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1.
Deoxyuracil in DNA in health and disease.
Chakraborty, J, Stover, PJ
Current opinion in clinical nutrition and metabolic care. 2020;(4):247-252
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Abstract
PURPOSE OF REVIEW Genome instability has long been implicated as a primary causal factor in cancer and diseases of aging. The genome is constantly under attack from extrinsic and intrinsic damaging agents. Uracil misincorporation in DNA and its repair is an intrinsic factor resulting in genomic instability and DNA mutations. Additionally, the presence of uracil in DNA can modify gene expression by interfering with promoter binding and transcription inhibition or upregulation of apoptotic proteins. In immune cells, uracil in DNA drives beneficial genomic diversity for antigen-driven immunity. This review addresses diseases that are linked to uracil accumulation in DNA, its causes, consequences, and the associated biomarkers of risk factors. RECENT FINDINGS Elevated genomic uracil is associated with megaloblastic anemia, neural tube defects, and retroviral immunity. Current evidence supporting causal mechanisms and nutritional interventions that rescue impaired pathways associated with uracil accumulation in DNA are summarized in this review. SUMMARY Nutritional deficiencies in B vitamins can cause uracil misincorporation into DNA leading to genome instability and associated diseases. Nutritional approaches to preventing uracil accumulation in DNA show some promise to address its associated diseases, but additional randomized controlled trials are needed.
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Dissecting efficiency of a 5' rapid amplification of cDNA ends (5'-RACE) approach for profiling T-cell receptor beta repertoire.
Lin, YH, Hung, SJ, Chen, YL, Lin, CH, Kung, TF, Yeh, YC, Tseng, JT, Liu, T
PloS one. 2020;(7):e0236366
Abstract
Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-β sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-β sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-β sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.
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The Trinity of cGAS, TLR9, and ALRs Guardians of the Cellular Galaxy Against Host-Derived Self-DNA.
Kumar, V
Frontiers in immunology. 2020;:624597
Abstract
The immune system has evolved to protect the host from the pathogens and allergens surrounding their environment. The immune system develops in such a way to recognize self and non-self and develops self-tolerance against self-proteins, nucleic acids, and other larger molecules. However, the broken immunological self-tolerance leads to the development of autoimmune or autoinflammatory diseases. Pattern-recognition receptors (PRRs) are expressed by immunological cells on their cell membrane and in the cytosol. Different Toll-like receptors (TLRs), Nod-like receptors (NLRs) and absent in melanoma-2 (AIM-2)-like receptors (ALRs) forming inflammasomes in the cytosol, RIG (retinoic acid-inducible gene)-1-like receptors (RLRs), and C-type lectin receptors (CLRs) are some of the PRRs. The DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) is another PRR present in the cytosol and the nucleus. The present review describes the role of ALRs (AIM2), TLR9, and cGAS in recognizing the host cell DNA as a potent damage/danger-associated molecular pattern (DAMP), which moves out to the cytosol from its housing organelles (nucleus and mitochondria). The introduction opens with the concept that the immune system has evolved to recognize pathogens, the idea of horror autotoxicus, and its failure due to the emergence of autoimmune diseases (ADs), and the discovery of PRRs revolutionizing immunology. The second section describes the cGAS-STING signaling pathway mediated cytosolic self-DNA recognition, its evolution, characteristics of self-DNAs activating it, and its role in different inflammatory conditions. The third section describes the role of TLR9 in recognizing self-DNA in the endolysosomes during infections depending on the self-DNA characteristics and various inflammatory diseases. The fourth section discusses about AIM2 (an ALR), which also binds cytosolic self-DNA (with 80-300 base pairs or bp) that inhibits cGAS-STING-dependent type 1 IFN generation but induces inflammation and pyroptosis during different inflammatory conditions. Hence, this trinity of PRRs has evolved to recognize self-DNA as a potential DAMP and comes into action to guard the cellular galaxy. However, their dysregulation proves dangerous to the host and leads to several inflammatory conditions, including sterile-inflammatory conditions autoinflammatory and ADs.
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Association of Rare Predicted Loss-of-Function Variants in Cellular Pathways with Sub-Phenotypes in Age-Related Macular Degeneration.
Pietraszkiewicz, A, van Asten, F, Kwong, A, Ratnapriya, R, Abecasis, G, Swaroop, A, Chew, EY
Ophthalmology. 2018;(3):398-406
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Abstract
PURPOSE To investigate the association of rare predicted loss-of-function (pLoF) variants within age-related macular degeneration (AMD) risk loci and AMD sub-phenotypes. DESIGN Case-control study. PARTICIPANTS Participants of AREDS, AREDS2, and Michigan Genomics Initiative. METHODS Whole genome sequencing data were analyzed for rare pLoF variants (frequency <0.1%) in the regions of previously identified 52 independent risk variants known to be associated with AMD. Frequency of the rare pLoF variants in cases with intermediate or advanced AMD was compared with controls. Variants were assigned to the complement, extracellular matrix (ECM), lipid, cell survival, immune system, metabolism, or unknown/other pathway. Associations of rare pLoF variant pathways with AMD sub-phenotypes were analyzed using logistic and linear regression, and Cox proportional hazards models. MAIN OUTCOME MEASURES Differences in rare pLoF variant pathway burden and association of rare pLoF variant pathways with sub-phenotypes within the population with AMD were evaluated. RESULTS Rare pLoF variants were found in 298 of 1689 cases (17.6%) and 237 of 1518 controls (15.6%) (odds ratio [OR], 1.11; 95% confidence interval [CI], 0.91-1.36; P = 0.310). An enrichment of rare pLoF variants in the complement pathway in cases versus controls (OR, 2.94; 95% CI, 1.49-5.79; P = 0.002) was observed. Within cases, associations between all rare pLoF variants and choroidal neovascularization (CNV) (OR, 1.34; 95% CI, 1.04-1.73; P = 0.023), calcified drusen (OR, 1.33; 95% CI, 1.04-1.72; P = 0.025), higher scores on the AREDS Extended AMD Severity Scale (Standardized Coefficient Beta (β)=0.346 [0.086-0.605], P = 0.009), and progression to advanced disease (hazard ratio, 1.25; 95% CI, 1.01-1.55; P = 0.042) were observed. At the pathway level, there were associations between the complement pathway and geographic atrophy (GA) (OR, 2.17; 95% CI, 1.12-4.24; P = 0.023), the complement pathway and calcified drusen (OR, 3.75; 95% CI, 1.79-7.86; P < 0.001), and the ECM pathway and more severe levels in the AREDS Extended AMD Severity Scale (β = 0.62; 95% CI, 0.04-1.20; P = 0.035). CONCLUSIONS Rare pLoF variants are associated with disease progression. Variants in the complement pathway modify the clinical course of AMD and increase the risk of developing specific sub-phenotypes.
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Tricho-hepato-enteric syndrome (THE-S): two cases and review of the literature.
Chong, JH, Jamuar, SS, Ong, C, Thoon, KC, Tan, ES, Lai, A, Aan, MK, Tan, WL, Foo, R, Tan, EC, et al
European journal of pediatrics. 2015;(10):1405-11
Abstract
UNLABELLED Tricho-hepato-enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction. We report two cases of East Asian descent with THE-S who had remained undiagnosed despite extensive investigations but were diagnosed on whole exome sequencing (WES). Both cases presented with chronic diarrhea, failure to thrive, and recurrent infections. Case 1 had posteriorly rotated low set ears, mild retrognathia, and fine curly hypopigmented hair. She was managed with prolonged total parenteral nutrition and intravenous immunoglobulin infusions. Case 2 had sparse coarse brown hair as well as multiple lentigines and café-au-lait macules. She was managed with amino acid-based formula. For both cases, routine investigations were inconclusive. WES in both cases showed biallelic truncating mutations in TTC37 (c.3507T>G;p.Y1169X and c.3601C>T;p.R1201X in case 1 and c.3507T>G;p.Y1169X and c.154G>T;p.E52X in case 2), suggesting a diagnosis of THE-S. CONCLUSION We present novel mutations in the TTC37 gene in two individuals of East Asian descent with the rare THE-S, detected by WES. Future identification of patients with THE-S and establishing genotype-phenotype correlations will aid in counseling the patients and their families. WHAT IS KNOWN • Tricho-Hepato-Enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction. • Complex patients with diagnostic dilemmas undergo extensive investigations. What is New: • This is a report of novel mutations in TTC37 in individuals of East Asian descent. • Whole exome sequencing (WES) can be useful in certain complex cases with diagnostic dilemmas.
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Innovative approaches to the use of polyamines for DNA nanoparticle preparation for gene therapy.
Vijayanathan, V, Agostinelli, E, Thomas, T, Thomas, TJ
Amino acids. 2014;(3):499-509
Abstract
Advances in genomic technologies, such as next generation sequencing and disease specific gene targeting through anti-sense, anti-gene, siRNA and microRNA approaches require the transport of nucleic acid drugs through the cell membrane. Membrane transport of DNA/RNA drugs is an inefficient process, and the mechanism(s) by which this process occurs is not clear. A pre-requisite for effective transport of DNA and RNA in cells is their condensation to nanoparticles of ~100 nm size. Although viral vectors are effective in gene therapy, the immune response elicited by viral proteins poses a major challenge. Multivalent cations, such as natural polyamines are excellent promoters of DNA/RNA condensation to nanoparticles. During the past 20 years, our laboratory has synthesized and tested several analogs of the natural polyamine, spermine, for their efficacy to provoke DNA condensation to nanoparticles. We determined the thermodynamics of polyamine-mediated DNA condensation, measured the structural specificity effects of polyamine analogs in facilitating the cellular uptake of oligonucleotides, and evaluated the gene silencing activity of DNA nanoparticles in breast cancer cells. Polyamine-complexed oligonucleotides showed a synergistic effect on target gene inhibition at the mRNA level compared to the use of polyamines and oligonucleotides as single agents. Ionic and structural specificity effects were evident in DNA condensation and cellular transportation effects of polyamines. In condensed DNA structures, correlation exists between the attractive and repulsive forces with structurally different polyamines and cobalt hexamine, indicating the existence of a common force in stabilizing the condensed structures. Future studies aimed at defining the mechanism(s) of DNA compaction and structural features of DNA nanoparticles might aid in the development of novel gene delivery vehicles.
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An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA.
Fischer, S, Maier, LK, Stoll, B, Brendel, J, Fischer, E, Pfeiffer, F, Dyall-Smith, M, Marchfelder, A
The Journal of biological chemistry. 2012;(40):33351-63
Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.
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Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.
Breitbach, S, Tug, S, Simon, P
Sports medicine (Auckland, N.Z.). 2012;(7):565-86
Abstract
The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer. In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15-20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis or apoptosis of cells in acute exercise settings. Recently, rapid DNA release mechanisms of activated immune-competent cells like NETosis (pathogen-induced cell death including the release of neutrophil extracellular traps [NETs]) have been discovered. cfDNA accumulations might comprise a similar kind of cell death including trap formation or an active release of cfDNA. Just like chronic diseases, chronic high-intensity resistance training protocols induced persistent increases of cfDNA levels. Chronic, strenuous exercise protocols, either long-duration endurance exercise or regular high-intensity workouts, induce chronic inflammation that might lead to a slow, constant release of DNA. This could be due to mechanisms of cell death like apoptosis or necrosis. Yet, it has neither been implicated nor proven sufficiently whether cfDNA can serve as a marker for overtraining. The relevance of cfDNA with regard to overtraining status, performance level, and the degree of physical exhaustion still remains unclear. Longitudinal studies are required that take into account standardized and controlled exercise, serial blood sampling, and large and homogeneous cohorts of different athletic achievement. Furthermore, it is important to establish standardized laboratory procedures for the measurement of genomic cfDNA concentrations by quantitative real-time polymerase chain reaction (PCR). We introduce a new hypothesis based on acute exercise and chronic exposure to stress, and rapid active and passive chronic release of cfDNA fragments into the circulation.
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Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis.
Nahon, P, Zucman-Rossi, J
Journal of hepatology. 2012;(3):663-74
Abstract
Liver carcinogenesis is a complex and multi-factorial process, in which both environmental and genetic features interfere and contribute to malignant transformation. Patients with cirrhosis are particularly exposed and justify periodical screenings in order to detect the early development of hepatocellular carcinoma (HCC). The risk of HCC is, however, not identical from one patient to another. The identification of host factors that may also play an important role in HCC development may improve our understanding of the implications of the various biological pathways involved in liver carcinogenesis; such progress may as well help refine the selection of patients who could benefit from specific preventative measures or could be given adapted screening policies. Numerous candidate-gene studies have reported associations between single nucleotide polymorphisms (SNPs) and the presence of HCC. Some of these publications unfortunately suffer from major methodological drawbacks because of their case-control, retrospective and monocentric aspect. Prospective cohort studies conducted in large homogeneous populations and comprising a sufficient number of events during follow-up may overcome these pitfalls, but require a long time to be conducted and are still scarce. More recently, the first Genome Wide Association studies (GWAs) have enabled the identification of unsuspected loci that may be involved in various steps implicated in liver tumourigenesis. Taken together, these studies highlight variants that modulate oxidative stress, iron metabolism, inflammatory and immune responses, DNA repair mechanisms or systems involved in cell-cycle regulation as genetic traits susceptible to modify the natural history of cirrhotic patients and partly explain the observed differences in the risk of HCC occurrence. However, large genetic epidemiology studies in the field of cancer diseases have suggested the limited ability of polymorphic traits, alone, to refine individual prognosis. The integration of various panels of genes into clinical scores may in the near future define a "genomic risk prediction" specific to liver cancer developed in cirrhotic patients.