Search Results (25)

All beta- and gamma-herpesviruses utilize a set of six viral proteins to facilitate transcription from specific promoters that become active late in the viral life cycle. These proteins form a complex that interacts with a TA-rich sequence upstream of the late transcription start sites and recruits RNA polymerase II (Pol II). The structure of any of the late transcription factors (LTFs) alone or in complexes has not been solved by standard means yet, but a fair amount is known about how the proteins interact and where the complex is positioned over the late promoters. Here, AlphaFold3 was used to predict and analyze the LTF complex using proteins from the beta-herpesviruses HCMV, MCMV, HHV6, and HHV7, and from the gamma-herpesviruses EBV and KSHV. The predicted structures had high levels of confidence and were remarkably similar even though there is little sequence conservation in the LTFs across the viruses. The results are consistent with most of the previously determined information concerning the interaction of the factors with each other and with DNA. A conserved threonine phosphorylation in one of the subunits that is critical to the function of the LTFs is predicted to be at the junction of five subunits. AlphaFold 3 predicts seven metal ion binding sites in each of the four beta-herpesviruses and either five or six in the gamma-herpesviruses created by conserved residues in three of the subunits. The structures also provide insights into the function of the subunits and which host general transcription factors (GTFs) may or may not be utilized during initiation. Full article

Background: The nuclear-encoded enzyme polymerase gamma (Pol-γ) is crucial in the replication of the mitochondrial genome (mtDNA), which in turn is vital for mitochondria and hence numerous metabolic processes and energy production in eukaryotic cells. Variants in the POLG gene, which encodes the catalytic subunit of Pol-γ, can significantly impair Pol-γ enzyme function. Pol-γ-associated disorders are referred to as POLG-spectrum disorders (POLG-SDs) and are mainly autosomal-recessively inherited. Clinical manifestations include muscle weakness and fatigue, and severe forms of the disease can lead to premature death in infancy, childhood, and early adulthood, often associated with seizures, liver failure, or intractable epilepsy. Here, we analyzed fibroblasts from a compound heterozygous patient with the established pathogenic variant c.2419C>T; p.(Arg807Cys) and a previously undescribed variant c.678G>C; p.(Gln226His) with a clinical manifestation compatible with POLG-SDs, sensory ataxic neuropathy, and infantile muscular atrophy. We conducted a battery of functional studies for Pol-γ and mitochondrial dysfunction on the patient’s fibroblasts, to test whether the novel variant c.678G>C; p.(Gln226His) may be causative in human disease. Aims/Methods: We analyzed skin-derived fibroblasts in comparison to a first-degree relative (the mother of the patient), an asymptomatic carrier harboring only the established c.2419C>T; p.(Arg807Cys) mutation. Assessments of mitochondrial function included measurements of mtDNA content, mRNA levels of mitochondrial genes, mitochondrial mass, and mitochondrial morphology. Case Presentation and Results: A 13-year-old male presented with symptoms starting at three years of age, including muscle weakness and atrophy in the lower extremities and facial muscles, which later extended to the upper limbs, voice, and back muscles, without further progression. The patient also reported fatigue and muscle pain after physical activity, with no sensory deficits. Extensive diagnostic tests such as electromyography, nerve conduction studies, muscle biopsy, and MRI were unremarkable. Exome sequencing revealed that he carried the compound heterozygous variants in POLG c.678G>C; p.(Gln226His) and c.2419C>T; p.(Arg807Cys), but no other potential genetic pathogenic causes. In comparison to a first-degree relative (his mother) who only carried the c.2419C>T; p.(Arg807Cys) pathogenic mutation, in vitro analyses revealed a significant reduction in mtDNA content (~50%) and mRNA levels of mtDNA-encoded proteins. Mitochondrial mass was reduced by approximately 20%, and mitochondrial interconnectivity within cells was impaired, as determined by fluorescence microscopy and mitochondrial staining. Conclusions: Our findings suggest that the c.678G>C; p.(Gln226His) variant, in conjunction with the c.2419C>T; p.(Arg807Cys) mutation, may compromise mtDNA replication and mitochondrial function and could result in clinically significant mitochondriopathy. As this study is based on one patient compared to a first-degree relative (but with an identical mitochondrial genome), the pathogenicity of c.678G>C; p.(Gln226His) of POLG should be confirmed in future studies, in particular, in conjunction with other POLG-variants. Full article

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a variety of the human gamma-herpesvirus that often leads to the occurrence of malignant tumors. In addition, the occurrence of Kaposi’s sarcoma is a major cause of death among AIDS patients. Ganciclovir (GCV) is the most widely used drug against KSHV infection in the clinic. GCV can restrict the in vivo synthesis of DNA polymerase in KSHV, thereby inhibiting the replication of the herpesvirus. However, GCV still suffers from poor specificity and transmembrane capabilities, leading to many toxic side effects. Therefore, developing a drug delivery system that increases GCV concentrations in target cells remains a significant clinical challenge. In this study, zeolite imidazole salt framework-8 (ZIF-8), a biocompatible porous material constructed by coordinating zinc ions and 2-methylimidazole, was used to load GCV. A nano-delivery system with a microneedle structure was also constructed using a polydimethylsiloxane (PDMS) microneedle mold to fabricate MN/GCV@ZIF-8 arrays. These arrays not only offered good skin-piercing capabilities but also significantly inhibited the cleavage and replication of the virus in vivo, exerting an anti-KSHV function. For these reasons, the arrays were able penetrate the skin’s stratum corneum at the tumor site to deliver GCV and play an anti-KSHV role. Full article

Background: High variability in the age at cancer diagnosis in Lynch syndrome (LS) patients is widely observed, even among relatives with the same germline pathogenic variant (PV) in the mismatch repair (MMR) genes. Genetic polymorphisms and lifestyle factors are thought to contribute to this variability. We investigated the influence of previously reported genetic polymorphisms on the age at cancer diagnosis in a homogenous LS cohort with a South African founder germline PV c.1528C>T in the MLH1 gene. Methods: A total of 359 LS variant heterozygotes (LSVH) from 60 different families were genotyped for specific genetic polymorphisms in GSTM1, GSTT1, CYP1A1, CYP17, PPP2R2B, KIF20A, TGFB1, XRCC5, TNF, BCL2, CHFR, CDC25C, ATM, TTC28, CDC25C, HFE, and hTERT genes using Multiplex Polymerase Chain Reaction and MassArray methods. Kaplan–Meier survival analysis, univariate and multivariate Cox proportional hazards gamma shared frailty models adjusted for sex were used to estimate the association between age at cancer diagnosis and polymorphism genotypes. A p-value < 0.05 after correcting for multiple testing using the Benjamini–Hochberg method was considered significant at a 95% confidence interval. Results: We identified three genotypes in the cell-cycle regulation, DNA repair, and xenobiotic-metabolism genes significantly associated with age at cancer diagnosis in this cohort. The CYP1A1 rs4646903 risk (GG) and CDC25C rs3734166 polymorphic (GA+AA) genotypes were significantly associated with an increased risk of a younger age at cancer diagnosis (Adj HR: 2.03 [1.01–4.08], p = 0.034 and Adj HR: 1.53 [1.09–2.14], p = 0.015, respectively). LSVH who were heterozygous for the XRCC5 rs1051685 SNP showed significant protection against younger age at cancer diagnosis (Adj HR: 0.69 [CI, 0.48–0.99], p = 0.043). The risk of a younger age at any cancer diagnosis was significantly high in LS carriers of one to two risk genotypes (Adj HR: 1.49 [CI: 1.06–2.09], corrected p = 0.030), while having one to two protective genotypes significantly reduced the risk of developing any cancer and CRC at a younger age (Adj HR: 0.52 [CI: 0.37–0.73], and Adj HR: 0.51 [CI: 0.36–0.74], both corrected p < 0.001). Conclusions: Polymorphism genotypes in the cell-cycle regulation, DNA repair, and xenobiotic metabolizing genes may influence the age at cancer diagnosis in a homogenous LS cohort with a South African founder germline PV c.1528C>T in the MLH1 gene. Full article

