Enzymatic incorporations of LNA nucleotides into DNA strands

Unlocking uses of locked nucleic acids: LNA nucleoside 5′-triphosphates have been synthesized, and their ability to serve as substrates for polymerases have been investigated. Phusion high-fidelity DNA polymerase was found to be an efficient enzyme for incorporating LNA nucleoside 5′-triphosphates into DNA strands

Using UV Mutagenesis for Generation and Isolation of Novel Cell Division Cycle Mutants in Kluyveromyces lactis

Cell division cycle mutants, cells with malfunctioning genes that disrupt cell replication and division, have been studied and used in research to better understand the genetic aspect of cancer. The goal of the this research was to create and study novel temperature-sensitive cell division cycle (ts/cdc) mutants that have not been previously identified or studied in the budding yeast Kluyveromyces lactis. Previous work from Redlands students has yielded ten cdc mutants, created by ethyl methane sulfonate (EMS) mutagenesis, which arrested with large buds and were placed into eight complementation groups. To expand the number and variety of cdc mutants, ultraviolet (UV) light was used to create additional ts/cdc mutants in K. lactis because UV has a broad mutagenic spectrum, unlike ethyl methane sulfonate. The screening for ts conditional mutants was performed by overlaying the images of replica plates at the restrictive temperature onto replica plate images at the permissive temperature. Using UV mutagenesis, 61 K. lactis ts mutants were generated. Screening nine of our ts mutants and eight previously identified ts mutants for the characteristic uniform morphology, bud-size and nuclear configuration revealed five mutants of the cell division cycle. Four of the five cdc mutants found revealed novel phenotypes in this experiment. One, UV1BS4.32, displayed the previously seen large-budded phenotype. Two mutants, UV1BS4.32 and UV1BS5.36, showed the novel phenotype of small-budded morphology. The other two cdc mutants UV1BS1.4 and RCY1110 had unbudded phenotypes, which are also new to this research, and possibly new to this species

Reinforcing the pulmonary artery autograft in the aortic position with a textile mesh: a histological evaluation

OBJECTIVES The Ross procedure involves replacing a patient’s diseased aortic valve with their own pulmonary valve. The most common failure mode is dilatation of the autograft. Various strategies to reinforce the autograft have been proposed. Personalized external aortic root support has been shown to be effective in stabilizing the aortic root in Marfan patients. In this study, the use of a similar external mesh to support a pulmonary artery autograft was evaluated. METHODS The pulmonary artery was translocated as an interposition autograft in the descending thoracic aortas of 10 sheep. The autograft was reinforced with a polyethylene terephthalate mesh (n = 7) or left unreinforced (n = 3). After 6 months, a computed tomography scan was taken, and the descending aorta was excised and histologically examined using the haematoxylin–eosin and Elastica van Gieson stains. RESULTS The autograft/aortic diameter ratio was 1.59 in the unreinforced group but much less in the reinforced group (1.11) (P < 0.05). A fibrotic sheet, variable in thickness and containing fibroblasts, neovessels and foreign body giant cells, was incorporated in the mesh. Histological examination of the reinforced autograft and the adjacent aorta revealed thinning of the vessel wall due to atrophy of the smooth muscle cells. Potential spaces between the vessel wall and the mesh were filled with oedema. CONCLUSIONS Reinforcing an interposition pulmonary autograft in the descending aorta with a macroporous mesh showed promising results in limiting autograft dilatation in this sheep model. Histological evaluation revealed atrophy of the smooth muscle cell and consequently thinning of the vessel wall within the mesh support

Biomechanical evaluation of a personalized external aortic root support applied in the Ross procedure

A commonly heard concern in the Ross procedure, where a diseased aortic valve is replaced by the patient's own pulmonary valve, is the possibility of pulmonary autograft dilatation. We performed a biomechanical investigation of the use of a personalized external aortic root support or exostent as a possibility for supporting the autograft. In ten sheep a short length of pulmonary artery was interposed in the descending aorta, serving as a simplified version of the Ross procedure. In seven of these cases, the autograft was supported by an external mesh or so-called exostent. Three sheep served as control, of which one was excluded from the mechanical testing. The sheep were sacrificed six months after the procedure. Samples of the relevant tissues were obtained for subsequent mechanical testing: normal aorta, normal pulmonary artery, aorta with exostent, pulmonary artery with exostent, and pulmonary artery in aortic position for six months. After mechanical testing, the material parameters of the Gasser-Ogden-Holzapfel model were determined for the different tissue types. Stress-strain curves of the different tissue types show significantly different mechanical behavior. At baseline, stress-strain curves of the pulmonary artery are lower than aortic stress-strain curves, but at the strain levels at which the collagen fibers are recruited, the pulmonary artery behaves stiffer than the aorta. After being in aortic position for six months, the pulmonary artery tends towards aorta-like behavior, indicating that growth and remodeling processes have taken place. When adding an exostent around the pulmonary autograft, the mechanical behavior of the composite artery (exostent + artery) differs from the artery alone, the non-linearity being more evident in the former

Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo− ) DNA polymerase

In our previous communication we reported the enzymatic recognition of unnatural imidazopyridopyrimidine:naphthyridine (Im:Na) base pairs, i.e. ImON:NaNO and ImNO:NaON, using the Klenow fragment exo− [KF (exo−)]. We describe herein the successful results of (i) improved enzymatic recognition for ImNO:NaON base pairs and (ii) further primer extension reactions after the Im:Na base pairs by Deep Vent DNA polymerase exo− [Deep Vent (exo−)]. Since KF (exo−) did not catalyze primer extension reactions after the Im:Na base pair, we carried out a screening of DNA polymerases to promote the primer extension reaction as well as to improve the selectivity of base pair recognition. As a result, a family B DNA polymerase, especially Deep Vent (exo−), seemed most promising for this purpose. In the ImON:NaNO base pair, incorporation of NaNOTP against ImON in the template was preferable to that of the natural dNTPs, while incorporation of dATP as well as dGTP competed with that of ImONTP when NaNO was placed in the template. Thus, the selectivity of base pair recognition by Deep Vent (exo−) was less than that by KF (exo−) in the case of the ImON:NaNO base pair. On the other hand, incorporation of NaONTP against ImNO in the template and that of ImNOTP against NaON were both quite selective. Thus, the selectivity of base pair recognition was improved by Deep Vent (exo−) in the ImNO:NaON base pair. Moreover, this enzyme catalyzed further primer extension reactions after the ImNO:NaON base pair to afford a faithful replicate, which was confirmed by MALDI-TOF mass spectrometry as well as the kinetics data for extension fidelity next to the ImNO:NaON base pair. The results presented in this paper revealed that the ImNO:NaON base pair might be a third base pair beyond the Watson–Crick base pairs

Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and l-α-threofuranosyl ring systems: interactions with P2Y receptors

The ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of the P2Y1 receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y2 and P2Y4 receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) L-α-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y1 receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a Ki value of 22.5nM in binding to the human P2Y1 receptor, and antagonized the stimulation of PLC by the potent P2Y1 receptor agonist 2-methylthio-ADP (30nM) with an IC50 of 650nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A3). Thus, this ring system afforded some P2Y receptor selectivity. A L-α-threofuranosyl bisphosphate derivative 9 displayed an IC50 of 15.3μM for inhibition of 2-methylthio-ADP-stimulated PLC activity. L-α-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y2 (EC50 = 9.9μM) versus P2Y4 receptors. The P2Y1 receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking

Xenobiology: A new form of life as the ultimate biosafety tool

Synthetic biologists try to engineer useful biological systems that do not exist in nature. One of their goals is to design an orthogonal chromosome different from DNA and RNA, termed XNA for xeno nucleic acids. XNA exhibits a variety of structural chemical changes relative to its natural counterparts. These changes make this novel information-storing biopolymer “invisible” to natural biological systems. The lack of cognition to the natural world, however, is seen as an opportunity to implement a genetic firewall that impedes exchange of genetic information with the natural world, which means it could be the ultimate biosafety tool. Here I discuss, why it is necessary to go ahead designing xenobiological systems like XNA and its XNA binding proteins; what the biosafety specifications should look like for this genetic enclave; which steps should be carried out to boot up the first XNA life form; and what it means for the society at large

Pyridoxamine Limits Cardiac Dysfunction in a Rat Model of Doxorubicin-Induced Cardiotoxicity.

peer reviewedThe use of doxorubicin (DOX) chemotherapy is restricted due to dose-dependent cardiotoxicity. Pyridoxamine (PM) is a vitamin B6 derivative with favorable effects on diverse cardiovascular diseases, suggesting a cardioprotective effect on DOX-induced cardiotoxicity. The cardioprotective nature of PM was investigated in a rat model of DOX-induced cardiotoxicity. Six-week-old female Sprague Dawley rats were treated intravenously with 2 mg/kg DOX or saline (CTRL) weekly for eight weeks. Two other groups received PM via the drinking water next to DOX (DOX+PM) or saline (CTRL+PM). Echocardiography, strain analysis, and hemodynamic measurements were performed to evaluate cardiac function. Fibrotic remodeling, myocardial inflammation, oxidative stress, apoptosis, and ferroptosis were evaluated by various in vitro techniques. PM significantly attenuated DOX-induced left ventricular (LV) dilated cardiomyopathy and limited TGF-β1-related LV fibrotic remodeling and macrophage-driven myocardial inflammation. PM protected against DOX-induced ferroptosis, as evidenced by restored DOX-induced disturbance of redox balance, improved cytosolic and mitochondrial iron regulation, and reduced mitochondrial damage at the gene level. In conclusion, PM attenuated the development of cardiac damage after DOX treatment by reducing myocardial fibrosis, inflammation, and mitochondrial damage and by restoring redox and iron regulation at the gene level, suggesting that PM may be a novel cardioprotective strategy for DOX-induced cardiomyopathy

α,β-D-Constrained Nucleic Acids Are Strong Terminators of Thermostable DNA Polymerases in Polymerase Chain Reaction

(SC5′, RP) α,β-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5′C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases