Molecular Dynamics Simulation to Understand the Ability of Anionic Polymers to Alter the Morphology of Calcite

Molecular dynamics was utilized to investigate the ability of anionic macromolecules to drastically change the morphology of calcite in the presence of magnesium ions. Anionic poly(acrylic acid) and poly(methacrylic acid) were compared with cationic poly(ethylene imine) in their binding behavior on calcite (104) and (110) surfaces. Poly(acrylic acid) and poly(methacrylic acid) showed preferential binding on (110) with strong electrostatic attractions, whereas poly(ethylene imine) was only weakly attracted to (104). The extent of the charge imbalance on the surfaces appeared responsible for the current results, which originated from the deficient number of the coordinating oxygen atoms of carbonate around the surface calcium. The results of the current study were in accordance with the previous experimental observations, where the {hk0} surfaces of calcite were elongated under the coexistence of the anionic polymers and magnesium ions. These results could be generally utilized in the polymer-controlled crystallization with broad implications in the specific interactions with crystal surfaces

Autopsy Brain Removal Training Using Virtual Reality Simulation

Hospital autopsy is important for diagnosing neurodegenerative disease in deceased patients. Despite its importance, training autopsy brain removal is challenging for autopsy assistants due to lack of availability of real specimens, initial hesitation to perform the procedure because of proximity to the face, and limited teaching tools. To address these deficits, a virtual reality (VR) simulation was created to teach proper methods to perform the steps of the procedure. This simulation features real-time visual feedback of user performance during the step of opening the cranium with an oscillating saw in order to assist in skill improvement. It also provides an immersive VR interactive experience using realistic virtual patient models, sound effects, and haptic responses

Melting Diagrams of Adefovir Dipivoxil and Dicarboxylic Acids: An Approach to Assess Cocrystal Compositions

Pharmaceutical cocrystallization is a useful method to regulate the physical properties of active pharmaceutical ingredients (APIs). Since the cocrystals may form in various API/coformer ratios, identification of the cocrystal composition is the critical first step of any further analysis. However, the composition identification is not always unambiguous if cocrystallization is performed in solid state with unsuccessful solution crystallization. Single melting point and some new X-ray diffraction peaks are necessary but not sufficient conditions. In the present study, the use of melting diagrams coupled with the X-ray diffraction data was tested to identify cocrystal compositions. Adefovir dipivoxil (AD) was used as a model API, and succinic acid (SUC), suberic acid (SUB), and glutaric acid (GLU) were coformers. Compositions of AD/SUC and AD/SUB had been previously identified as 2:1 and 1:1, but that of AD/GLU was not unambiguously identified because of the difficulty of solution crystallization. Melting diagrams were constructed with differential scanning calorimetry, and their interpretation was assisted by powder X-ray diffraction. The cocrystal formation was exhibited as new compositions with congruent melting in the phase diagrams. This method correctly indicated the previously known cocrystal compositions of AD/SUC and AD/SUB, and it successfully identified the AD/GLU cocrystal composition as 1:1. The current approach is a simple and useful method to assess the cocrystal compositions when the crystallization is only possible in solid state

Ball Milling to Build the Hybrid Mesocrystals of Ibuprofen and Aragonite

Mesocrystal formation is one of the new paradigms of the nonclassical crystallization, where the assembly of crystal domains is observed. Also, it has been recently employed in studies on drug formulation to utilize controlled dissolution of the drug domains. In this report, ibuprofen was attempted to form hybrid mesocrystals with calcium carbonate crystals. Two polymorphs of calcium carbonate (aragonite and calcite) were used during the solid-state process of ball milling. Structural analyses confirmed the mesocrystal formation of ibuprofen with aragonite but not with calcite. The origin of the observed behavior was found from the higher affinity of ibuprofen to aragonite, especially its (0 1 0) surface, compared to calcite. The hybrid mesocrystals of ibuprofen and aragonite showed the environment-responsive release behavior, where the stability of aragonite was the controlling factor for the release kinetics of ibuprofen