Using Chlamydomonas as a model organism, we attempted to eliminate mitochondrial DNA (mtDNA) similar to rho⁰ or rho⁻ cells (completely or partially mtDNA-eliminated cells) in yeast. We successfully generated partially mtDNA-eliminated cells named as crm- cells, causing the inactivation of mitochondrial activity. We used three different chemicals to eliminate mtDNA including acriflavine (AF), ethidium bromide (EB) and dideoxycytidine (ddC) which prevents replication, inhibits POLG (DNA polymerase gamma) and terminates the mtDNA chain, respectively. The qPCR method was used to detect the mtDNA copy number and the selected rrnL6 gene for the detection of mitochondria, as well as the selected Chlamydomonas CC-124 strain. A reduction in the mitochondrial copy number led to a higher expression of AOX1, UCP1, PGRL1 and ICL1, which indicates the disturbance of the mitochondria–chloroplast ATP and NADPH balance. We selected AOX genes to further study this family and carried out a genome-wide search to identify AOX genes in green algae (C. reinhardtii). Our results revealed that C. reinhardtii contains four AOX genes, i.e., CrAOX1, CrAOX2, CrAOX3 and CrAOX4, which are distributed on Chr 3, Chr7 and Chr9. All CrAOX genes were predicted to localize in mitochondria using bioinformatics tools. Phylogenetic analysis suggests that these CrAOXs are subdivided into four groups and genes existing in the same group could perform identical functions. Collinearity analysis describes the strong evolutionary relationships of AOXs between the unicellular green algae Chlamydomonas reinhardtii and the multicellular green algae Volvox carteri. GO (gene ontology) annotation analysis predicted that CrAOXs played an integral part in carrying out alternate oxidative and respirative activities. Three putative miRNAs, cre-miR1162-3p, cre-miR1171 and cre-miR914, targeting the CrAOX2 gene were identified. Our studies have laid a foundation for the further use of partially mtDNA-eliminated cells and elucidating the functional characteristics of the AOX gene family. Full article

Parkinson’s disease (PD) is a multisystem and multifactorial disorder and, therefore, the application of modern genetic techniques may assist in unraveling its complex pathophysiology. We conducted a clinical–demographic evaluation of 126 patients with PD, all of whom were Caucasian and of Sicilian ancestry. DNA was extracted from the peripheral blood for each patient, followed by sequencing using a Next-Generation Sequencing system. This system was based on a custom gene panel comprising 162 genes. The sample underwent further filtering, taking into account the allele frequencies of genetic variants, their presence in the Human Gene Mutation Database, and their association in the literature with PD or other movement/neurodegenerative disorders. The largest number of variants was identified in the leucine-rich repeat kinase 2 (LRRK2) gene. However, variants in other genes, such as acid beta-glucosidase (GBA), DNA polymerase gamma catalytic subunit (POLG), and parkin RBR E3 ubiquitin protein ligase (PRKN), were also discovered. Interestingly, some of these variants had not been previously associated with PD. Enhancing our understanding of the genetic basis of PD and identifying new variants possibly linked to the disease will contribute to improved diagnostic accuracy, therapeutic developments, and prognostic insights for affected individuals. Full article

Metabolic activities within the gut microbiome are intimately linked to human health and disease, especially within the context of environmental exposure and its potential ramifications. Perturbations within this microbiome, termed “gut microbiome perturbations”, have emerged as plausible intermediaries in the onset or exacerbation of diseases following environmental chemical exposures, with fluoride being a compound of particular concern. Despite the well-documented adverse impacts of excessive fluoride on various human physiological systems—ranging from skeletal to neurological—the nuanced dynamics between fluoride exposure, the gut microbiome, and the resulting dose–response relationship remains a scientific enigma. Leveraging the precision of 16S rRNA high-throughput sequencing, this study meticulously examines the ramifications of diverse fluoride concentrations on the gut microbiome’s composition and functional capabilities within Wistar rats. Our findings indicate a profound shift in the intestinal microbial composition following fluoride exposure, marked by a dose-dependent modulation in the abundance of key genera, including Pelagibacterium, Bilophila, Turicibacter, and Roseburia. Moreover, discernible alterations were observed in critical functional and metabolic pathways of the microbiome, such as D-lyxose ketol-isomerase and DNA polymerase III subunit gamma/tau, underscoring the broad-reaching implications of fluoride exposure. Intriguingly, correlation analyses elucidated strong associations between specific bacterial co-abundance groups (CAGs) and these shifted metabolic pathways. In essence, fluoride exposure not only perturbs the compositional equilibrium of the gut microbiota but also instigates profound shifts in its metabolic landscape. These intricate alterations may provide a mechanistic foundation for understanding fluoride’s potential toxicological effects mediated via gut microbiome modulation. Full article

