Subsequent Substitutions Research Articles

Early diagenesis and recrystallization of bone

One of the most challenging problems in paleobiology is determining how bone transforms from a living tissue into a fossil. The geologic record is replete with vertebrate fossils preserved from a range of depositional environments, including wetland systems. However, thermodynamic models suggest that bone (modeled as hydroxylapatite) is generally unstable in a range of varying geochemical conditions and should readily dissolve if it does not alter to a more thermodynamically stable phase, such as a fluorine-enriched apatite. Here, we assess diagenesis of alligator bone from fleshed, articulated skeletons buried in wetland soils and from de-fleshed bones in experimental mesocosms with and without microbial colonization. When microbial colonization of bone was inhibited, bioapatite recrystallization to a more stable apatite phase occurred after one month of burial. Ca-Fe-phosphate phases in bone developed after several months to years due to ion substitutions from the protonation of the hydroxyl ion. These rapid changes demonstrate a continuum of structural and bonding transformations to bone that have not been observed previously. When bones were directly in contact with sediment and microbial cells, rapid bioerosion and compositional alteration occurred after one week, but slowed after one month because biofilms reduced exposed surfaces and subsequent bioapatite lattice substitutions. Microbial contributions are likely essential in forming stable apatite phases during early diagenesis and for enabling bone preservation and fossilization.

Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

Many genomes contain families of paralogs--proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD's ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors' response element-binding specificity were far from the proteins' DNA-binding interface and interacted epistatically to change the DBD's function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

What Is the Future of Generics in Transplantation?

Generic immunosuppressive drugs are available in Europe, Canada, and the United States. Between countries, there are large differences in the market penetration of generic drugs in general, and for immunosuppressive drugs in particular. The registration criteria for generic immunosuppressive drugs are often criticized. However, it is unlikely that the criteria for registration of narrow therapeutic index drugs are going to change, and bioequivalence studies, performed in healthy volunteers, will remain the backbone of the registration process. It would be good if the registration authorities would demand that all generic variants of an innovator drug have the same pill appearance to reduce errors and promote drug adherence.To allow for safe substitution, a number of criteria need to be fulfilled. Generic substitution should not be taken out of the hands of the treating physicians. Generic substitution can only be done safely if initiated by the prescriber, and in well-informed and prepared patients. Payers should refrain from forcing pharmacists to dispense generic drugs in patients on maintenance treatment with innovator drug. Instead, together with transplant societies, they should design guidelines on how to implement generic immunosuppressive drugs into clinical practice. Substitutions must be followed by control visits to check if the patient is taking the medication correctly and if drug exposure remains stable. Inadvertent, uncontrolled substitutions from 1 generic to another, initiated outside the scope of the prescriber, must be avoided as they are unsafe. Repetitive subsequent generic substitutions result in minimal additional cost savings and have an inherent risk of medication errors.

Contingency and entrenchment in protein evolution under purifying selection

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.

Gold-Catalyzed Benzylic Azidation of Phthalans and Isochromans and Subsequent FeCl3-Catalyzed Nucleophilic Substitutions.

The benzylic positions of the phthalan and isochroman derivatives (1) as benzene-fused cyclic ethers effectively underwent gold-catalyzed direct azidation using trimethylsilylazide (TMSN3) to give the corresponding 1-azidated products (2) possessing the N,O-acetal partial structure. The azido group of the N,O-acetal behaved as a leaving group in the presence of catalytic iron(III) chloride, and 1-aryl or allyl phthalan and isochroman derivatives were obtained by nucleophilic arylation or allylation, respectively. Meanwhile, a double nucleophilic substitution toward the 1-azidated products (2) occurred at the 1-position using indole derivatives as a nucleophile accompanied by elimination of the azido group and subsequent ring opening of the cyclic ether nucleus produced the bisindolylarylmethane derivatives.