Effects of <i>N</i>²‑Alkylguanine, <i>O</i>⁶‑Alkylguanine, and Abasic Lesions on DNA Binding and Bypass Synthesis by the Euryarchaeal B‑Family DNA Polymerase Vent (exo^–)

Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo^–) from the euryarchaeon <i>Thermococcus litoralis</i> was studied with three types of DNA lesions<i>N</i>²-alkylG, <i>O</i>⁶-alkylG, and an abasic (AP) sitein comparison with a model Y-family pol Dpo4 from <i>Sulfolobus solfataricus</i>, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo^–) readily bypassed <i>N</i>²-methyl­(Me)­G and <i>O</i>⁶-MeG, but was strongly blocked at <i>O</i>⁶-benzyl­(Bz)­G and <i>N</i>²-BzG, whereas Dpo4 efficiently bypassed <i>N</i>²-MeG and <i>N</i>²-BzG and partially bypassed <i>O</i>⁶-MeG and <i>O</i>⁶-BzG. Vent (exo^–) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo^–) showed ∼110-, 180-, and 300-fold decreases in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) for nucleotide insertion opposite an AP site, <i>N</i>²-MeG, and <i>O</i>⁶-MeG but ∼1800- and 5000-fold decreases opposite <i>O</i>⁶-BzG and <i>N</i>²-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ∼13-fold decreases opposite <i>N</i>²-MeG and <i>N</i>²-BzG but ∼260–370-fold decreases opposite <i>O</i>⁶-MeG, <i>O</i>⁶-BzG, and the AP site. Vent (exo^–) preferentially misinserted G opposite <i>N</i>²-MeG, T opposite <i>O</i>⁶-MeG, and A opposite an AP site and <i>N</i>²-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo^–) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo^–) is as or more efficient as Dpo4 in synthesis opposite <i>O</i>⁶-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) <i>N</i>²-alkylGs than Vent (exo^–), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo^–) accepts nonbulky DNA lesions (e.g., <i>N</i>²- or <i>O</i>⁶-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove <i>N</i>²-alkylG lesions over major-groove or noninstructive lesions

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase kappa

DNA polymerase (pol) κ efficiently catalyzes error-free translesion DNA synthesis (TLS) opposite bulky N2-guanyl lesions induced by carcinogens such as polycyclic aromatic hydrocarbons. We investigated the biochemical effects of nine human nonsynonymous germline POLK variations on the TLS properties of pol κ, utilizing recombinant pol κ (residues 1-526) enzymes and DNA templates containing an N2-CH2(9-anthracenyl)G (N2-AnthG), 8-oxo-7,8-dihydroguanine (8-oxoG), O6-methyl(Me)G, or an abasic site. In steady-state kinetic analyses, the R246X, R298H, T473A, and R512W variants displayed 7- to 18-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to that of the wild-type, and further showed 5- to 190-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Km for correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to that of the wild-type. These five hypoactive variants also showed similar patterns of attenuation of TLS activity opposite 8-oxoG, O6-MeG, and abasic lesions. By contrast, the T44M variant exhibited 7- to 11-fold decreases in kcat/Km for dCTP insertion opposite N2-AnthG and O6-MeG (as well as for dATP insertion opposite an abasic site) but not opposite both G and 8-oxoG, nor beyond N2-AnthG, compared to that of the wild-type. These results suggest that the R246X, R298H, T473A, R512W, and A471V variants cause a general catalytic impairment of pol κ opposite G and all four lesions, whereas the T44M variant induces opposite lesion-dependent catalytic impairment, i.e., only opposite O6-MeG, abasic, and bulky N2-G lesions but not opposite G and 8-oxoG, in pol κ, which might indicate that these hypoactive pol κ variants are genetic factors in modifying individual susceptibility to genotoxic carcinogens in certain subsets of populations. © 2016 American Chemical Society.

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²‑Guanyl DNA Adducts

DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i>²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i>²-CH₂(9-anthracenyl)­G (<i>N</i>²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i>_cat/<i>K</i>_m for dCTP insertion opposite G and <i>N</i>²-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i>_cat/<i>K</i>_m for next-base extension from C paired with <i>N</i>²-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i>²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens