Adenine Guanine Research Articles

Disrupted cerebellar structural connectome in spinocerebellar ataxia type 3 and its association with transcriptional profiles

Abstract Spinocerebellar ataxia type 3 (SCA3) is primarily characterized by progressive cerebellar degeneration, including gray matter atrophy and disrupted anatomical and functional connectivity. The alterations of cerebellar white matter structural network in SCA3 and the underlying neurobiological mechanism remain unknown. Using a cohort of 20 patients with SCA3 and 20 healthy controls, we constructed cerebellar structural networks from diffusion MRI and investigated alterations of topological organization. Then, we mapped the alterations with transcriptome data from the Allen Human Brain Atlas to identify possible biological mechanisms for regional selective vulnerability to white matter damage. Compared with healthy controls, SCA3 patients exhibited reduced global and nodal efficiency, along with a widespread decrease in edge strength, particularly affecting edges connected to hub regions. The strength of inter-module connections was lower in SCA3 group and negatively correlated with the Scale for the Assessment and Rating of Ataxia score, International Cooperative Ataxia Rating Scale score, and cytosine–adenine–guanine repeat number. Moreover, the transcriptome–connectome association study identified the expression of genes involved in synapse-related and metabolic biological processes. These findings suggest a mechanism of white matter vulnerability and a potential image biomarker for the disease severity, providing insights into neurodegeneration and pathogenesis in this disease.

The phase coherence of the neurovascular unit is reduced in Huntington’s disease

Abstract Huntington’s disease is a neurodegenerative disorder in which neuronal death leads to chorea and cognitive decline. Individuals with ≥40 cytosine–adenine–guanine repeats on the interesting transcript 15 gene develop Huntington’s disease due to a mutated huntingtin protein. While the associated structural and molecular changes are well characterized, the alterations in neurovascular function that lead to the symptoms are not yet fully understood. Recently, the neurovascular unit has gained attention as a key player in neurodegenerative diseases. The mutant huntingtin protein is known to be present in the major parts of the neurovascular unit in individuals with Huntington’s disease. However, a non-invasive assessment of neurovascular unit function in Huntington’s disease has not yet been performed. Here, we investigate neurovascular interactions in presymptomatic (N = 13) and symptomatic (N = 15) Huntington’s disease participants compared to healthy controls (N = 36). To assess the dynamics of oxygen transport to the brain, functional near-infrared spectroscopy, ECG and respiration effort were recorded. Simultaneously, neuronal activity was assessed using EEG. The resultant time series were analysed using methods for discerning time-resolved multiscale dynamics, such as wavelet transform power and wavelet phase coherence. Neurovascular phase coherence in the interval around 0.1 Hz is significantly reduced in both Huntington’s disease groups. The presymptomatic Huntington’s disease group has a lower power of oxygenation oscillations compared to controls. The spatial coherence of the oxygenation oscillations is lower in the symptomatic Huntington’s disease group compared to the controls. The EEG phase coherence, especially in the α band, is reduced in both Huntington’s disease groups and, to a significantly greater extent, in the symptomatic group. Our results show a reduced efficiency of the neurovascular unit in Huntington’s disease both in the presymptomatic and symptomatic stages of the disease. The vasculature is already significantly impaired in the presymptomatic stage of the disease, resulting in reduced cerebral blood flow control. The results indicate vascular remodelling, which is most likely a compensatory mechanism. In contrast, the declines in α and γ coherence indicate a gradual deterioration of neuronal activity. The results raise the question of whether functional changes in the vasculature precede the functional changes in neuronal activity, which requires further investigation. The observation of altered dynamics paves the way for a simple method to monitor the progression of Huntington’s disease non-invasively and evaluate the efficacy of treatments.

Radiological Evaluation of Huntington’s Disease: A Case Report

Huntington’s Disease (HD) is a rare neurodegenerative condition inherited in an autosomal dominant pattern, where Gamma Amino Butyric Acid-ergic (GABAergic) neurons in the basal ganglia progressively deteriorate. Symptoms include subcortical dementia, behavioural changes, midlife psychosis, and involuntary choreoathetosis movements. Imaging techniques like Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) show shrinkage of the caudate nucleus and putamen, alongside enlarged lateral ventricles. Diagnosis is confirmed through molecular analysis identifying the hallmark amplified Cytosine Adenine Guanine (CAG) triplet. The case report describes a 57-year-old female diagnosed with HD, presenting symptoms of accidental falls caused by involuntary jerky movements, along with behavioural changes and cognitive decline over three years. Initial Non Contrast Computed Tomography (NCCT) of the brain revealed bilateral basal ganglia and diffuse cerebral atrophy. Quantitative analysis indicated a decreased frontal horn width to intercaudate distance (FH/CC) ratio of 1.24 and an increased Intercaudate distance to inner table width (CC/IT) ratio of 0.30. Multivoxel Magnetic Resonance Spectroscopy (MRS) showed reduced N-Acetyl Alanine (NAA) and creatine (Cr) levels with an NAA/Cr ratio of 0.84. Magnetic Resonance Tractography and Diffusion Tensor Imaging (DTI) revealed elevated Fractional Anisotropy (FA) and diffusivity in the basal ganglia, and reduced FA with increased diffusivity in white matter tracts. Genetic analysis by Polymerase Chain Reaction (PCR) confirmed HD. Newer MRI like DTI allows early identification of disease and better assessment of disease progression and response to therapy. A multidisciplinary approach involving neurologists, psychiatrists, and other healthcare professionals is essential. Genetic counseling and psychosocial support are crucial for patients and their families. Early and accurate identification of HD is vital for timely interventions and improved symptom control.

Exercise attenuates polyglutamine-mediated neuromuscular degeneration in a mouse model of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a hereditary neuromuscular disorder caused by the expansion of trinucleotide cytosine-adenine-guanine (CAG) repeats, which encodes a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. Recent evidence suggests that, in addition to motor neuron degeneration, defective skeletal muscles are also the primary contributors to the pathogenesis in SBMA. While benefits of physical exercise have been suggested in SBMA, underlying mechanism remains elusive. We investigated the effect of running exercise in a transgenic mouse model of SBMA carrying human AR with 97 expanded CAGs (AR97Q). We assigned AR97Q mice to exercise and sedentary control groups, and mice in the exercise group received 1-h forced running wheel (5m/min) 5days a week for 4weeks during the early stage of the disease. Motor function (grip strength and rotarod performance) and survival of each group were analysed, and histopathological and biological features in skeletal muscles and motor neurons were evaluated. AR97Q mice in the exercise group showed improvement in motor function (~40% and ~50% increase in grip strength and rotarod performance, respectively, P<0.05) and survival (median survival 23.6 vs. 16.7weeks, P<0.05) with amelioration of neuronal and muscular histopathology (~1.4-fold and ~2.8-fold increase in motor neuron and muscle fibre size, respectively, P<0.001) compared to those in the sedentary group. Nuclear accumulation of polyQ-expanded AR in skeletal muscles and motor neurons was suppressed in the mice with exercise compared to the sedentary mice (~50% and ~30% reduction in 1C2-positive cells in skeletal muscles and motor neurons, respectively, P<0.05). We found that the exercise activated 5'-adenosine monophosphate-activated protein kinase (AMPK) signalling and inhibited mammalian target of rapamycin pathway that regulates protein synthesis in skeletal muscles of SBMA mice. Pharmacological activation of AMPK inhibited protein synthesis and reduced polyQ-expanded AR proteins in C2C12 muscle cells. Our findings suggest the therapeutic potential of exercise-induced effect via AMPK activation in SBMA.

Prevalence of Glu323Lys Mutation of the TIGR/MYOC Gene and Risk Factors amongst Primary Open-angle Glaucoma Patients in Ouagadougou, Burkina Faso.

