Dynamic Passivation with BSA Overcomes LTCC Mediated Inhibition of PCR

The increasing use of low temperature co-fired ceramic (LTCC) for the fabrication of biological microfluidic devices necessitates further research on LTCC biocompatibility. In this study we explore the inhibitory effect of DuPont\u27s 951 LTCC on Polymerase Chain Reaction (PCR), and demonstrate a novel mechanism to increase biocompatibility between LTCC and PCR with the addition of a common passivation substance, bovine serum albumin (BSA). We show that DuPont\u27s 951 LTCC binds negatively charged proteins including BSA and ovalbumin (OVA). This is a significant discovery as proteins (enzymes) are an essential component of most biological reactions, and a frequent addition to microfluidic devices. A proposed model for LTCC inhibition of PCR by enzyme adsorption is presented

Structure of Arabidopsis thaliana 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog

<p>Abstract</p> <p>Background</p> <p>Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics.</p> <p>Results</p> <p>The structure of <it>Arabidopsis thaliana </it>MTR kinase co-crystallized with ATPγS and MTR has been determined at 1.9 Å resolution. The structure is similar to <it>B. subtilis </it>MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of <it>A. thaliana </it>MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of <it>A. thaliana </it>and <it>B. subtilis </it>MTR kinase adopt different conformations despite high sequence similarity. The ATPγS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the <it>A. thaliana </it>enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR.</p> <p>Conclusion</p> <p>The structure of <it>A. thaliana </it>MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.</p

Preparation and Characterization of Photoactive Antimicrobial Graphitic Carbon Nitride (g-C\u3csub\u3e3\u3c/sub\u3eN\u3csub\u3e4\u3c/sub\u3e) Films

Photoactive films derived from nanostructured samples of the metal-free, intermediate band gap semiconductor graphitic carbon nitride (ns-g-C3N4) have been synthesized and characterized for their particle properties and antimicrobial activity. Physical characterization reveals that these materials are composed of discrete nanoparticles whose dimensions range from 200 nm to 700 nm. Investigation of the photochemical reactivity of ns-g-C3N4 using coumarin-3- carboxylic acid (3-CCA) indicates that this material produces reactive oxygen species (ROS) under visible radiation. When irradiated with 0.31J visible light, ns-g-C3N4-based materials reduced the viability of both gram-negative Escherichia coli O157:H7 and gram-positive Staphylococcus aureus by approximately 50%. Nearly complete inactivation of both strains of microorganisms was achieved upon administration of a 0.62J dose of visible radiation. Importantly, no biocidal activity was observed for non-irradiated samples, indicating that the g-C3N4-derived films are not inherently toxic in the absence of visible light. The results of this study suggest that materials and, by extention, films and coatings derived from g-C3N4 may present a novel route for controlling pathogenic microorganisms on surfaces in the environment, and could be useful in reducing incidents of hospital-acquired infections

Structure of \u3cem\u3eArabidopsis thaliana\u3c/em\u3e 5-Methylthioribose Kinase Reveals a More Occluded Active Site Than its Bacterial Homolog

Background: Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. Results: The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPγS and MTR has been determined at 1.9 Å resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPγS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. Conclusion: The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase

Modification of Cellular DNA by Synthetic Aziridinomitosenes

Two synthetic aziridinomitosenes (AZMs), Me-AZM and H-AZM, structurally related to mitomycin C (MC) were evaluated for their anticancer activity against six cancer cell lines (HeLa, Jurkat, T47D, HepG2, HL-60, and HuT-78) and tested for their DNA-modifying abilities in Jurkat cells. Cytotoxicity assays showed that Me-AZM is up to 72-fold and 520-fold more potent than MC and H-AZM, respectively. Me-AZM also demonstrated increased DNA modification over MC and H-AZM in alkaline COMET and Hoechst fluorescence assays that measured crosslinks in cellular DNA. Me-AZM and H-AZM treatment of Jurkat cells was found to sponsor significant DNA-protein crosslinks using a K-SDS assay. The results clearly indicate that the AZM C6/C7 substitution pattern plays an important role in drug activity and supports both DNA-DNA and DNA-protein adduct formation as mechanisms for inducing cytotoxic effects

1,4,5,8-Naphthalene Tetracarboxylate Dianhydride/g-C\u3csub\u3e3\u3c/sub\u3eN\u3csub\u3e4\u3c/sub\u3e van der Waals Heterojunctions Exhibit Enhanced Photochemical H\u3csub\u3e2\u3c/sub\u3eO\u3csub\u3e2\u3c/sub\u3e Production and Antimicrobial Activity

