Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell

The toposiomerase IIIalpha-RMI1-RMI2 complex orients human Bloom’s syndrome helicase for efficient disruption of D-loops

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom’s syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM’s multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM’s interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms

Az Ortinno Hip&Knee rehabilitációs berendezés hatékonyságának értékelése járásvizsgáló rendszerrel cerebrál paretikus betegek esetén

 Az évi ötezer térdprotézis műtét (magyarországi adat) mellett egy másik, igen jelentős betegcsoport is a térd teljes nyújthatóságának elmaradásával küzd. Ezek az ún. cerebrál paretikus (CP) betegek, akiknél általában a perinatális időszakban bekövetkezett különböző súlyosságú agykárosodás okoz testszerte tónuszavart, ami a térdízület esetében a szemiflektált helyzet állandósulását jelenti. Huzamos ideig fennálló szemiflektált helyzet egy idő után a rossz izombalansz mellett a folyamatot súlyosbító ízületi tok, ízületközeli ín, és akár a térd körüli bőr zsugorodását is okozza. A problémát számos szerző elemezte, azonban általánosan elfogadott megoldás mindeddig nem született. Mindkét betegcsoport kezelésére, illetve rehabilitációjára egy olyan mechanizmust találtunk ki, amely a térd teljes nyújtását a gravitáció segítségül hívásával javítja. A térdízületi extenziós deficit leküzdésére egy olyan rehabilitációs berendezést készítettünk, amely az ülő, vagy fekvő esetben fenekével az ágy szélén fekvő beteg alsó végtagját a boka-láb régióban egy speciális saroktartóban rögzítve és így felfüggesztve tartja. A felfüggesztést úgy alakítottuk ki, hogy önbeálló módon a talpél függőleges, és ennek megfelelően a térdhajlítás haránttengelye vízszintes lesz. Amennyiben ez a helyzet megvalósul, és az így felfüggesztett végtagot ciklikusan megemeljük, majd ejtjük, az ejtés végpontját elérve a térdízület hátsó passzív rögzítő struktúráiban, illetve a fokozott izomtónusú térdhajlító izmokban egy nyújtó hatás lép fel. Ennek repetitív alkalmazása elősegíti a térdízület kívánatos teljes nyújtásának elérését. Az Ortinno Hip&Knee megalkotásánál eredendően az alapvető cél a térdprotézis műtétet követő flexiós kontraktúra megelőzése, kezelése volt. A térdprotézis műtétek Covid-járvány kialakulása miatti leállítása okán került át figyelmünk a cerebral pareticus betegek irányába. A méréssorozatot egy pilot study előzte meg, melynek során szerzett tapasztalatokra alapozva 2021 tavaszán kezdtük el a kezeléseket és a műszeres utánkövetést. A mérések eredményei alapján általánosságban elmondhatjuk, hogy az Ortinno Hip&Knee egy jól használható rehabilitációs berendezés, amely ilyen rövid, mindössze kéthetes kezelési idő alatt is kedvező eredményeket hozott a kezelt betegek alsóvégtagi mozgástartományát illetően. Ezt mind a fizikális vizsgálatok, mind a Diers 4D Motionlab rendszerrel vett járásadatok is alátámasztják. Ami azonban még ennél is fontosabb, hogy szinte kivétel nélkül minden kommunikációképes beteg és ápolóik, szüleik kedvező hatásokról számolnak be a készülék kapcsán. A járáskép javulása mellett mentális pozitív hatásai is vannak a berendezésnek, esetenként még a beszédkészség javulásával is összefüggésbe hozható a kezelés hatása. Ugyanakkor a vizsgálatsorozat arra is rámutatott, hogy a kezelés felfüggesztése sok esetben az állapot romlását idézte elő, azaz nagyon fontosnak tűnik a kezelés folyamatos biztosítása a beteg részére.&nbsp

CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality

Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2′-deoxyuridine 5′-triphosphate (dUTP) into 2′-deoxyuridine 5′-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/– animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut −/− embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements

p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis

Primary Founder Mutations in the PRKDC Gene Increase Tumor Mutation Load in Colorectal Cancer

