Search Results (24)

Background: Aicardi-Goutières Syndrome (AGS) is a rare neuroinflammatory condition characterized by early-onset symptoms that extend outside the nervous system. Due to the rarity of the disease, the pathogenesis is not well understood, and its diagnosis and treatment remain elusive. We recently demonstrated mitochondrial abnormalities and increased reactive oxygen species (ROS) levels in lymphoblastoid cell lines (LCLs) derived from RNASEH2B- and RNASEH2A-mutated AGS patients. On this background, we turned our attention to metformin, the first-choice drug for type 2 diabetes, as a possible treatment acting on oxidative stress in RNASEH2-mutant AGS cells. Methods and Results: By means of flow cytometry, we found that metformin treatment significantly decreases ROS production in RNASEH2B- and RNASEH2A-mutated AGS LCLs. Of note, metformin treatment reduces the green JC-1 monomeric signal and, concurrently, increases the red JC-1 signal in both mutated LCLs, accounting for restoration of the mitochondrial membrane potential. Immunofluorescence staining shows a decrease in 8-oxoG levels only in RNASEH2B- mutated AGS LCLs. Finally, the significant upregulation of Forkhead Box O3 (FOXO3), cytochrome C somatic (CYCS), and superoxide dismutase 2 (SOD2) mRNA levels in RNASEH2B-mutated AGS LCLs after metformin treatment points to FOXO3 signaling as a possible mechanism to reduce oxidative stress. Conclusions: In conclusion, even if these pilot results need to be confirmed on a larger cohort, we shed light on metformin treatment as a valid approach to ameliorate oxidative stress-related inflammation in AGS patients. Full article

In a study involving 385 Large White pigs, a genome-wide association study (GWAS) was conducted to investigate reproductive traits, specifically the number of healthy litters (NHs) and the number of weaned litters (NWs). Several SNP loci, including ALGA0098819, ALGA0037969, and H3GA0032302, were significantly associated with these traits. In the combined-parity analysis, candidate genes, such as BLVRA, STK17A, PSMA2, and C7orf25, were identified. GO and KEGG pathway enrichment analyses revealed that these genes are involved in key biological processes, including organic synthesis, the regulation of sperm activity, spermatogenesis, and meiosis. In the by-parity analysis, the PLCXD3 gene was significantly associated with the NW trait in the second and fourth parities, while RNASEH1, PYM1, and SEPTIN9 were linked to cell proliferation, DNA repair, and metabolism, suggesting their potential role in regulating reproductive traits. These findings provide new molecular markers for the genetic study of reproductive traits in Large White pigs. For the phenotypic prediction of NH and NW traits, several machine learning models (GBDT, RF, LightGBM, and Adaboost.R2), as well as traditional models (GBLUP, BRR, and BL), were evaluated using SNP data in varying proportions. After PCA processing, the GBDT model achieved the highest PCC for NH (0.141), while LightGBM reached the highest PCC for NW (0.146). The MAE, MSE, and RMSE results showed that the traditional models exhibited stable error rates, while the machine learning models performed comparatively better across the different SNP ratios. Overall, PCA processing provided some improvement in the predictive performance of all of the models, though the overall increase in accuracy was limited. Full article

The objective of this study was to identify differentially expressed genes and their potential influence on the carcinogenesis of serous-type ovarian cancer tumors. Serous cancer is an epithelial ovarian cancer subtype and is the most common type of ovarian cancer. Transcriptomic profiles of serous cancer and non-cancerous datasets were obtained from the Gene Expression Omnibus (GEO-NCBI). Differentially expressed genes were then derived from those profiles; the identified genes were consistently upregulated in three or more transcriptomic profiles. These genes were considered as the serous ovarian cancer gene set for further study. The serous gene set derived from the transcriptomic profiles was then evaluated for ontological functional analysis using the Molecular Signatures Database. Next, we examined the mutational impact of this serous gene set on the transcriptomic profile of high-grade serous ovarian (HGSO) adenocarcinoma using the cBioPortal database. Results from OncoPrint revealed that 26 genes were amplified in more than 5% of HGSO cancer patients. Interestingly, several of these genes are involved in cell cycle processes, including genes ATPase family AAA domain containing 2 (ATAD2), recQ-like helicase 4 (RECQL4), cyclin E1 (CCNE1), anti-silencing function 1B histone chaperone (ASF1B), ribonuclease H2 subunit A (RNASEH2A), structural maintenance of chromosome 4 (SMC4), cell division cycle associated 20 (CDC20), and cell division cycle associated 8 (CDCA8). The receiver operating characteristic (ROC) curve results also revealed higher specificity and sensitivity for this subtype of tumors. Furthermore, these genes may affect the recurrence of serous ovarian carcinogenesis. Overall, our analytical study identifies cell cycle-related genes that can potentially be targeted as diagnostic and prognostic markers for serous ovarian cancer. Full article

Objective: To report a series of atypical presentations of Aicardi–Goutières syndrome. Methods: Clinical, neuroimaging, and genetic data. Results: We report a series of six unrelated patients (five males) with a subacute loss of developmental milestones, pyramidal signs, and regression of communication abilities, with onset at ages ranging from 7 to 20 months, reaching a nadir after 4 to 24 weeks. A remarkable improvement of lost abilities occurred in the follow-up, and they remained with residual spasticity and dysarthria but preserved cognitive function. Immunization or febrile illness occurred before disease onset in all patients. CSF was normal in two patients, and in four, borderline or mild lymphocytosis was present. A brain CT scan disclosed a subtle basal ganglia calcification in one of six patients. Brain MRI showed asymmetric signal abnormalities of white matter with centrum semi-ovale involvement in five patients and a diffuse white matter abnormality with contrast enhancement in one. Four patients were diagnosed and treated for acute demyelinating encephalomyelitis (ADEM). Brain imaging was markedly improved with one year or more of follow-up (average of 7 years), but patients remained with residual spasticity and dysarthria without cognitive impairment. Demyelination relapse occurred in a single patient four years after the first event. Whole-exome sequencing (WES) was performed in all patients: four of them disclosed biallelic pathogenic variants in RNASEH2B (three homozygous p.Ala177Thr and one compound heterozygous p.Ala177Thr/p.Gln58*) and in two of them the same homozygous deleterious variants in RNASEH2A (p.Ala249Val). Conclusions: This report expands the phenotype of AGS to include subacute developmental regression with partial clinical and neuroimaging improvement. Those clinical features might be misdiagnosed as ADEM. Full article

As the key enzyme mediating ribonucleotide excision repair, RNase H2 is essential for the removal of single ribonucleotides from DNA in order to prevent genome damage. Loss of RNase H2 activity directly contributes to the pathogenesis of autoinflammatory and autoimmune diseases and might further play a role in ageing and neurodegeneration. Moreover, RNase H2 activity is a potential diagnostic and prognostic marker in several types of cancer. Until today, no method for quantification of RNase H2 activity has been validated for the clinical setting. Herein, validation and benchmarks of a FRET-based whole-cell lysate RNase H2 activity assay are presented, including standard conditions and procedures to calculate standardized RNase H2 activity. Spanning a wide working range, the assay is applicable to various human cell or tissue samples with overall methodological assay variability from 8.6% to 16%. Using our assay, we found RNase H2 activity was reduced in lymphocytes of two patients with systemic lupus erythematosus and one with systemic sclerosis carrying heterozygous mutations in one of the RNASEH2 genes. Implementation of larger control groups will help to assess the diagnostic and prognostic value of clinical screening for RNase H2 activity in the future. Full article

Aicardi–Goutières syndrome (AGS) is a rare encephalopathy characterized by neurological and immunological features. Mitochondrial dysfunctions may lead to mitochondrial DNA (mtDNA) release and consequent immune system activation. We investigated the role of mitochondria and mtDNA in AGS pathogenesis by studying patients mutated in RNASEH2B and RNASEH2A genes. Lymphoblastoid cell lines (LCLs) from RNASEH2A- and RNASEH2B-mutated patients and healthy control were used. Transmission Electron Microscopy (TEM) and flow cytometry were used to assess morphological alterations, reactive oxygen species (ROS) production and mitochondrial membrane potential variations. Seahorse Analyzer was used to investigate metabolic alterations, and mtDNA oxidation and VDAC1 oligomerization were assessed by immunofluorescence. Western blot and RT-qPCR were used to quantify mtTFA protein and mtDNA release. Morphological alterations of mitochondria were observed in both mutated LCLs, and loss of physiological membrane potential was mainly identified in RNASEH2A LCLs. ROS production and 8-oxoGuanine levels were increased in RNASEH2B LCLs. Additionally, the VDAC1 signal was increased, suggesting a mitochondrial pore formation possibly determining mtDNA release. Indeed, higher cytoplasmic mtDNA levels were found in RNASEH2B LCLs. Metabolic alterations confirmed mitochondrial damage in both LCLs. Data highlighted mitochondrial alterations in AGS patients’ LCLs suggesting a pivotal role in AGS pathogenesis. Full article