Synthesis and amino acids complexation of tripodal hexasubstituted benzene chiral receptors

The parent 1,3,5-triacetyl-2,4,6-trihydroxybenzene was prepared in up to 91% yield using a one-pot, one step reaction catalyzed by aluminum chloride. Its alkylations with 1,5-dibromopentane generated a symmetric tripodal hexasubstituted benzene precursor in the alternated conformer predicted by a theoretical calculation. Subsequent substitutions and reductions provided the corresponding tris-amine in 59% yield. Aminations of the tripodal precursor with (R)-(+)-1-phenylethylamine obtained a chiral tris-amine ligand in 44% yield. 1H NMR titrations of this ligand with each of three l-amino acid derivatives as guest molecules confirmed the presence of their complexes, in which the complex with alanine derivative displayed the strongest interactions with the ligand. Job plots suggested that all complexes composed of 1:2 ratios of the ligand and these guests. Theoretical calculations additionally revealed the structures and the associated binding parameters of the complexes.

The Sequence Saturation Mutagenesis (SeSaM) method.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method developed to overcome the limitations of existing error-prone PCR (epPCR) protocols. SeSaM is advantageous with respect to (1) elimination of mutagenic "hot spots", (2) increase in frequency of subsequent nucleotide substitutions, (3) control over the mutational bias through the utilization of universal base analogs, and, consequently, (4) the prospect of generating transversion-enriched mutant libraries. These advanced features lead to chemically diverse mutant libraries on the protein level, essentially making SeSaM a complementary technology to transition biased epPCR mutagenesis methods.

Precise Spectroscopic [IR, Raman and NMR] Investigation and Gaussian Hybrid Computational Analysis (UV-Visible, NIR, MEP Maps and Kubo Gap) on L-Valine

In the present methodical study, FT-IR, FT-Raman and NMR spectra of the L-Valine are recorded and the observed vibrational frequencies are assigned. The hybrid computational calculations are carried out by HF and DFT (B3LYP and B3PW91) methods with 6-31+G(d,p) and 6-311++G(d,p) basis sets and the corresponding results are tabulated. The alternation of structure of amino acid due to the subsequent substitutions of CH 3 is investigated. The vibrational sequence pattern of the molecule related to the zwitter ion motion is analyzed. Moreover, 13 C NMR and 1 H NMR are calculated by using the gauge independent atomic orbital (GIAO) method with B3LYP methods and the 6-311++G(d,p) basis set and their spectra are simulated and the chemical shifts related to TMS are compared. A study on the electronic and optical properties; absorption wavelengths, excitation energy, dipole moment and frontier molecular orbital energies, are performed by HF and DFT methods. The calculated HOMO and LUMO energies and the kubo gap analysis show that the occurring of charge transformation within the molecule. Besides frontier molecular orbitals (FMO), molecular electrostatic potential (MEP) was performed. NLO properties related to Polarizability and hyperpolarizability is also discussed.

Spectroscopic [IR and Raman] Analysis and Gaussian Hybrid Computational Investigation- NMR, UV-Visible, MEP Maps and Kubo Gap on 2,4,6-Nitrophenol

In the present methodical study, FT-IR and FT-Raman of the 2,4,6-Nitrophenol (TNP) called as picric acid are recorded and the observed vibrational frequencies are assigned. The hybrid computational calculations are carried out by HF and DFT (B3LYP and B3PW91) methods with 6-31+G(d,p) and 6-311++G(d,p) basis sets and the corresponding results are tabulated. The alternation of structure of nitro phenol due to the subsequent substitutions of NO2 is investigated. The vibrational sequence pattern of the molecule related to the substitutions is analyzed. Moreover, 13C NMR and 1H NMR are calculated by using the gauge independent atomic orbital (GIAO) method with B3LYP methods and the 6-311++G(d,p) basis set and their spectra are simulated and the chemical shifts related to TMS are compared. A study on the electronic properties; absorption wavelengths, excitation energy, dipole moment and frontier molecular orbital energies, are performed by HF and DFT methods. The calculated HOMO and LUMO energies and the kubo gap analysis show that the occurring of charge transformation within the molecule. Besides frontier molecular orbitals (FMO), molecular electrostatic potential (MEP) was performed. NLO properties related to Polarizability and hyperpolarizability are also discussed. The thermodynamic properties (thermal energy, heat capacity and entropy) of the title compound are calculated in gas phase and are interpreted with different types of phenols.

Engineering a model protein cavity to catalyze the Kemp elimination

Synthetic cavitands and protein cavities have been widely studied as models for ligand recognition. Here we investigate the Met102→His substitution in the artificial L99A cavity in T4 lysozyme as a Kemp eliminase. The resulting enzyme had k(cat)/K(M)=0.43M(-1)s(-1) and a (k(cat)/K(M))/k(uncat)=10(7) at pH5.0. The crystal structure of this enzyme was determined at 1.30Å, as were the structures of four complexes of substrate and product analogs. The absence of ordered waters or hydrogen bonding interactions, and the presence of a common catalytic base (His102) in an otherwise hydrophobic, buried cavity, facilitated detailed analysis of the reaction mechanism and its optimization. Subsequent substitutions increased eliminase activity by an additional four-fold. As activity-enhancing substitutions were engineered into the cavity, protein stability decreased, consistent with the stability-function trade-off hypothesis. This and related model cavities may provide templates for studying protein design principles in radically simplified environments.

Enhanced High-Temperature Thermoelectric Performance of Yb14–xCaxMnSb11

The high temperature p-type thermoelectric material Yb(14)MnSb(11) has been of increasing research interest since its high temperature thermoelectric properties were first measured in 2006. Subsequent substitutions of Zn, Al, and La into the structure have shown that this material can be further optimized by altering the carrier concentration or by reduction of spin-disorder scattering. Here the properties of the Yb(14-x)Ca(x)MnSb(11) solid solution series where isovalent Ca(2+) is substituted for Yb(2+) will be presented. Crystals of the Yb(14-x)Ca(x)MnSb(11) solid solution series were made by Sn-flux (x = 2, 4, 6, 8) with the following ratio of elements: (14-x)Yb: xCa: 6 Mn: 11Sb: 86Sn, and their structures determined by single crystal X-ray diffraction. The density of the material significantly decreases by over 2 g/cm(3) as more Ca is added (from x = 1 to 8), because of the lighter mass of Ca. The resulting lower density is beneficial from a device manufacturing perspective where there is often a trade-off with the specific power per kilogram. The compounds crystallize in the Ca(14)AlSb(11) structure type. The Ca substitution contributes to systematic lengthening the Mn-Sb bond while shortening the Sb-Sb bond in the 3 atom linear unit with increasing amounts of Ca. Temperature dependent thermoelectric properties, Seebeck, electrical resistivity, and thermal conductivity were measured from room temperature to 1273 K. Substitution of Yb with Ca improves the Seebeck coefficient while decreasing the thermal conductivity, along with decreasing the carrier concentration in this p-type material resulting in an enhanced thermoelectric figure of merit, zT, compared to Yb(14)MnSb(11).

S gene mutations of HBV in children with HBV-associated glomerulonephritis

BackgroundHepatitis B virus-associated glomerulonephritis (HBV-GN) is a kind of immune complex-induced glomerulonephritis. The present study was designed to determine whether mutation of Hepatitis B virus (HBV) S gene is associated with glomerulonephritis in Chinese children.MethodsTotal 53 subjects, including 30 HBV-GN, 5 nephrosis with HBV carriers (control group 1), and 18 HBV carriers (control group 2) were included in this study. Polymerase chain reaction (PCR) was used to detect the HBV-GN S gene mutation.Results(1) The serotype of HBV was adw in the majority (52/53) of subjects, and was adr in only 1 subject in the control group 2; (2) the genotype of HBV was the type B in 51 subjects, the type E in 1 HBV-GN child, and the type C in 1 HBV carrier; (3) Seventeen point mutations in the S gene of HBV were identified in 21 of 30 (70%) HBV-GN patients. Among them, 16 of 21 (76.2%) mutations may cause amino acid substitutions of the HBV proteins, which occur predominantly (11/16 mutations) at threonine, serine or tyrosine phosphorylation sites of mitogen-activated protein kinase (MAPK) or protein tyrosine kinase (PTK). (4) In addition, single nucleotide mutations without amino acid substitutions (same sense mutation) were found in 2 subjects in each control group and 5 subjects in HBV-GN group.ConclusionsHBV S gene mutations and the subsequent amino acid substitutions in HBV proteins were found in most children with HBV-GN, suggesting that these mutations may play an important role in the pathogenesis of HBV-GN.

