Developing Somatic Hypermutation as a Protein Engineering Tool to Study Angiopoietin Binding

The angiopoietin (Ang) and Tie families play an important role in the latter stages of vascular development and in adult vasculature. A variety of studies on Ang1 ligand have provided compelling evidence of its therapeutic potential and along with Ang2 plays a major role in various protective and pathological conditions. Understanding the Ang-Tie interaction by manipulation of the angiopoietins is a very desirable prospect for developing it as a protein therapeutic drug. The aim of this project was to examine the molecular basis of Ang binding using a combination of rational mutagenesis and directed evolution. Directed evolution is a powerful strategy for protein engineering. A new method of directed evolution using mammalian surface display combined with mutagenesis driven by somatic hypermutation (SHM) was investigated for engineering Ang proteins with altered binding characteristics (to Tie1 and Tie2). The Ang receptor binding domains (RBD) were cloned linked to the asialoglycoprotein receptor (ASGPR) transmembrane domain. The fusion protein produced was capable of expression on the extracellular cell surface. Activation-induced (cytidine) deaminase (AID) expressing B cells were used for expression of this fusion protein. SHM driven by AID is capable of mutating highly expressed transgenes; this was used to generate the mutant library. The library was screened for enhanced Tie2 binding affinity and acquired Tie1 binding function by Fluorescence Activated Cell Sorting (FACS). Unstable expression of AID in the cell line and non-specific binding of Tie1 to the cell surface proteins in the binding screen prevented efficient selection of desired mutants. However, Ang2 FReD (fibrinogen-related domain) mutants were generated by SHM and rational mutagenesis. These were analysed and compared to the wild-type Ang2 FReD protein for expression by Western blotting and binding affinity determined by flow cytometry

DataSheet1.xlsx

<p>Staphylococcus epidermidis is a prominent commensal member of human skin microbiome and an emerging nosocomial pathogen, making it a good model organism to provide genomic insights, correlating its transition between commensalism and pathogenicity. While there are numerous studies to understand differences in commensal and pathogenic isolates, systematic efforts to understand variation and evolutionary pattern in multiple strains isolated from healthy individuals are lacking. In the present study, using whole genome sequencing and analysis, we report presence of diverse lineages of S. epidermidis isolates in healthy individuals from two geographically diverse locations of India and North America. Further, there is distinct pattern in the distribution of candidate gene(s) for pathogenicity and commensalism. The pattern is not only reflected in lineages but is also based on geographic origin of the isolates. This is evident by the fact that North American isolates under this study are more genomically dynamic and harbor pathogenicity markers in higher frequency. On the other hand, isolates of Indian origin are less genomically dynamic, harbor less pathogenicity marker genes and possess two unique antimicrobial peptide gene clusters. This study provides a basis to understand the nature of selection pressure in a key human skin commensal bacterium with implications in its management as an opportunistic pathogen.</p

Competitive Pseudopericyclic [3,3]- and [3,5]-Sigmatropic Rearrangements of Trichloroacetimidates

The Woodward–Hoffmann rules predict whether concerted pericyclic reactions are allowed or forbidden based on the number of electrons involved and whether the cyclic orbital overlap involves suprafacial or antarafacial orbital overlap. Pseudopericyclic reactions constitute a third class of reactions in which orthogonal orbitals make them orbital symmetry allowed, regardless of the number of electrons involved in the reaction. Based on the recent report of eight-centered ester rearrangements, it is predicted that the isoelectronic eight-centered rearrangements of imidates would also be allowed. We now report that these rearrangements occur, and indeed, an eight-centered rearrangement is slightly favored in at least one case over the well-known six-centered Overman rearrangements, in a trichloroacetimidoylcyclohexadienone, a molecular system where both rearrangements are possible

Glycolamide-functionalized ionic liquid: Synthesis and actinide ion extraction studies