Organic semiconductors, including graphitic carbon nitride (g-C3N4, CN), represent an important class of materials for the development of novel antimicrobial or biomedical technologies. Of principal interest is the ability of these materials to catalyze the reduction of elemental oxygen to generate reactive oxygen species (ROS), including hydrogen peroxide (H2O2). Here, we describe the fabrication of photoactive van der Waals heterojunctions incorporating 1,4,5,8-naphthalene tetracarboxylic dianhydride (NTCDA) and CN. The composite heterojunction systems were characterized by a combination of physical (TEM, SEM, pXRD), spectroscopic (FT-IR, XPS, DRUV, photoluminescence, TCSPC) and kinetic experiments. Electronic interactions between the two components of the heterojunction increase the rate of photochemical production of H2O2 from elemental oxygen by 410%, relative to samples of pure CN. Mechanistic analysis reveals that interaction of NTCDA with the surface of CN modifies the mechanism of H2O2 formation in the heterojunction photocatalysts. The photochemical production of H2O2 by irradiation of the most active heterojunction composition is sufficient to reduce the viability of E. coli O157:H7, S. aureus and Ps. aeruginosa PAO1 by 99%. Importantly, H2O2 production by the NTCDA/CN heterojunctions suppresses Ps. aeruginosa biofilm formation, even at light exposure doses that had a lesser impact on overall planktonic cell growth

Structural Basis for α-Conotoxin Potency and Selectivity

Parkinson\u27s disease is a debilitating movement disorder characterized by altered levels of α6β2* nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. α-Conotoxin MII (α-CTx MII) is a highly useful ligand to probe α6ß2 nAChRs structure and function, but it does not discriminate among closely related α6* nAChR subtypes. Modification of the α-CTx MII primary sequence led to the identification of α-CTx MII[E11A], an analog with 500-5300 fold discrimination between α6* subtypes found in both human and non-human primates. α-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity α6* nAChR, a subtype that is selectively lost in Parkinson\u27s disease. Here we present the three-dimensional solution structure for α-CTx MII[E11A] as determined by two-dimensional 1H NMR spectroscopy to 0.13 +/- 0.09 Ǻ backbone and 0.45 +/- 0.08 Ǻ heavy atom root mean square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of α-CTx MII[E11A] relative to other members of the α-CTx family may be responsible for its exceptionally high affinity for α6α4β2* nAChR as well as discrimination between α6ß2 and α3β2 containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for α6* nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson\u27s disease

Whole-Exome sequencing analysis identified TMSB10/TRABD2A locus to be associated with carfilzomib-related cardiotoxicity among patients with multiple myeloma

BackgroundProteasome inhibitor Carfilzomib (CFZ) is effective in treating patients with refractory or relapsed multiple myeloma (MM) but has been associated with cardiovascular adverse events (CVAE) such as hypertension, cardiomyopathy, and heart failure. This study aimed to investigate the contribution of germline genetic variants in protein-coding genes in CFZ-CVAE among MM patients using whole-exome sequencing (WES) analysis.MethodsExome-wide single-variant association analysis, gene-based analysis, and rare variant analyses were performed on 603,920 variants in 247 patients with MM who have been treated with CFZ and enrolled in the Oncology Research Information Exchange Network (ORIEN) at the Moffitt Cancer Center. Separate analyses were performed in European Americans and African Americans followed by a trans-ethnic meta-analysis.ResultsThe most significant variant in the exome-wide single variant analysis was a missense variant rs7148 in the thymosin beta-10/TraB Domain Containing 2A (TMSB10/TRABD2A) locus. The effect allele of rs7148 was associated with a higher risk of CVAE [odds ratio (OR) = 9.3 with a 95% confidence interval of 3.9—22.3, p = 5.42*10−7]. MM patients with rs7148 AG or AA genotype had a higher risk of CVAE (50%) than those with GG genotype (10%). rs7148 is an expression quantitative trait locus (eQTL) for TRABD2A and TMSB10. The gene-based analysis also showed TRABD2A as the most significant gene associated with CFZ-CVAE (p = 1.06*10−6).ConclusionsWe identified a missense SNP rs7148 in the TMSB10/TRABD2A as associated with CFZ-CVAE in MM patients. More investigation is needed to understand the underlying mechanisms of these associations