of equivalent of complete generations (1,41). Results of research of busha Ne by pedigree analysis give useful informations in monitoring of genetic diversity, but also confirm the fact that pedigree data can often be defective in NE estimation, i.e. genetic variability. In this cases, only methods of molecular genetic provide more reliable information about genetic variability on DNA structure level, and that is also the case with autochthonous cattle breed busha

ESTIMATION OF GENETIC DIVERSITY USING PROBABILITIES OF GENE ORIGIN OF AUTOCHTHONOUS CATTLE BREED BUSHA

Registar izvorne pasmine goveda buša utemeljen je 2003. i danas broji 1020 grla. U cjelokupnom rodovniku utvrđeno je 329 foundera i 249 polufoundera. Broj foundera u referentnoj populaciji iznosi 285, polufoundera 219 i 255 ancestora, a 50% genetske varijabilnosti objašnjava 17 ancestora i 20 foundera. Najveći broj poznatih generacija predaka u rodovniku je pet, s prosječnim ekvivalentom kompletnih generacija 1,41 koji ukazuje na slabu informativnost i kompletnost rodovnika. Dio značajnih predaka je dao svoj doprinos genetskoj varijabilnosti kao founder, ali ne i kao ancestor, što ukazuje na gubitak dijela genetske varijabilnosti. Povećanje stupnja inbreedinga i povezanosti, omjeri pojedinih parametara porijekla gena (fe/fa, fg/fe, fe/f) i smanjenje doprinosa broja foundera u referentnoj populaciju u odnosu na cjelokupan rodovnik, ukazuju na gubitak genetske varijabilnosti u populaciji. Rezultati analize rodovnika ukazuju da je u cilju očuvanja genetske varijabilnosti, nužno i žurno dizajniranje kvalitetnog uzgojnog programa, plana pripusta i konzervacijske strategije, temeljenih na analizama rodovnika i metodama molekularne genetike (DNA).Register of autochthonous cattle breed busha was founded in 2003. and till today it is consisted of 1020 heads. In whole pedigree 329 founders and 249 half-founders are determined. The number of founders in reference population is 285, half-founders 219, and ancestors 255, and 50% of genetic variability explain 17 ancestors and 20 founders. The largest number of complete generations 1,41 which indicates poor informativity and completness of pedigree. Part of significant ancestors gave its contribution to genetic variability as founder, but not as ancestor which indicates loss of part of genetic variability. Increasing of inbreeding rate and connections, ratios of certain parameters of gene origin (fe/fa, fg/fe, fe/f) and decreasing of contribution of number of founders in reference population compared to the whole pedigree, indicate the loss of genetic variability in population. Pedigree analysis results indicate that in purpose of preservation of genetic variability, it is necessary and urgent creating of quality breeding progam, breeding plan and conservation strategy, based on pedigree analysis and method of molecular genetic

BREEDING SYSTEMATIZATION OF AUTOCHTHONOUS CATTLE BREED BUSHA

Buša je jedna od tri hrvatske izvorne pasmine goveda i prema veličini populacije druga je izvorna pasmina goveda u Republici Hrvatskoj. Za vjerodostojno i uspješno plansko vođenje uzgoja i provedbu uzgojnog programa, nužno je učiniti sistematizaciju uzgoja temeljem rodovničkih podataka, a plansko vođenje uzgoja (planski pripust) organizirati prema linijama bikova i rodovima krava. Iz tog razloga provedena je sistematizacija uzgoja buše (izgradnja rodovnika), a rezultat sistematizacije je definiranje 24 linije bikova i 154 rodova krava. Sistematiziran uzgoj prema linijama i rodovima omogućava plansko vođenje uzgoja u cilju sprečavanja reduciranja genetske varijabilnosti, vjerodostojnije utvrđivanje demografskih parametara, procjenu kompletne genetske varijabilnosti pasmine, efikasniju provedbu molekularnih istraživanja uz znatno manje troškove, smanjuje troškove pohrane genetskog materijala u banku gena te omogućava kontrolu uzgoja obzirom na izlučenje grla iz uzgoja (klanje).Busha is one of three Croatian autochthonous breeds of cattle and by population size is the second autochthonous cattle bree in the Republic of Croatia. For reliable and successful planned managing of breeding and implementation of breeding program, it is necessary to systemize breeding on the basis of pedigree data, and organize planned mating by sire lines and dam lines. For this reason, systematization of Busha breed has been conducted (pedigree building), and the result of systematization is defining 24 sire lines and 154 dam lines. Systemized breed by sire lines and dam lines allows planned leading of breeding with goal of avoiding reduction of genetic variability, more reliable determination of demographic parameters, evaluation of complete genetic variability of the breed, more efficient application of molecular research with significantly decreased costs, storage of genetic material in the gene bank, and provides control of breeding regarding to exclusion of heads out of the breeding (slaughtering)