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus. Full article

Centromere Protein I (CENP-I) is a member of the CENP-H/I/K complex. CENP-H/I/K is a major component of the inner kinetochore and aids in ensuring proper chromosomal segregation during mitosis. In addition to this chromosomal segregation function, CENP-I also plays a role in DNA double-strand break (DSB) repair. Loss of CENP-I leads to increased endogenous 53BP1 foci and R-loop formation, while reducing cellular survival after ionizing radiation and Niraparib, a PARP1 small molecule inhibitor, exposures. Cells lacking CENP-I display delayed 53BP1 foci regression, an indication that DSB repair is impaired. Additionally, loss of CENP-I impairs the homologous recombination DSB repair pathway, while having no effect on the non-homologous end-joining pathway. Interestingly, we find that RNaseH1 expression restores HR capacity in CENP-I deficient cells. Importantly, CENP-I expression is elevated in glioma tissue as compared to normal brain tissue. This elevated expression also correlates with poor overall patient survival. These data highlight the multi-functional role CENP-I plays in maintaining genetic, as well as chromosomal, stability and tumor survival. Full article

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3-[(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC₅₀ = 4.5 mM)- and RDDP (IC₅₀ = 8.0 mM) HIV RT-associated functions. Full article

Local three-stranded DNA/RNA hybrid regions of genomes (R-loops) have been detected either by binding of a monoclonal antibody (DRIP assay) or by enzymatic recognition by RNaseH. Such a structure has been postulated for mouse and human telomeres, clearly suggested by the identification of the complementary RNA Telomeric repeat-containing RNA “TERRA”. However, the tremendous disparity in the information obtained with antibody-based technology drove us to investigate a new strategy. Based on the observation that DNA/RNA hybrids in a triplex complex genome co-purify with the double-stranded chromosomal DNA fraction, we developed a direct preparative approach from total protein-free cellular extract without antibody that allows their physical isolation and determination of their RNA nucleotide sequence. We then define in the normal mouse and human sperm genomes the notion of stable DNA associated RNA terminal R-loop complexes, including TERRA molecules synthesized from local promoters of every chromosome. Furthermore, the first strong evidence of all telomeric structures, applied additionally to the whole murine sperm genome compared to the testes, showed reproducible R-loop complexes of the whole genome and suggesting a defined profile in the sperm genome for the next generation. Full article

The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp—DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome. Full article

Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2′-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi–Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences. Full article

Ribonuclease (RNase) H2 is a key enzyme for the removal of RNA found in DNA-RNA hybrids, playing a fundamental role in biological processes such as DNA replication, telomere maintenance, and DNA damage repair. RNase H2 is a trimer composed of three subunits, RNASEH2A being the catalytic subunit. RNASEH2A expression levels have been shown to be upregulated in transformed and cancer cells. In this study, we used a bioinformatics approach to identify RNASEH2A co-expressed genes in different human tissues to underscore biological processes associated with RNASEH2A expression. Our analysis shows functional networks for RNASEH2A involvement such as DNA replication and DNA damage response and a novel putative functional network of cell cycle regulation. Further bioinformatics investigation showed increased gene expression in different types of actively cycling cells and tissues, particularly in several cancers, supporting a biological role for RNASEH2A but not for the other two subunits of RNase H2 in cell proliferation. Mass spectrometry analysis of RNASEH2A-bound proteins identified players functioning in cell cycle regulation. Additional bioinformatic analysis showed that RNASEH2A correlates with cancer progression and cell cycle related genes in Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) Pan Cancer datasets and supported our mass spectrometry findings. Full article

Mitochondrial encephalomyopathies comprise a group of heterogeneous disorders resulting from impaired oxidative phosphorylation (OxPhos). Among a variety of symptoms progressive external ophthalmoplegia (PEO) seems to be the most common. The aim of this study is to present clinical and genetic characteristics of Polish patients with PEO. Clinical, electrophysiological, neuroradiological, and morphological data of 84 patients were analyzed. Genetic studies of mitochondrial DNA (mtDNA) were performed in all patients. Among nuclear DNA (nDNA) genes POLG was sequenced in 41 patients, TWNK (C10orf2) in 13 patients, and RNASEH1 in 2 patients. Total of 27 patients were included in the chronic progressive external ophthalmoplegia (CPEO) group, 24 in the CPEO+ group. Twenty-six patients had mitochondrial encephalomyopathy (ME), six patients Kearns–Sayre syndrome (KSS), and one patient sensory ataxic neuropathy, dysarthria, ophthalmoparesis (SANDO) syndrome. Genetic analysis of nDNA genes revealed the presence of pathogenic or possibly pathogenic variants in the POLG gene in nine patients, the TWNK gene in five patients and the RNASEH1 gene in two patients. Detailed patients’ history and careful assessment of family history are essential in the diagnostic work-up. Genetic studies of both mtDNA and nDNA are necessary for the final diagnosis of progressive external ophthalmoplegia and for genetic counseling. Full article

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNA^Lys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNA^Lys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNA^Lys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNA^Lys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNA^Lys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions. Full article