Glaucoma is a group of degenerative diseases of the optic nerve whose predisposing factors may be genetic. The objective of this study was to estimate the frequency of the Glu323Lys mutation as a genetic risk factor for glaucoma. A cross-sectional study over 6 months from October 2020 to March 2021 in Ouagadougou, Burkina Faso. A total of 89 samples of patients with primary open-angle glaucoma (POAG) were collected. The frequency of the Glu323Lys mutation of the myocilin, trabecular meshwork inducible glucocorticoid response (TIGR/MYOC) gene by polymerase chain reaction (PCR)-restriction fragment length polymorphism. In glaucoma patients, only homozygous nonmutated guanine-guanine (GG) and heterozygous mutated adenine-guanine (AG) genotypes were found in 96.63 and 3.37% of cases, respectively. Around 69.66% of patients had a family history of glaucoma, 28.09% had a history of hypertension, and 7.86% had a history of diabetes. The frequency of the Glu323Lys mutation of the TIGR/MYOC gene was 3.37% in the glaucoma population in Ouagadougou. A case-control study is necessary to know the contribution of the Glu323Lys mutation as a genetic risk factor for glaucoma in our study population. This study constituted the beginning of genetic investigations of glaucoma in our context and showed a low Glu323Lys mutation. Traoré L, Sanou J, Bakyono BS, et al. Prevalence of Glu323Lys Mutation of the TIGR/MYOC Gene and Risk Factors amongst Primary Open-angle Glaucoma Patients in Ouagadougou, Burkina Faso. J Curr Glaucoma Pract 2023;17(2):79-84.

A complete telomere-to-telomere assembly of the maize genome

A complete telomere-to-telomere (T2T) finished genome has been the long pursuit of genomic research. Through generating deep coverage ultralong Oxford Nanopore Technology (ONT) and PacBio HiFi reads, we report here a complete genome assembly of maize with each chromosome entirely traversed in a single contig. The 2,178.6 Mb T2T Mo17 genome with a base accuracy of over 99.99% unveiled the structural features of all repetitive regions of the genome. There were several super-long simple-sequence-repeat arrays having consecutive thymine–adenine–guanine (TAG) tri-nucleotide repeats up to 235 kb. The assembly of the entire nucleolar organizer region of the 26.8 Mb array with 2,974 45S rDNA copies revealed the enormously complex patterns of rDNA duplications and transposon insertions. Additionally, complete assemblies of all ten centromeres enabled us to precisely dissect the repeat compositions of both CentC-rich and CentC-poor centromeres. The complete Mo17 genome represents a major step forward in understanding the complexity of the highly recalcitrant repetitive regions of higher plant genomes.

Immunohistochemical study of proline, glutamic acid and leucine-rich protein 1 (PELP1) in correlation with guanine adenine thymine adenine family member 3 (GATA-3) receptors expression in breast carcinomas

Abstract Background Breast cancer is the most common cancer affecting Egyptian women. Guanine adenine thymine adenine family member 3 (GATA-3) and Proline, glutamic acid and leucine-rich protein 1 (PELP1) are both expressed in breast carcinomas. Aim To evaluate GATA-3 and PELP1 proteins expression in breast carcinoma, assess association of both immunohistochemical markers with variable prognostic factors, correlate between GATA-3 and PELP1 expression and assess the diagnostic utility of both markers in breast carcinomas. Subjects and methods The current study was a cross-sectional descriptive study, performed in the Pathology laboratory of Suez Canal University Hospital on 86 samples of formalin fixed paraffin embedded blocks of breast carcinoma during the period from January 2016 to May 2019. Results GATA-3 and PELP1 showed positive nuclear expression in breast carcinoma cases (74.4% and 82.6%, respectively). PELP1 expression showed statistically significant association with older age cases. GATA-3 expression showed statistically significant association with positive ER, PR and HER-2 expression and molecular subtypes. A statistically significant correlation was found between GATA-3 and PELP1 expression in breast carcinomas (n=86, P value &lt;0.05). PELP1 showed the highest sensitivity in stage III breast cancer samples (84.9%) compared to GATA-3 (75.8%), ER (66.7%), PR (54.6%) and HER2 (39.4%). PELP1 is more sensitive (80%) than GATA-3 (60%) in grade III breast carcinomas. GATA-3 is more sensitive (89.7%) than PELP1 (86.2%) in luminal breast cancer subtype. the combined parallel use of GATA-3 and PELP1 raised their sensitivity to 92% in Grade III and 98.6% in luminal breast cancers. Conclusions GATA-3 and PELP1 may be a promising diagnostic markers of breast carcinoma. Further studies are needed to fully evaluate the prognostic significance of GATA-3 and PELP1 expression in invasive breast carcinomas.