INBREEDING OF AUTOCHTHONOUS CATTLE BREED BUSHA

Svrha studije je temeljem rodovničkih podataka utvrditi aktualno i predvidjeti trend budućeg stupnja inbreedinga u uzgoju izvorne pasmine goveda buša. Prosječan generacijski interval iznosi 5,96 godina što potvrđuje kasnu zrelost kao pasminsku karakteristiku. Koeficijent inbreedinga i povezanosti jedinki u rodovniku iznose 1,27% i 1,78%, a smatramo ih niskim u odnosu na komparativna istraživanja. No, rezultati analize udjela i prosječnog koeficijenta inbreedinga inbred jedinki u referentnoj populaciji daju dovoljno informacija za predviđanje intenzivnog porasta koeficijenta inbreedinga, odnosno značajnijeg smanjenja efektivne veličine populacije u uzgoju buše. Zaključak podupiru pozitivne i uske korelacije između koeficijenta inbreedinga i povezanosti s brojem i kompletnosti poznatih generacija predaka, te relativno mala veličina populacije buše od 341 rasplodne jedinke (HPA, 2012). Pretpostavljamo da je stupanj inbreedinga u populaciji buše znatno veći, ali ga nije moguće utvrditi temeljem analize rodovnika zbog njegove slabe informativnosti (ekvivalent kompletnih generacija = 1,41). Radi dobivanja vjerodostojnije informacije o stupnju inbreedinga u populaciji buše nužno je njegovo utvrđivanje metodama molekularne genetike na razni strukture DNA. Obzirom da se radi o maloj populaciji potrebno je primijeniti posebne mjere strategije planskog pripusta u cilju svođenja inbreedinga na najmanju moguću mjeru.The purpose oft he study is to determine actual and predict trend of the future rate of inbredding in breeding of autochthonous cattle breed busha based on pedigree data. Average generation interval is 5,96 years, which confirms late maturity as breed characteristics. Inbreeding factor and relatedness of units are 1,27% and 1,78%, and are considered low with respect to comparative researches. But results of proportion and average inbreeding factor of inbreed units in reference population give enough information for prediction of intensive growth of inbreeding factor, i.e. significant decrease of effective population size in breeding busha. The conclusion is supported by positive and narrow correlations between traced ancestors generation, and relatively small busha population with 341 breeding heads (CAA 2012). We assume that the rate of inbreeding in busha population is considerably larger, but it is not possible to determine it based on pedigree analysis because of its poor informativity (equivalent of complete generations = 1,41). In order to obtain more reliable information on inbreeding rate in busha population, it is necessary to determine it by methods of molecular genetic on the DNA structure leve. With consideration on the small population, special measures of planned breeding strategies must be applied, with goal of brining inbreeding to the minimum possible rate

Environmentally sensitive hotspots in the methylome of the early human embryo

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin-specific methylation and germline differentially methylated regions, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 embryonic stem cells and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental 'hotspots' providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease

Association of MAPT haplotype-tagging polymorphisms with cerebrospinal fluid biomarkers of Alzheimer's disease: a preliminary study in a Croatian cohort