The clonal composition of a malignant tumor strongly depends on cellular dynamics influenced by the asynchronized loss of DNA repair mechanisms. Here, our aim was to identify founder mutations leading to subsequent boosts in mutation load. The overall mutation burden in 591 colorectal cancer tumors was analyzed, including the mutation status of DNA-repair genes. The number of mutations was first determined across all patients and the proportion of genes having mutation in each percentile was ranked. Early mutations in DNA repair genes preceding a mutational expansion were designated as founder mutations. Survival analysis for gene expression was performed using microarray data with available relapse-free survival. Of the 180 genes involved in DNA repair, the top five founder mutations were in PRKDC (n = 31), ATM (n = 26), POLE (n = 18), SRCAP (n = 18), and BRCA2 (n = 15). PRKDC expression was 6.4-fold higher in tumors compared to normal samples, and higher expression led to longer relapse-free survival in 1211 patients (HR = 0.72, p = 4.4 × 10−3). In an experimental setting, the mutational load resulting from UV radiation combined with inhibition of PRKDC was analyzed. Upon treatments, the mutational load exposed a significant two-fold increase. Our results suggest PRKDC as a new key gene driving tumor heterogeneity

The Year in Hungarian Cardiology 2022: the top 10 papers in cardiovascular imaging

In 2022, the Hungarian Cardiovascular Imaging community contributed to several scientifi c papers that were published in international journals. Clinical research activities employed novel echocardiographic methods including two- and three-dimensional, strain and tissue Doppler imaging of all cardiac chambers. These echocardiographic modalities were applied in diff erent patient populations such as patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or post-SARS-CoV-2 aff ected individuals, athletes, patients with systemic autoimmune diseases, cardiomyopathies, hematologic diseases, metabolic diseases, congenital heart disease or valvular interventions. Beyond resting echocardiography, original research results revealed important information in the fi eld of exercise stress echocardiography as well. Hungarian studies using cardiac magnetic resonance imaging evaluated patients with diff erent pathologies such as cardiac amyloidosis, cardiomyopathies, myocarditis due to SARS-CoV-2 vaccinations, and athletes in post-SARS-CoV-2 states. Hungarian research activities in the fi eld of cardiac computed tomographic imaging covered the evaluation of moderate coronary lesions, correlates of myocardial ischemia, coronary plaques, coronary calcium, recurrence of atrial fi brillation after radiofrequency ablation and morphological changes after valvular interventions. Novel technologies have been applied in cardiac computed tomographic imaging by Hungarian researchers, with higher spatial resolution and enhanced spectral capabilities in coronary artery assessment

NLS copy number variation governs efficiency of nuclear import: case study on dUTPases

Nucleocytoplasmic trafficking of large macromolecules requires an active transport machinery. In many cases, this is initiated by binding of the nuclear localization signal (NLS) peptide of cargo proteins to importin-a molecules. Fine orchestration of nucleocytoplasmic trafficking is of particularly high importance for proteins involved in maintenance of genome integrity, such as dUTPases, which are responsible for prevention of uracil incorporation into the genome. In most eukaryotes, dUTPases have two homotrimeric isoforms: one of these contains three NLSs and is present in the cell nucleus, while the other is located in the cytoplasm or the mitochondria. Here we focus on the unusual occurrence of a pseudo-heterotrimeric dUTPase in Drosophila virilis that contains one NLS, and investigate its localization pattern compared to the homotrimeric dUTPase isoforms of Drosophila melanogaster. Although the interaction of individual NLSs with importin-a has been well characterized, the question of how multiple NLSs of oligomeric cargo proteins affect their trafficking has been less frequently addressed in adequate detail. Using the D. virilis dUTPase as a fully relevant physiologically occurring model protein, we show that NLS copy number influences the efficiency of nuclear import in both insect and mammalian cell lines, as well as in D. melanogaster and D. virilis tissues. Biophysical data indicate that NLS copy number determines the stoichiometry of complexation between importin-a and dUTPases. The main conclusion of our study is that, in D. virilis, a single dUTPase isoform efficiently reproduces the cellular dUTPase distribution pattern that requires two isoforms in D. melanogaster