Rapid creation of CENH3-mediated haploid induction lines using a cytosine base editor (CBE).

Haploid induction (HI) can create true-breeding lines in a single generation, which can significantly accelerates the breeding process. In recent years, scientists have developed a variety of new techniques to induce haploids through manipulation of CENH3, a variant of the centromere-specific histone H3. One alternative approach is based on CENH3 point mutations derived from EMS/TILLING, which is not lethal and yet is responsible for inducing haploids. However, most residues have been obtained by EMS mutagenesis over a long period of time. Recently, a new approach called 'base editing' was developed for plants. Here, we report a new method that uses a cytosine base editor (CBE) to create a point mutation of CENH3 as a haploid induction line, which substitutes adenine (A) for guanine (G). As proof of the extreme simplicity of this approach to create haploid-induced lines, we identified an L130F substitution within the histone fold domain in Arabidopsis thaliana. Subsequently, we tested the haploid-inducing potential of homozygous L130F plants by pollinating them with Col-0, and obtained 2.9% paternal haploid plants. In brief, our innovative technology provides a new perspective for the promotion of CENH3-mediated haploid induction in crops, and also provides a variety of options for breeders. Such conserved point mutations as L130F could be developed into a general instrument for haploid induction in a wide range of plant species. Extending these systems would represent a major advance over haploid production.

Molecular insights into the interaction of CAG trinucleotide RNA repeats with nucleolin and its implication in polyglutamine diseases.

Polyglutamine (polyQ) diseases are a type of inherited neurodegenerative disorders caused by cytosine–adenine–guanine (CAG) trinucleotide expansion within the coding region of the disease-associated genes. We previously demonstrated that a pathogenic interaction between expanded CAG RNA and the nucleolin (NCL) protein triggers the nucleolar stress and neuronal cell death in polyQ diseases. However, mechanisms behind the molecular interaction remain unknown. Here, we report a 1.45 Å crystal structure of the r(CAG)5 oligo that comprises a full A′-form helical turn with widened grooves. Based on this structure, we simulated a model of r(CAG)5 RNA complexed with the RNA recognition motif 2 (RRM2) of NCL and identified NCL residues that are critical for its binding to CAG RNA. Combined with in vitro and in vivo site-directed mutagenesis studies, our model reveals that CAG RNA binds to NCL sites that are not important for other cellular functions like gene expression and rRNA synthesis regulation, indicating that toxic CAG RNA interferes with NCL functions by sequestering it. Accordingly, an NCL mutant that is aberrant in CAG RNA-binding could rescue RNA-induced cytotoxicity effectively. Taken together, our study provides new molecular insights into the pathogenic mechanism of polyQ diseases mediated by NCL–CAG RNA interaction.