INTRODUCTION: Alzheimer's disease (AD) is the world leading cause of dementia. Early detection of AD is essential for faster and more efficacious usage of therapeutics and preventive measures. Even though it is well known that one ε4 allele of apolipoprotein E gene increases the risk for sporadic AD five times, and that two ε4 alleles increase the risk 20 times, reliable genetic markers for AD are not yet available. Previous studies have shown that microtubule-associated protein tau (MAPT) gene polymorphisms could be associated with increased risk for AD. ----- METHODS: The present study included 113 AD patients and 53 patients with mild cognitive impairment (MCI), as well as nine healthy controls (HC) and 53 patients with other primary causes of dementia. The study assessed whether six MAPT haplotype-tagging polymorphisms (rs1467967, rs242557, rs3785883, rs2471738, del-In9, and rs7521) and MAPT haplotypes are associated with AD pathology, as measured by cerebrospinal fluid (CSF) AD biomarkers amyloid β1-42 (Aβ1-42 ), total tau (t-tau), tau phosphorylated at epitopes 181 (p-tau181 ), 199 (p-tau199 ), and 231 (p-tau231 ), and visinin-like protein 1 (VILIP-1). ----- RESULTS: Significant increases in t-tau and p-tau CSF levels were found in patients with AG and AA MAPT rs1467967 genotype, CC MAPT rs2471738 genotype and in patients with H1H2 or H2H2 MAPT haplotype. ----- CONCLUSIONS: These results indicate that MAPT haplotype-tagging polymorphisms and MAPT haplotypes should be further tested as potential genetic biomarkers of AD

Abdominal organ motion during inhalation and exhalation breath-holds: pancreatic motion at different lung volumes compared

Contrary to what is commonly assumed, organs continue to move during breath-holding. We investigated the influence of lung volume on motion magnitude during breath-holding and changes in velocity over the duration of breath-holding. Sixteen healthy subjects performed 60-second inhalation breath-holds in room-air, with lung volumes of ∼100% and ∼70% of the inspiratory capacity, and exhalation breath-holds, with lung volumes of ∼30% and ∼0% of the inspiratory capacity. During breath-holding, we obtained dynamic single-slice magnetic-resonance images with a time-resolution of 0.6s. We used 2-dimensional image correlation to obtain the diaphragmatic and pancreatic velocity and displacement during breath-holding. Organ velocity was largest in the inferior-superior direction and was greatest during the first 10s of breath-holding, with diaphragm velocities of 0.41mm/s, 0.29mm/s, 0.16mm/s and 0.15mm/s during BH100%, BH70%, BH30% and BH0%, respectively. Organ motion magnitudes were larger during inhalation breath-holds (diaphragm moved 9.8 and 9.0mm during BH100% and BH70%, respectively) than during exhalation breath-holds (5.6 and 4.3mm during BH30% and BH0%, respectively). Using exhalation breath-holds rather than inhalation breath-holds and delaying irradiation until after the first 10s of breath-holding may be advantageous for irradiation of abdominal tumor

Evaluation of cerebrospinal fluid phosphorylated tau231 as a biomarker in the differential diagnosis of Alzheimer's disease and vascular dementia

BACKGROUND: The diagnosis of either Alzheimer's disease (AD) or vascular dementia (VaD) is still largely based on clinical guidelines and exclusion of other diseases that may lead to dementia. ----- AIMS: In this study, we assessed whether the use of sensitive and specific biomarkers such as phosphorylated tau proteins could contribute to an earlier and more accurate diagnosis of AD and VaD, as well as to their differentiation. ----- MATERIAL AND METHODS: A total of 198 patients, of which 152 had AD, 28 VaD, and 18 were healthy controls (HC), were included in the analyses. We analyzed cerebrospinal fluid (CSF) levels of total tau protein (t-tau), tau protein phosphorylated at threonine 231 (p-tau231), and factor score (FS) determined by combination of p-tau231 and Mini-Mental State Examination (MMSE) in patients with AD and VaD, as well as in HC. We tested the diagnostic accuracy of these biomarkers in the CSF and FS (p-tau231, MMSE) in differentiating AD from VaD and HC. ----- RESULTS: Total tau levels were significantly elevated in subjects with AD compared to HC, as well as in VaD subjects compared to HC. ----- DISCUSSION: p-tau231 levels were significantly higher in patients with ADvsHC as well in patients with VaD vsHC. p-tau231 levels did not distinguish AD from VaD patients. Importantly, FS(p-tau231 and MMSE) showed statistically significant differences in the distribution of subjects with AD and VaD. ----- CONCLUSION: These results indicate that FS (p-tau231 and MMSE) has a strong potential to provide an early distinction between AD and VaD