European Society for Organ Transplantation Advisory Committee Recommendations on Generic Substitution of Immunosuppressive Drugs

European Society for Organ Transplantation Advisory Committee Recommendations on Generic Substitution of Immunosuppressive Drugs

A Human-Specific De Novo Protein-Coding Gene Associated with Human Brain Functions

To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203). Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

Divergent evolution in the cytoplasmic domains of PRLR and GHR genes in Artiodactyla

BackgroundProlactin receptor (PRLR) and growth hormone receptor (GHR) belong to the large superfamily of class 1 cytokine receptors. Both of them have been identified as candidate genes affecting key quantitative traits, like growth and reproduction in livestock. We have previously studied the molecular anatomy of the cytoplasmic domain of GHR in different cattle breeds and artiodactyl species. In this study we have analysed the corresponding cytoplasmic signalling region of PRLR.ResultsWe sequenced PRLR gene exon 10, coding for the major part of the cytoplasmic domain, from cattle, American bison, European bison, yak, sheep, pig and wild boar individuals. We found different patterns of variation in the two receptors within and between ruminants and pigs. Pigs and bison species have no variation within GHR exon 10, but show high haplotype diversity for the PRLR exon 10. In cattle, PRLR shows lower diversity than GHR. The Bovinae PRLR haplotype network fits better the known phylogenetic relationships between the species than that of the GHR, where differences within cattle breeds are larger than between the different species in the subfamily. By comparison with the wild boar haplotypes, a high number of subsequent nonsynonymous substitutions seem to have accumulated in the pig PRLR exon 10 after domestication.ConclusionBoth genes affect a multitude of traits that have been targets of selection after domestication. The genes seem to have responded differently to different selection pressures imposed by human artificial selection. The results suggest possible effects of selective sweeps in GHR before domestication in the pig lineage or species divergence in the Bison lineage. The PRLR results may be explained by strong directional selection in pigs or functional switching.

Regioselectivity of fluorine substitution by alkoxides on unsymmetrical difluoroarenes

An efficient approach to unsymmetrical halogenated resorcinol diethers has been developed. This synthesis consists of two subsequent nucleophilic aromatic substitutions (S NAr) of unsymmetrical difluoroarenes by alkoxides. The novelty of this approach is its control of regioselectivity during the first S NAr, which occurs at room temperature. Interestingly, the reactivity of competing fluorines was correlated to their chemical shift in 19F NMR.

A Diels–Alder approach to biaryls (DAB): synthesis of the western portion of TMC-95

The rapid synthesis of the western biaryl portion of TMC-95 is disclosed via the use of a Diels–Alder reaction with o-nitrostyrene derivative and 1-silyloxy diene with excellent regiochemical control. Subsequent sequential substitutions of a p-iodo-phenol derivative followed by an o-bromo-nitrobenzene intermediate are employed to incorporate the western carbon framework of TMC-95.

Study of the Replacement of Weak Ligands on Square-Planar Organometallic Nickel(II) Complexes. Organo-Nickel Aquacomplexes

When trans-[NiRf2L2] (Rf = 3,5-C6Cl2F3; L = group 15 soft monodentate weak ligand such as SbPh3 or AsPh3) is dissolved in wet (CD3)2CO, isomerization (to give cis-[NiRf2L2]) and subsequent substitutions of L by (CD3)2CO or by water occur, and several complexes containing acetone and aqua ligands are formed. The isomerization takes place in a few seconds at room temperature. The substitution reactions on the cis isomer formed are faster. The kinetics of the equilibria between all of the participating species have been studied by 19F exchange spectroscopy experiments at 217 K, and the exchange rates and rate constants have been calculated. These data reflect the weakness of acetone compared to water and AsPh3. The data obtained are the first available for square-planar nickel(II) aquacomplexes. The bulkier AsCyPh2 ligand slows down the exchange processes while the displacement of AsMePh2 is clearly disfavored. Activation entropy studies support an associative ligand substitution. All of these data fit well with the previously reported relative activity of these complexes as catalysts in norbornene polymerization.

Synthesis and gas transport property of polyimide from 2,2′-disubstituted biphenyltetracarboxylic dianhydrides (BPDA)

A series of dianhydride monomers, 2,2′-disubstituted-4,4′,5,5′-biphenyltetracarboxylic dianhydride (substituents = phenoxy, p-methylphenoxy, p- tert-butylphenoxy, nitro, and methoxy) were synthesized by the nitration of an N-methyl protected 3,3′,4,4′-biphenyltetracarboxylic dianhydride (BPDA) and subsequent aromatic nucleophilic substitutions with aroxides (NaOAr) or methoxide. These dianhydrides were polymerized with various aromatic diamines in refluxing m-cresol containing isoquinoline to afford a series of aromatic polyimides. The effects of varying 2,2′-substituents of the dianhydride (BPDA) moiety on the properties of polyimides were investigated. It was found that polyimides from the dianhydrides containing phenoxy, p-methylphenoxy, and p- tert-butylphenoxy side groups possessed excellent solubility and film forming capability whereas polyimides from 2,2′-dinitro-BPDA and 2,2′-dimethoxy-BPDA were less soluble in organic solvent. The soluble polymers formed flexible, tough and transparent films. The films had a tensile strength, elongation at break, and Young’s modulus in the ranges 102–168 MPa, 8–21%, 2.02–2.38 GPa, respectively. The polymer gas permeability coefficients ( P) and ideal selectivities for N 2, O 2, CO 2 and CH 4 were determined for the –OAr substituted polyimides. The oxygen permeability coefficient ( P O2) and permselectivity of oxygen to nitrogen ( P O2/N2) of the films were in the ranges 3.4–11.3 barrer and 3.8–4.6, respectively. The gas permeability typically increased with increasing free volume in the order of tert-butylphenoxy substituted PI > methylphenoxy substituted PI > phenoxy substituted PI.

Detecting recombination in evolving nucleotide sequences

BackgroundGenetic recombination can produce heterogeneous phylogenetic histories within a set of homologous genes. These recombination events can be obscured by subsequent residue substitutions, which consequently complicate their detection. While there are many algorithms for the identification of recombination events, little is known about the effects of subsequent substitutions on the accuracy of available recombination-detection approaches.ResultsWe assessed the effect of subsequent substitutions on the detection of simulated recombination events within sets of four nucleotide sequences under a homogeneous evolutionary model. The amount of subsequent substitutions per site, prior evolutionary history of the sequences, and reciprocality or non-reciprocality of the recombination event all affected the accuracy of the recombination-detecting programs examined. Bayesian phylogenetic-based approaches showed high accuracy in detecting evidence of recombination event and in identifying recombination breakpoints. These approaches were less sensitive to parameter settings than other methods we tested, making them easier to apply to various data sets in a consistent manner.ConclusionPost-recombination substitutions tend to diminish the predictive accuracy of recombination-detecting programs. The best method for detecting recombined regions is not necessarily the most accurate in identifying recombination breakpoints. For difficult detection problems involving highly divergent sequences or large data sets, different types of approach can be run in succession to increase efficiency, and can potentially yield better predictive accuracy than any single method used in isolation.