<p>A glycolamide-functionalized ionic liquid (G-FIL) was synthesized for the first time and was evaluated for the extraction of actinide ions such as Am³⁺, Pu⁴⁺ and UO₂²⁺ and fission product element ions such as Eu³⁺, Sr²⁺ and Cs⁺. The extraction of the trivalent metal ions was found to be exceptionally high at low acid concentrations, which rapidly decreased with increasing acidity. In view of the high viscosity of the G-FIL, the studies were carried out using its diluted solution in a commercial ionic liquid, <i>viz</i>. 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C₄mim][Tf₂N]).</p

Competitive Pseudopericyclic [3,3]- and [3,5]-Sigmatropic Rearrangements of Trichloroacetimidates

The Woodward–Hoffmann rules predict whether concerted pericyclic reactions are allowed or forbidden based on the number of electrons involved and whether the cyclic orbital overlap involves suprafacial or antarafacial orbital overlap. Pseudopericyclic reactions constitute a third class of reactions in which orthogonal orbitals make them orbital symmetry allowed, regardless of the number of electrons involved in the reaction. Based on the recent report of eight-centered ester rearrangements, it is predicted that the isoelectronic eight-centered rearrangements of imidates would also be allowed. We now report that these rearrangements occur, and indeed, an eight-centered rearrangement is slightly favored in at least one case over the well-known six-centered Overman rearrangements, in a trichloroacetimidoylcyclohexadienone, a molecular system where both rearrangements are possible

Experimental and Computational Studies on the [3,3]- and [3,5]-Sigmatropic Rearrangements of Acetoxycyclohexadienones: A Non-ionic Mechanism for Acyl Migration

Flash vacuum pyrolysis studies of substituted 6-acetoxy-2,4-cyclohexadienones (<b>3</b> and <b>10</b>) from 300 to 500 °C provide strong experimental evidence that direct [3,5]-sigmatropic rearrangements in these molecules are favored over the more familiar [3,3]-sigmatropic rearrangements. The preference holds when the results are extrapolated to 0.0% conversion, indicating that this is a concerted process. Pyrolysis of 6,6-diacetoxy-2-methyl-2,4-cyclohexadienone (<b>9</b>) at 350 °C gives a modest yield of the initial [3,5]-sigmatropic rearrangement product, 2,6-diacetoxy-6-methyl-2,4-cyclohexadienone (<b>11</b>). Qualitative arguments and electronic structure theory calculations are in agreement that the lowest energy pathway for each [3,5]-sigmatropic rearrangement is via an allowed, concerted pseudopericyclic transition state. The crystal structures of compounds <b>3</b>, <b>9</b>, and <b>10</b> prefigure these transition states. The selectivity for the [3,5] products increases with an increasing temperature. This unexpected selectivity is explained by a concerted, intramolecular, and pseudopericyclic transition state (<b>TS-5</b>) that forms a tetrahedral interemediate (<i>ortho</i>-acid ester <b>4′</b>), followed by similar ring openings to isomeric phenols, which shifts the equilibrium toward the phenols from the [3,5] (but not the [3,3]) products

High-Performance Recyclable Magnetic CuFe_2–<i>x</i>Cr_<i>x</i>O₄ Nanocatalysts for Facile Reduction of Nitrophenols and Photooxidative Degradation of Organic Dyes

Remediating pollutants in water sources is essential for preserving and protecting our water resources. Water pollution can have severe environmental, economic, and public health consequences. Water scarcity for domestic purposes can eventually be addressed by improving wastewater management. In the present study, highly active and magnetically separable Cr-doped copper ferrites CuFe2–xCrxO4 (x = 0, 0.2, 0.4, 0.6, 0.8, and 1) were synthesized via a sol–gel technique and employed for the conversion of nitrophenols to aminophenols and photooxidative deterioration of various dyes such as methyl orange (MO), rhodamine B (RhB), and methylene blue (MB), as well as their ternary solution (RhB + MO + MB). Powder X-ray diffraction investigation indicates that the prepared phases are well indexed in the Fd3̅ m space group except for CuFe2O4, which crystallizes in the I41/amd space group. The M–H hysteresis loop results support the ferromagnetic behavior of the nanophases, which makes them magnetically separable. The optical-band-gap energies of prepared nanostructures were found to be in the range of 1.70–1.39 eV, which makes them appropriate and reliable photocatalysts. CuFe2O4 was found to be the most efficient nanocatalyst toward the conversion of nitrophenols to aminophenols, while CuCrFeO4 showed a maximum degradation efficiency for the deterioration of dyes. The kinetic models correctly explain both reduction and photodegradation processes using pseudo-first-order kinetics. Further, several recyclability runs demonstrated that the nanocatalysts were extremely stable and reusable. This study reflects the possibility of employing mesoporous nanocatalysts for practical applications in the treatment of wastewater

Experimental and Computational Studies on the [3,3]- and [3,5]-Sigmatropic Rearrangements of Acetoxycyclohexadienones: A Non-ionic Mechanism for Acyl Migration

Flash vacuum pyrolysis studies of substituted 6-acetoxy-2,4-cyclohexadienones (<b>3</b> and <b>10</b>) from 300 to 500 °C provide strong experimental evidence that direct [3,5]-sigmatropic rearrangements in these molecules are favored over the more familiar [3,3]-sigmatropic rearrangements. The preference holds when the results are extrapolated to 0.0% conversion, indicating that this is a concerted process. Pyrolysis of 6,6-diacetoxy-2-methyl-2,4-cyclohexadienone (<b>9</b>) at 350 °C gives a modest yield of the initial [3,5]-sigmatropic rearrangement product, 2,6-diacetoxy-6-methyl-2,4-cyclohexadienone (<b>11</b>). Qualitative arguments and electronic structure theory calculations are in agreement that the lowest energy pathway for each [3,5]-sigmatropic rearrangement is via an allowed, concerted pseudopericyclic transition state. The crystal structures of compounds <b>3</b>, <b>9</b>, and <b>10</b> prefigure these transition states. The selectivity for the [3,5] products increases with an increasing temperature. This unexpected selectivity is explained by a concerted, intramolecular, and pseudopericyclic transition state (<b>TS-5</b>) that forms a tetrahedral interemediate (<i>ortho</i>-acid ester <b>4′</b>), followed by similar ring openings to isomeric phenols, which shifts the equilibrium toward the phenols from the [3,5] (but not the [3,3]) products

The Role of Iron in Oxidant-Initiated Halogenation Reactions of Hydraulic Fracturing Additives

Produced water is the largest waste stream associated with hydraulic fracturing. The presence of unknown dissolved contaminants in produced water prevents its reuse. Many of these contaminants are transformation productscompounds that are generated during the fracturing process by chemical reactions between injected chemicals and reservoir components. Reactions among fracturing fluid additives, reservoir brine, and injected oxidants result in halogenation reactions that generate halogenated contaminants with high expected toxicity. Here, we investigate the role of subsurface iron in facilitating subsurface halogenation reactions. Experiments were performed using a model brine, cinnamaldehyde as a model fracturing fluid additive, two different oxidants used in hydraulic fracturing (ammonium persulfate and sodium hypochlorite), and three different iron species (iron­(II) dichloride, iron­(III) trichloride, and iron­(II) dichloride chelated with citric acid). We observed that iron­(II) dichloride increased iodination reactions in the presence of ammonium persulfate oxidant, while iron­(III) trichloride increased bromination reactions in the presence of sodium hypochlorite oxidant. Although the absence of replicate analyses in this study precludes the ability to assess the reproducibility of the data, we believe that these results can serve as inspiration for future researchers to consider the potential involvement of iron in chemical reactions of fracturing fluid additives

HDX-MS of RshA.

<p>(<b>A</b>) Sequence coverage map for RshA. Solid line denotes the peptic fragments analyzed in the study with total sequence coverage of 88%. (<b>B</b>) ESI-Q-TOF mass spectra for one pepsin digest fragment of RshA (35–57) m/z = 881.084, z = 3, which showed significant difference upon RshA binding. (i) Undeuterated RshA peptide (ii) The isotopic envelop for the same peptide from free RshA following 10min deuteration; (iii) The isotopic envelope for the same peptide from RshA and SigH complex following 10 min deuteration., The isotopic envelope for the same peptide. (<b>C</b>) The protein is shown in magenta. The region in red represents regions showing decreased exchange upon interactions with its partner, SigH.</p