The exposure of ionizing radiation during early gestation often leads to deleterious and even lethal effects; however, few extensive studies have been conducted on late gestational exposures. This research examined the behavior al effects of C57Bl/6J mouse offspring exposed to low dose ionizing gamma irradiation during the equivalent third trimester. Pregnant dams were randomly assigned to sham or exposed groups to either low dose or sublethal dose radiation (50, 300, or 1000 mGy) at gestational day 15. Adult offspring underwent a behavioral and genetic analysis after being raised under normal murine housing conditions. Our results indicate very little change in the behavioral tasks measuring general anxiety, social anxiety, and stress-management in animals exposed prenatally across the low dose radiation conditions. Quantitative real-time polymerase chain reactions were conducted on the cerebral cortex, hippocampus, and cerebellum of each animal; results indicate some dysregulation in markers of DNA damage, synaptic activity, reactive oxygen species (ROS) regulation, and methylation pathways in the offspring. Together, our results provide evidence in the C57Bl/6J strain, that exposure to sublethal dose radiation (<1000 mGy) during the last period of gestation leads to no observable changes in behaviour when assessed as adults, although some changes in gene expression were observed for specific brain regions. These results indicate that the level of oxidative stress occurring during late gestation for this mouse strain is not sufficient for a change in the assessed behavioral phenotype, but results in some modest dysregulation of the genetic profile of the brain. Full article

Marked cellular changes occur in human intervertebral disc (IVD) degeneration during disc degeneration with biochemical changes. Genome-wide analysis of the DNA methylation profile has identified 220 differentially methylated loci associated with human IVD degeneration. Among these, two cell-cycle–associated genes, growth arrest and DNA damage 45 gamma (GADD45G) and cytoplasmic activation/proliferation-associated protein-1 (CAPRIN1), were focused on. The expression of GADD45G and CAPRIN1 in human IVDs remains unknown. We aimed to examine the expression of GADD45G and CAPRIN1 in human nucleus pulposus (NP) cells and evaluate those in human NP tissues in the early and advanced stages of degeneration according to Pfirrmann magnetic resonance imaging (MRI) and histological classifications. Human NP cells were cultured as monolayers after isolation from NP tissues by sequential enzyme digestion. Total RNA was isolated, and the mRNA expression of GADD45G and CAPRIN1 was quantified using real-time polymerase chain reaction. To examine the effects of pro-inflammatory cytokines on mRNA expression, human NP cells were cultured in the presence of IL-1β. Protein expression was evaluated using Western blotting and immunohistochemistry. GADD45G and CAPRIN1 expression was identified in human NP cells at both mRNA and protein levels. The percentage of cells immunopositive for GADD45G and CAPRIN1 significantly increased according to the Pfirrmann grade. A significant correlation between the histological degeneration score and the percentage of GADD45G-immunopositive cells was identified, but not with that of CAPRIN1-immunopositive cells. The expression of cell-cycle-associated proteins (GADD45G and CAPRIN1) was enhanced in human NP cells at an advanced stage of degeneration, suggesting that it may be regulated during the progression of IVD degeneration to maintain the integrity of human NP tissues by controlling cell proliferation and apoptosis under epigenetic alteration. Full article

The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested. Full article

Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are the backbone of HIV antiretroviral therapy (ART). ART use in pregnancy has been associated with adverse birth outcomes, in part due to NRTI-induced mitochondrial toxicity. Direct comparison on the effects of commonly used dual-NRTI regimens on placental mitochondria toxicity in pregnancy is lacking. We compared zidovudine/lamivudine, abacavir/lamivudine, and tenofovir/emtricitabine using a mouse model and examined markers of placental mitochondrial function and oxidative stress. Zidovudine/lamivudine and abacavir/lamivudine were associated with lower fetal and placental weights compared to controls, whereas tenofovir/emtricitabine was associated with the least fetal and placental weight reduction, as well as lower resorption rates. Placental mitochondrial DNA content, as well as placental expression of cytochrome c-oxidase subunit-II, DNA polymerase gamma, and citrate synthase, was higher in tenofovir/emtricitabine-treated mice compared to other groups. Zidovudine/lamivudine-treated mice had elevated malondialdehyde levels (oxidative stress marker) compared to other groups and lower mRNA levels of manganese superoxide dismutase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha in the placenta compared to tenofovir/emtricitabine-treated mice. We observed differences in effects between NRTI regimens on placental mitochondrial function and birth outcomes. Tenofovir/emtricitabine was associated with larger fetuses, increased mtDNA content, and higher expression of mitochondrial-specific antioxidant enzymes and mitochondrial biogenesis enzymes, whereas zidovudine/lamivudine was associated with markers of placental oxidative stress. Full article