Brain microvascular endothelial cell dysfunction in an isogenic juvenile iPSC model of Huntington’s disease

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by expansion of cytosine–adenine–guanine (CAG) repeats in the huntingtin gene, which leads to neuronal loss and decline in cognitive and motor function. Increasing evidence suggests that blood–brain barrier (BBB) dysfunction may contribute to progression of the disease. Studies in animal models, in vitro models, and post-mortem tissue find that disease progression is associated with increased microvascular density, altered cerebral blood flow, and loss of paracellular and transcellular barrier function. Here, we report on changes in BBB phenotype due to expansion of CAG repeats using an isogenic pair of induced pluripotent stem cells (iPSCs) differentiated into brain microvascular endothelial-like cells (iBMECs). We show that CAG expansion associated with juvenile HD alters the trajectory of iBMEC differentiation, producing cells with ~ two-fold lower percentage of adherent endothelial cells. CAG expansion is associated with diminished transendothelial electrical resistance and reduced tight junction protein expression, but no significant changes in paracellular permeability. While mutant huntingtin protein (mHTT) aggregates were not observed in HD iBMECs, widespread transcriptional dysregulation was observed in iBMECs compared to iPSCs. In addition, CAG expansion in iBMECs results in distinct responses to pathological and therapeutic perturbations including angiogenic factors, oxidative stress, and osmotic stress. In a tissue-engineered BBB model, iBMECs show subtle changes in phenotype, including differences in cell turnover and immune cell adhesion. Our results further support that CAG expansion in BMECs contributes to BBB dysfunction during HD.

Selected Histone Deacetylase Inhibitors Reverse the Frataxin Transcriptional Defect in a Novel Friedreich's Ataxia Induced Pluripotent Stem Cell-Derived Neuronal Reporter System.

Friedreich’s ataxia (FRDA) is a neurodegenerative disorder caused by the expansion of guanine–adenine–adenine repeats within the first intron of the frataxin (FXN) gene. The location and nature of the expansion have been proven to contribute to transcriptional repression of FXN by decreasing the rate of polymerase II (RNA polymerase II) progression and increasing the presence of histone modifications associated with a heterochromatin-like state. Targeting impaired FXN transcription appears as a feasible option for therapeutic intervention, while no cure currently exists. We created a novel reporter cell line containing an FXN-Nanoluciferase (FXN-NLuc) fusion in induced pluripotent stem cells (iPSCs) reprogrammed from the fibroblasts of patients with FRDA, thus allowing quantification of endogenous FXN expression. The use of iPSCs provides the opportunity to differentiate these cells into disease-relevant neural progenitor cells (NPCs). NPCs derived from the FXN-NLuc line responded to treatments with a known FXN inducer, RG109. Results were validated by quantitative PCR and Western blot in multiple FRDA NPC lines. We then screened a commercially available library of compounds consisting of molecules targeting various enzymes and pathways critical for silencing or activation of gene expression. Only selected histone deacetylase inhibitors were capable of partial reactivation of FXN expression. This endogenous, FRDA iPSC-derived reporter can be utilized for high-throughput campaigns performed in cells most relevant to disease pathology in search of FXN transcription activators.

IGF-1 as a Potential Therapy for Spinocerebellar Ataxia Type 3.

Although the effects of growth hormone (GH) therapy on spinocerebellar ataxia type 3 (SCA3) have been examined in transgenic SCA3 mice, it still poses a nonnegligible risk of cancer when used for a long term. This study investigated the efficacy of IGF-1, a downstream mediator of GH, in vivo for SCA3 treatment. IGF-1 (50 mg/kg) or saline, once a week, was intraperitoneally injected to SCA3 84Q transgenic mice harboring a human ATXN3 gene with a pathogenic expanded 84 cytosine–adenine–guanine (CAG) repeat motif at 9 months of age. Compared with the control mice harboring a 15 CAG repeat motif, the SCA3 84Q mice treated with IGF-1 for 9 months exhibited the improvement only in locomotor function and minimized degeneration of the cerebellar cortex as indicated by the survival of more Purkinje cells with a more favorable mitochondrial function along with a decrease in oxidative stress caused by DNA damage. These findings could be attributable to the inhibition of mitochondrial fission, resulting in mitochondrial fusion, and decreased immunofluorescence staining in aggresome formation and ataxin-3 mutant protein levels, possibly through the enhancement of autophagy. The findings of this study show the therapeutic potential effect of IGF-1 injection for SCA3 to prevent the exacerbation of disease progress.

Assessment of myeloperoxidase (Mpo) gene polymorphism in cervical cancer

Background/Aim: Cervical cancer (CC) is the most common gynecological malignancy in women. In spite of a variety of treatment protocols, it is necessary to carefully investigate all factors that play a role in the pathogenesis of these tumors which may have mortal progression. In this context, in our study we aimed to assess the myeloperoxidase (MPO) gene polymorphism, an important inflammatory enzyme, among cervix cancer cases. Methods: In this cross-sectional study, 79 cases diagnosed as cervical carcinoma between 1992-2012 is included. The cases without archival paraffin blocks and clinical follow-ups are excluded. All slides with tumor involvement are reviewed and the ones which demonstrate tumor’s characteristics are determined. After block determination 3 sections with 10-micron thickness obtained from paraffin blocks and MPO gene polymorphism was shown using acil restriction endonuclease enzyme with the restriction fragment length polymorphism (RFLP) method after polymerase chain reaction (PCR). The histopathological parameters including tumor stage and type, lymph node metastasis, ovary and endometrium involvement, recurrence and late metastasis are compared with genotype using chi-square and Fisher’s exact test. Results: The mean age of cases was 51.3 (10.9) years. Of 79 cases, 29 (36.7%) had AG (adenine-guanine) and 50 (633%) had GG (guanine-guanine) genotypes. Only endometrium involvement was identified to have a statistically difference with MPO gene polymorphism among the assessed histopathologic parameters (P=0.015). When clinical parameters are assessed, there was no difference identified between genotype and mortality (P=0.622). Conclusion: Cervical cancer is thought to have progressive and regressive characteristics of tumor development due to the inflammatory response of the host. Within this framework, in our study assessing gene polymorphism of one of the inflammatory response foundation stones of MPO, we identified more endometrial involvement for cases with AG genotype. We believe this significance will be encountered for more parameters in broad case series.

Androgen receptor gene polymorphism and female sexual functionin Egyptian women

BackgroundAndrogen receptor (AR) polymorphism in cytosine–adenine–guanine (CAG) repeat has an effect on the functional capacity of AR in males. However, little researches in this field are available regarding female sexual function.ObjectivesTo investigate the possible link between polymorphism in CAG repeat of AR gene and female sexual function in a sample of Egyptian women.Patients and methodsIn all, 500 married Egyptian women completed a questionnaire regarding sociodemographic, reproductive, and sexual data. AR CAG repeat length was analyzed for those having female sexual dysfunctions using real-time PCR.ResultsThe most sensitive domain to AR CAG repeat length was orgasm domain that showed significant positive correlations with short allele (P = 0.001), long allele (P = 0.015), biallelic mean (P = 0.000), and X-weighted biallelic mean (P = 0.000). Satisfaction domain had significant positive correlations with biallelic mean (P = 0.035) and X-weighted biallelic mean (P = 0.032). However, the pain domain was of significant negative correlations with AR polymorphism of short allele (P = 0.002), biallelic mean (P = 0.013), and X-weighted biallelic mean (P = 0.011).ConclusionsAR polymorphism could represent a nonnegligible aspect in female sexual function. The lower AR CAG repeat polymorphism was of significant impact on female sexual dysfunction affecting mainly female orgasm, followed by pain disorders that finally reflected on her sexual satisfaction.

Bioinformatics and wet laboratory analysis of rs1800562 to predict genetic aetiology of iron overload in Nigeria

Abstract Introduction/Objective The most reported single nucleotide polymorphism (SNP) of the HFE gene is rs1800562, representing the substitution of Adenine for Guanine at position 847 of the HFE gene. This has been widely implicated in hereditary haemochromatosis and other conditions like altered cholesterol balance, Alzheimer’s disease and cutaneous photosensitivity. Abnormal HFE protein resulting from the mutant HFE gene leads to formation of excess iron which has been postulated as likely mechanism for these diseases. Although there is evidence of iron overload in Africans, only few studies have explored possible genetic causes, and prevalence of rs1800562 is not known in West African population. Hence the need to determine the prevalence of rs1800562 in Nigeria using computational and wet laboratory approach. Methods/Case Report Details of rs1800562 were retrieved from Ensembl Genome Browser version 99. Severity of the consequences of this SNP on protein product was determined using bioinformatics tools including SIFT, Polyphen, Mutation Assessor, HOPE, I-mutant and MutPred2. Genotyping of rs1800562 was done In silico using restriction fragment length polymorphism (RFLP). Primer3plus was used for primer design, NCBI BLAST and SMS were used for primer validation. We used Webcutter 2.0 to determine suitable restriction enzymes. The genotyping was simulated using USCS virtual PCR and RestrictionMapper. Whole blood samples were obtained from 200 participants selected randomly from a pool of blood donors. DNA was extracted and flanking region of rs1800562 was amplified. The amplified product was digested by RSA1and fragments examined on agarose gel electrophoresis. Results (if a Case Study enter NA) The MAF was found to be 0.01 globally and 0.02 in Africa. In the two Nigerian population examined (Yoruba and Esan population), MAF was 0.00. Mutation Assessor and SIFT Polyphen consistently predicted the mutation to be of severe consequences. Analysis on HOPE, I-mutant and Mutpred2 revealed loss of protein stability, change in net charges affecting the HFE protein localization and its interaction with other proteins. All the participants in the wet laboratory analysis were homozygous for the wild type allele of rs1800562 (MAF=0). Conclusion This study confirmed the In silico prediction of the absence of rs1800562 in Nigeria. Future studies should focus on other SNPs of the HFE gene as well as other gene involved in iron metabolism.

Versatile cell-based assay for measuring DNA alkylation damage and its repair

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

Ranking the Predictive Power of Clinical and Biological Features Associated With Disease Progression in Huntington's Disease.

Huntington's disease (HD) is characterised by a triad of cognitive, behavioural, and motor symptoms which lead to functional decline and loss of independence. With potential disease-modifying therapies in development, there is interest in accurately measuring HD progression and characterising prognostic variables to improve efficiency of clinical trials. Using the large, prospective Enroll-HD cohort, we investigated the relative contribution and ranking of potential prognostic variables in patients with manifest HD. A random forest regression model was trained to predict change of clinical outcomes based on the variables, which were ranked based on their contribution to the prediction. The highest-ranked variables included novel predictors of progression—being accompanied at clinical visit, cognitive impairment, age at diagnosis and tetrabenazine or antipsychotics use—in addition to established predictors, cytosine adenine guanine (CAG) repeat length and CAG-age product. The novel prognostic variables improved the ability of the model to predict clinical outcomes and may be candidates for statistical control in HD clinical studies.

FOXO1 controls protein synthesis and transcript abundance of mutant polyglutamine proteins, preventing protein aggregation.

FOXO1, a transcription factor downstream of the insulin/insulin like growth factor axis, has been linked to protein degradation. Elevated expression of FOXO orthologs can also prevent the aggregation of cytosine adenine guanine (CAG)-repeat disease causing polyglutamine (polyQ) proteins but whether FOXO1 targets mutant proteins for degradation is unclear. Here, we show that increased expression of FOXO1 prevents toxic polyQ aggregation in human cells while reducing FOXO1 levels has the opposite effect and accelerates it. Although FOXO1 indeed stimulates autophagy, its effect on polyQ aggregation is independent of autophagy, ubiquitin–proteasome system (UPS) mediated protein degradation and is not due to a change in mutant polyQ protein turnover. Instead, FOXO1 specifically downregulates protein synthesis rates from expanded pathogenic CAG repeat transcripts. FOXO1 orchestrates a change in the composition of proteins that occupy mutant expanded CAG transcripts, including the recruitment of IGF2BP3. This mRNA binding protein enables a FOXO1 driven decrease in pathogenic expanded CAG transcript- and protein levels, thereby reducing the initiation of amyloidogenesis. Our data thus demonstrate that FOXO1 not only preserves protein homeostasis at multiple levels, but also reduces the accumulation of aberrant RNA species that may co-contribute to the toxicity in CAG-repeat diseases.

Reverse Phase Protein Array Reveals Correlation of Retinoic Acid Metabolism With Cardiomyopathy in Friedreich's Ataxia

Identifying biomarkers is important for assessment of disease progression, prediction of symptom development, and determination of treatment effectiveness. While unbiased analyses of differential gene expression using next-generation sequencing methods are now routinely conducted, proteomics studies are more challenging because of traditional methods predominantly being low throughput and offering a limited dynamic range for simultaneous detection of hundreds of proteins that drastically differ in their intracellular abundance. We utilized a sensitive and high-throughput proteomic technique, reverse phase protein array (RPPA), to attain protein expression profiles of primary fibroblasts obtained from patients with Friedreich's ataxia (FRDA) and unaffected controls (CTRLs). The RPPA was designed to detect 217 proteins or phosphorylated proteins by individual antibody, and the specificity of each antibody was validated prior to the experiment. Among 62 fibroblast samples (44 FRDA and 18 CTRLs) analyzed, 30 proteins/phosphoproteins were significantly changed in FRDA fibroblasts compared with CTRL cells (p < 0.05), mostly representing signaling molecules and metabolic enzymes. As expected, frataxin was significantly downregulated in FRDA samples, thus serving as an internal CTRL for assay integrity. Extensive bioinformatics analyses were conducted to correlate differentially expressed proteins with critical disease parameters (e.g., selected symptoms, age of onset, guanine–adenine–adenine sizes, frataxin levels, and Functional Assessment Rating Scale scores). Members of the integrin family of proteins specifically associated with hearing loss in FRDA. Also, RPPA data, combined with results of transcriptome profiling, uncovered defects in the retinoic acid metabolism pathway in FRDA samples. Moreover, expression of aldehyde dehydrogenase family 1 member A3 differed significantly between cardiomyopathy-positive and cardiomyopathy-negative FRDA cohorts, demonstrating that metabolites such as retinol, retinal, or retinoic acid could become potential predictive biomarkers of cardiac presentation in FRDA.

Multiscale Conformational Sampling Reveals Excited-State Locality in DNA Self-Repair Mechanism.

Ultraviolet (UV) irradiation is known to be responsible for DNA damage. However, experimental studies in DNA oligonucleotides have shown that UV light can also induce sequence-specific self-repair. Following charge transfer from a guanine adenine sequence adjacent to a cyclobutane pyrimidine dimer (CPD), the covalent bond between the two thymines could be cleaved, recovering the intact base sequence. Mechanistic details promoting the self-repair remained unclear, however. In our theoretical study, we investigated whether optical excitation could directly lead to a charge-transfer state, thereby initiating the repair, or whether the initial excited state remains localized on a single nucleobase. We performed conformational sampling of 200 geometries of the damaged DNA double strand solvated in water and used a hybrid quantum and molecular mechanics approach to compute excited states at the complete active space perturbation level of theory. Analysis of the conformational data set clearly revealed that the excited-state properties are uniformly distributed across the fluctuations of the nucleotide in its natural environment. From the electronic wavefunction, we learned that the electronic transitions remained predominantly local on either adenine or guanine, and no direct charge transfer occurred in the experimentally accessed energy range. The investigated base sequence is not only specific to the CPD repair mechanism but ubiquitously occurs in nucleic acids. Our results therefore give a very general insight into the charge locality of UV-excited DNA, a property that is regarded to have determining relevance in the structural consequences following absorption of UV photons.