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1.
A metric space for semantic containment: Towards the implementation of genetic firewalls.
Schmidt, M
Bio Systems. 2019;:104015
Abstract
Analysing or engineering the genetic code has mainly been considered as an approach to reduce or increase the mutational robustness of the genetic code, i.e. the error tolerance in DNA mutations, or to enable the incorporation of non-canonical amino acids. The approach of "semantic containment", however, is less interested in altering the mutational tolerance of the standard code, but to create synthetic alternative genetic codes that limit or all together impede horizontal gene transfer between a natural and genomically recoded organisms (GRO). A major claim or conjecture of semantic containment is: "the farther, the safer", meaning, the less similarity there is between two codes, the less chance of a horizontal gene transfer, and the stronger the genetic firewall. So far, no metrics were available to measure and quantify the "genetic distance" between different genetic codes. Such a metric, however, is iis paramount to allow the experimental testing and evaluation of the validity of semantic biocontainment for the first time. Here, we introduce a metric space to measure exactly the distance (dissimilarity) between different genetic codes, in order to provide a framework to evaluate the relation between distance and strength of a genetic firewall. Results are presented that incorporate bespoken metrics when producing alternative genetic codes according to predefined goals, specifications and limitations. Finally, as an outlook, implications and challenges for genetic firewall(s) are discussed for dual- and multi-code systems.
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2.
Enhancing Antioxidant Effect against Peroxyl Radical-Induced Oxidation of DNA: Linking with Ferrocene Moiety!
Liu, ZQ
Chemical record (New York, N.Y.). 2019;(12):2385-2397
Abstract
As a major member in the family of reactive oxygen species, peroxyl radical is able to abstract hydrogen atom from 4-position of ribose, leading to the collapse of DNA strand. Thus, inhibiting oxidative stress with exogenous antioxidants acts as a promising strategy to protect the integrity of DNA structure and is thereby suggested to be a pathway against developments of related diseases. Ferrocene as an organometallic scaffold is widely applied in the design of organometallic drugs, and redox of Fe(II)/Fe(III) in ferrocene offers advantage for providing electron to radicals. Presented herein are our ongoing studies on ferrocene-appended antioxidants, including McMurry reaction applied to construct ferrocifen; Aldol condensation used to prepare ferrocenyl curcumin; Povarov reaction employed to prepare ferrocenyl quinoline; Biginelli reaction used to construct ferrocenyl dihydropyrimidine; Groebke reaction used to synthesize ferrocenyl imidazo[1,2-a]pyridine; and Passerini three-component reaction as well as Ugi four-component reaction applied to synthesize α-acyloxycarboxamide and bisamide, respectively. It is found that ferrocene moiety is able to enhance antioxidative effect of the aforementioned scaffolds even without the aid of phenolic hydroxyl group. The role of ferrocene in enhancing antioxidative effect can be attributable to trapping radicals, decreasing oxidative potential, and increasing the affinity toward DNA strand. Therefore, ferrocene is worthy to be taken into consideration in the design of drugs in relation to DNA oxidation.
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3.
Autoimmune and inflammatory K+ channelopathies in cardiac arrhythmias: Clinical evidence and molecular mechanisms.
Capecchi, PL, Laghi-Pasini, F, El-Sherif, N, Qu, Y, Boutjdir, M, Lazzerini, PE
Heart rhythm. 2019;(8):1273-1280
Abstract
Cardiac K+ channelopathies account for a significant proportion of arrhythmias and sudden cardiac death (SCD) in subjects without structural heart disease. It is well recognized that genetic defects are key factors in many cases, and in practice, the term cardiac channelopathies currently coincides with inherited cardiac channelopathies. However, mounting evidence demonstrate that not only genetic alterations but also autoimmune and inflammatory factors can cause cardiac K+-channel dysfunction and arrhythmias in the setting of a structurally normal heart. In particular, it has been demonstrated that specific autoantibodies as well as inflammatory cytokines can modulate expression and/or function of different K+ channels in the heart, resulting in a disruption of the cardiac action potential and arrhythmias/sudden cardiac death. Awareness about the existence of these newly recognized forms is essential to identify and adequately manage affected patients. In the present review, we focus on autoimmune and inflammatory K+ channelopathies as a novel mechanism for cardiac arrhythmias and analyze the recent advancements in this topic, providing complementary basic, clinical, and population health perspectives.
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4.
Rate limiting factors for DNA transduction induced by weak electromagnetic field.