We have performed whole-genome sequencing to identify the genetic variants potentially contributing to the early-onset semantic dementia phenotype in a patient with family history of dementia and episodic memory deficit accompanied with profound semantic loss. Only very rare variants of unknown significance (VUS) have been identified: a nonsense variant c.366C>A/p.Cys122* in plasminogen activator, urokinase (PLAU) and a missense variant c.944C>T/p.Thr315Met in β-site APP-cleaving enzyme 1 (BACE1)—along with known disease-modifying variants of moderate penetrance. Patient-derived fibroblasts showed reduced PLAU and elevated BACE1 mRNA and protein levels compared to control fibroblasts. Successful rescue of PLAU mRNA levels by nonsense-mediated mRNA decay (NMD) inhibitor (puromycin) confirmed NMD as the underlying mechanism. This is the first report of the PLAU variant with the confirmed haploinsufficiency, associated with semantic dementia phenotype. Our results suggest that rare variants in the PLAU and BACE1 genes should be considered in future studies on early-onset dementias. Full article

Defects in the mitochondrial genome (mitochondrial DNA (mtDNA)) are associated with both congenital and acquired disorders in humans. Nuclear-encoded DNA polymerase subunit gamma (POLG) plays an important role in mtDNA replication, and proofreading and mutations in POLG have been linked with increased mtDNA deletions. SSBP1 is also a crucial gene for mtDNA replication. Here, we describe a patient diagnosed with Pearson syndrome with large mtDNA deletions that were not detected in the somatic cells of the mother. Exome sequencing was used to evaluate the nuclear factors associated with the patient and his family, which revealed a paternal POLG mutation (c.868C > T) and a maternal SSBP1 mutation (c.320G > A). The patient showed lower POLG and SSBP1 expression than his healthy brothers and the general population of a similar age. Notably, c.868C in the wild-type allele was highly methylated in the patient compared to the same site in both his healthy brothers. These results suggest that the co- deficient expression of POLG and SSBP1 genes could contribute to the development of mtDNA deletion. Full article

Background: The clinical spectrum associated with POLG1 gene mutations ranges from non-syndromic epilepsy or mild isolated neurological signs to neurodegenerative disorders. Our aim was to review diagnostic findings, therapeutic approaches and outcomes of reported cases of epilepsy related to POLG1 mutation. Methods: The articles for review were identified through a systematic research on PubMed and EMBASE databases from January 2003 to April 2020, searching for the terms “Epilepsy AND POLG OR polymerase gamma,” OR “POLG1”. Results: Forty-eight articles were selected for review, which included 195 patients. Two main peaks of age at epilepsy onset were found: at ages 1 and 13 years. The most frequent seizure type was myoclonic. The occurrence of Status Epilepticus was reported in 46.4% of cases. Epileptiform and slow abnormalities were most frequently seen over occipital regions. Brain Magnetic Resonance Imaging (MRI) revealed increased T2 signal intensities in thalamic regions. Genetic analysis revealed a prevalence of A467T, W748S and G848S (74.2% of patients) mutations. Survival at 5 years was estimated at very low levels (30.2% of patients). Conclusion: In this review, we included cases with both pediatric and adult epilepsy onset. The analysis of data regarding prognosis showed that survival is related to age at onset of epilepsy. Full article

The central paradigm of novel therapeutic approaches in cancer therapy is identifying and targeting molecular biomarkers. One such target is the nuclear DNA repair enzyme Poly-(ADP ribose) polymerase 1 (PARP1). Sensitivity to PARP inhibition in certain cancers such as gBRCA^mut breast and ovarian cancers has led to its exploitation as a target. The overexpression of PARP1 in several types of cancer further evoked interest in its use as an imaging target. While PARP1-targeted inhibitors have fast developed and approved in this past decade, determination of PARP1 expression might help to predict the response to PARP inhibitor treatment. This has the potential of improving prognosis and moving towards tailored therapy options and/or dosages. This review summarizes the recent pre-clinical advancements in imaging and theranostic PARP1 targeted tracers. To assess PARP1 levels, several imaging probes with fluorescent or beta/gamma emitting radionuclides have been proposed and three have advanced to ongoing clinical evaluation. Apart from its diagnostic value in detection of primary tumors as well as metastases, this shall also help in delivering therapeutic radionuclides to PARP1 overexpressing tumors. Henceforth nuclear medicine has now advanced towards conjugating theranostic radionuclides to PARP1 inhibitors. This paves the way for a future of PARP1-targeted theranostics and personalized therapy. Full article