Tang, BQ, Li, T, Bai, X, Zhao, M, Wang, B, Rein, G, Yang, Y, Gao, P, Zhang, X, Zhao, Y, et al
Electromagnetic biology and medicine. 2019;(1):55-65
Abstract
DNA transduction across aqueous solutions has been reported previously. In this study, we examined a few key factors affecting DNA transduction rate in an extremely low frequency electromagnetic field. These include: the chemical composition of the aqueous solutions, the type of experimental vessel, the dilution step, and the origin of the DNA fragments. The results indicate that partially introducing essential ingredients for DNA amplification (i.e. dNTPs and PCR buffer) to the aqueous solution enhanced the transduction rate greatly, and transduction vessels made of hydrophilic quartz yielded more favorable results than vessels made of hydrophobic plastic. In addition, performing a serial dilution to the transduction solution more than doubled the transduction rate compared to that without the dilution step. For the DNA fragments used in this study, there was one with a pathogenic origin and two with non-pathogenic origins. However, all three fragments achieved DNA transduction regardless of the difference in their origins. The experimental setup for eliminating the false positives caused by both biological and potentially physical contamination is also described.
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5.
APOE Alleles and Extreme Human Longevity.
Sebastiani, P, Gurinovich, A, Nygaard, M, Sasaki, T, Sweigart, B, Bae, H, Andersen, SL, Villa, F, Atzmon, G, Christensen, K, et al
The journals of gerontology. Series A, Biological sciences and medical sciences. 2019;(1):44-51
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Abstract
We assembled a collection of 28,297 participants from seven studies of longevity and healthy aging comprising New England Centenarian, Long Life Family, Longevity Gene Population, Southern Italian Centenarian, Japanese Centenarian, the Danish Longevity, and the Health and Retirement Studies to investigate the association between the APOE alleles ε2ε3 and ε4 and extreme human longevity and age at death. By using three different genetic models and two definitions of extreme longevity based on either a threshold model or age at death, we show that ε4 is associated with a substantially decreased odds for extreme longevity, and increased risk for death that persists even beyond ages reached by less than 1% of the population. We also show that carrying the ε2ε2 or ε2ε3 genotype is associated with significantly increased odds to reach extreme longevity, with decreased risk for death compared with carrying the genotype ε3ε3 but with only a modest reduction in risk for death beyond an age reached by less than 1% of the population.
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6.
Role of amino acid residues important for nucleic acid binding in human Translin.
Gupta, A, Pillai, VS, Chittela, RK
The international journal of biochemistry & cell biology. 2019;:105593
Abstract
Translin is a multifunctional DNA/RNA binding protein involved in DNA repair and RNA metabolism. It has two basic regions and involvement of some residues in these regions in nucleic acid binding is established experimentally. Here we report the functional role of four residues of basic region II, Y85, R86, H88, R92 and one residue of C terminal region, K193 in nucleic acid binding using substitution mutant variants. CD analysis of the mutant proteins showed that secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Octameric state was maintained in all the mutants of basic region as evidenced by TEM, DLS, native PAGE and gel filtration analyses. However, K193G mutation completely abolished the octameric state of Translin protein and consequently its ability to bind ssDNA/ssRNA. The mutants of the basic region II exhibited a differential effect on nucleic acid binding, with R86A and R92G as most deleterious. Interestingly, H88A mutant showed higher nucleic acid binding affinity in comparison to the wild type Translin. An in silico analysis of the mutant variant sequences predicted all the mutations to be destabilizing, causing increase in flexibility and also leading to disruption of local interactions. The differential effect of mutations on DNA/RNA binding where octameric state is maintained could be attributed to these predicted disturbances.
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7.
Atomistic mechanisms of the double proton transfer in the H-bonded nucleobase pairs: QM/QTAIM computational lessons.
Brovarets', OO, Hovorun, DM
Journal of biomolecular structure & dynamics. 2019;(7):1880-1907
Abstract
In this Review, we have summarized and generalized the results of the investigation of the microstructural mechanisms of the tautomerization by the counter movement of the protons along the neighboring intermolecular H-bonds in 22 biologically important pairs of nucleotide bases in the framework of the original method, which allows to trace the evolution of the physicochemical parameters, that characterize these processes along the intrinsic reaction coordinate (IRC). It was demonstrated the performance of the introduction of the conception of the key points (KPs) (from nine to five, depending on the symmetry and nature of system), which exhaustively characterize the flow of the tautomerization processes. It was proved that for all tautomerizing base pairs the extrema of the first derivative of the electron energy of the complex by IRC coincide with the second and penultimate KPs, in which the Laplacian of the electron density equals zero at the corresponding (3,-1) bond critical points of the H-bonds. It was established the linear dependence of the width of the transition state zone of the DPT tautomerization on the degree of its asynchrony. Authors emphasize that the tautomerization reaction through the DPT of the H-bonded pairs of nucleotide bases can be considered successful in those and only in those case if the tautomerized complex is a dynamically stable system, during lifetime of which low-frequency intermolecular vibrations could develop. Perspectives of the application of the obtained approaches to the thorough study of the proton transfer processes in the biologically important objects have been briefly discussed.
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8.
High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries.
Lietard, J, Schaudy, E, Hölz, K, Ameur, D, Somoza, MM
Journal of visualized experiments : JoVE. 2019;(150)
Abstract
Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid·ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of β-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.
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9.
Type 8 long QT syndrome: pathogenic variants in CACNA1C-encoded Cav1.2 cluster in STAC protein binding site.
Mellor, GJ, Panwar, P, Lee, AK, Steinberg, C, Hathaway, JA, Bartels, K, Christian, S, Balaji, S, Roberts, JD, Simpson, CS, et al
Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing, arrhythmias, and cardiac cellular electrophysiology of the European Society of Cardiology. 2019;(11):1725-1732
Abstract
AIMS: Pathogenic gain-of-function variants in CACAN1C cause type-8 long QT syndrome (LQT8). We sought to describe the electrocardiographic features in LQT8 and utilize molecular modelling to gain mechanistic insights into its genetic culprits. METHODS AND RESULTS Rare variants in CACNA1C were identified from genetic testing laboratories. Treating physicians provided clinical information. Variant pathogenicity was independently assessed according to recent guidelines. Pathogenic (P) and likely pathogenic (LP) variants were mapped onto a 3D modelled structure of the Cav1.2 protein. Nine P/LP variants, identified in 23 patients from 19 families with non-syndromic LQTS were identified. Six variants, found in 79% of families, clustered to a 4-residue section in the cytosolic II-III loop region which forms a region capable of binding STAC SH3 domains. Therefore, variants may affect binding of SH3-domain containing proteins. Arrhythmic events occurred in similar proportions of patients with II-III loop variants and with other P/LP variants (53% vs. 48%, P = 0.41) despite shorter QTc intervals (477 ± 31 ms vs. 515 ± 37 ms, P = 0.03). A history of sudden death was reported only in families with II-III loop variants (60% vs. 0%, P = 0.03). The predominant T-wave morphology was a late peaking T wave with a steep descending limb. Exercise testing demonstrated QTc prolongation on standing and at 4 min recovery after exercise. CONCLUSION The majority of P/LP variants in patients with CACNA1C-mediated LQT8 cluster in an SH3-binding domain of the cytosolic II-III loop. This represents a 'mutation hotspot' in LQT8. A late-peaking T wave with a steep descending limb and QT prolongation on exercise are commonly seen.
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10.
Heterozygous familial hypercholesterolaemia in a pair of identical twins: a case report and updated review.
Mohd Nor, NS, Al-Khateeb, AM, Chua, YA, Mohd Kasim, NA, Mohd Nawawi, H
BMC pediatrics. 2019;(1):106
Abstract
BACKGROUND Familial hypercholesterolaemia (FH) is the most common inherited metabolic disease with an autosomal dominant mode of inheritance. It is characterised by raised serum levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c), leading to premature coronary artery disease. Children with FH are subjected to early and enhanced atherosclerosis, leading to greater risk of coronary events, including premature coronary artery disease. To the best of our knowledge, this is the first report of a pair of monochorionic diamniotic identical twins with a diagnosis of heterozygous FH, resulting from mutations in both LDLR and ABCG8 genes. CASE PRESENTATION This is a rare case of a pair of 8-year-old monochorionic diamniotic identical twin, who on family cascade screening were diagnosed as definite FH, according to the Dutch Lipid Clinic Criteria (DLCC) with a score of 10. There were no lipid stigmata noted. Baseline lipid profiles revealed severe hypercholesterolaemia, (TC = 10.5 mmol/L, 10.6 mmol/L; LDL-c = 8.8 mmol/L, 8.6 mmol/L respectively). Their father is the index case who initially presented with premature CAD, and subsequently diagnosed as FH. Family cascade screening identified clinical FH in other family members including their paternal grandfather who also had premature CAD, and another elder brother, aged 10 years. Genetic analysis by targeted next-generation sequencing using MiSeq platform (Illumina) was performed to detect mutations in LDLR, APOB100, PCSK9, ABCG5, ABCG8, APOE and LDLRAP1 genes. Results revealed that the twin, their elder brother, father and grandfather are heterozygous for a missense mutation (c.530C > T) in LDLR that was previously reported as a pathogenic mutation. In addition, the twin has heterozygous ABCG8 gene mutation (c.55G > C). Their eldest brother aged 12 years and their mother both had normal lipid profiles with absence of LDLR gene mutation. CONCLUSION A rare case of Asian monochorionic diamniotic identical twin, with clinically diagnosed and molecularly confirmed heterozygous FH, due to LDLR and ABCG8 gene mutations have been reported. Childhood FH may not present with the classical physical manifestations including the pathognomonic lipid stigmata as in adults. Therefore, childhood FH can be diagnosed early using a combination of clinical criteria and molecular analyses.