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Sample records for additional mechanistic insights

  1. Helical-Peptide-Catalyzed Enantioselective Michael Addition Reactions and Their Mechanistic Insights.

    PubMed

    Ueda, Atsushi; Umeno, Tomohiro; Doi, Mitsunobu; Akagawa, Kengo; Kudo, Kazuaki; Tanaka, Masakazu

    2016-08-01

    Helical peptide foldamer catalyzed Michael addition reactions of nitroalkane or dialkyl malonate to α,β-unsaturated ketones are reported along with the mechanistic considerations of the enantio-induction. A wide variety of α,β-unsaturated ketones, including β-aryl, β-alkyl enones, and cyclic enones, were found to be catalyzed by the helical peptide to give Michael adducts with high enantioselectivities (up to 99%). On the basis of X-ray crystallographic analysis and depsipeptide study, the amide protons, N(2)-H and N(3)-H, at the N terminus in the α-helical peptide catalyst were crucial for activating Michael donors, while the N-terminal primary amine activated Michael acceptors through the formation of iminium ion intermediates. PMID:27384597

  2. To be or not to be butterfly: New mechanistic insights in the Aza-Michael asymmetric addition of lithium (R)-N-benzyl-N-(α-methylbenzyl)amide.

    PubMed

    Nieto, Carlos T; Díez, David; Garrido, Narciso M

    2014-09-30

    The asymmetric Aza-Michael addition of homochiral lithium benzylamides to α,β-unsaturated esters represents an extended protocol to obtain enantioenriched β-amino esters. An exhaustive mechanistic revision of the originally proposed mechanism is reported, developing a quantum mechanics/molecular mechanics protocol for the asymmetric Aza-Michael reaction of homochiral lithium benzylamides. Explicit and implicit solvent schemes were considered, together with a proper account of long-range dispersion forces, evaluated through a density functional theory benchmark of different functionals. Theoretical results showed that the diastereoselectivity is mainly controlled by the N-α-methylbenzyl moiety placing, deriving a Si/Re 99:1 diastereoselective ratio, in good agreement with reported experimental results. The main transition state geometries are two transition state conformers in a "V-stacked" orientation of the amide's phenyl rings, differing in the tetrahydrofuran molecule arrangement coordinated to the metal center. Extensive conformational sampling and quantum-level refinement give reasonable good speed/accuracy results, allowing this protocol to be extended to other similar Aza-Michael reaction systems. PMID:25052741

  3. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  4. Somatodendritic dopamine release: recent mechanistic insights.

    PubMed

    Rice, Margaret E; Patel, Jyoti C

    2015-07-01

    Dopamine (DA) is a key transmitter in motor, reward and cogitative pathways, with DA dysfunction implicated in disorders including Parkinson's disease and addiction. Located in midbrain, DA neurons of the substantia nigra pars compacta project via the medial forebrain bundle to the dorsal striatum (caudate putamen), and DA neurons in the adjacent ventral tegmental area project to the ventral striatum (nucleus accumbens) and prefrontal cortex. In addition to classical vesicular release from axons, midbrain DA neurons exhibit DA release from their cell bodies and dendrites. Somatodendritic DA release leads to activation of D2 DA autoreceptors on DA neurons that inhibit their firing via G-protein-coupled inwardly rectifying K(+) channels. This helps determine patterns of DA signalling at distant axonal release sites. Somatodendritically released DA also acts via volume transmission to extrasynaptic receptors that modulate local transmitter release and neuronal activity in the midbrain. Thus, somatodendritic release is a pivotal intrinsic feature of DA neurons that must be well defined in order to fully understand the physiology and pathophysiology of DA pathways. Here, we review recent mechanistic aspects of somatodendritic DA release, with particular emphasis on the Ca(2+) dependence of release and the potential role of exocytotic proteins. PMID:26009764

  5. Mechanistic insights into transient severe mitral regurgitation.

    PubMed

    Liang, Jackson J; Syed, Faisal F; Killu, Ammar M; Boilson, Barry A; Nishimura, Rick A; Pislaru, Sorin V

    2015-09-01

    Acute mitral regurgitation (AMR), a known complication of acute coronary syndromes, is usually associated with posterior papillary muscle dysfunction/rupture. In severe cases, management of AMR requires surgical intervention. Reversible severe AMR in patients in the absence of left ventricular systolic dysfunction and coronary artery stenosis may result from processes which cause transient subendocardial ischemia, such as intermittent episodes of hypotension or coronary artery vasospasm. We present two cases of reversible transient AMR due to subendocardial and/or endocardial ischemia, both of which offer insight into the mechanism of transient severe AMR. PMID:26982531

  6. Mechanistic Insights of Vitamin D Anticancer Effects.

    PubMed

    Ma, Yingyu; Johnson, Candace S; Trump, Donald L

    2016-01-01

    Vitamin D is a secosteroid hormone that regulates many biological functions in addition to its classical role in maintaining calcium homeostasis and bone metabolism. Vitamin D deficiency appears to predispose individuals to increased risk of developing a number of cancers. Compelling epidemiological and experimental evidence supports a role for vitamin D in cancer prevention and treatment in many types of cancers. Preclinical studies show that 1,25D3, the active metabolite of vitamin D, and its analogs have antitumor effects in vitro and in vivo through multiple mechanisms including the induction of cell cycle arrest, apoptosis, differentiation and the suppression of inflammation, angiogenesis, invasion, and metastasis. 1,25D3 also potentiates the effect of chemotherapeutic agents and other agents in the combination treatment. In this review, the antitumor effects of 1,25D3 and the potential underlying mechanisms will be discussed. The current findings support the application of 1,25D3 in cancer prevention and treatment. PMID:26827961

  7. Mechanistic insights into selective autophagy pathways: lessons from yeast.

    PubMed

    Farré, Jean-Claude; Subramani, Suresh

    2016-09-01

    Autophagy has burgeoned rapidly as a field of study because of its evolutionary conservation, the diversity of intracellular cargoes degraded and recycled by this machinery, the mechanisms involved, as well as its physiological relevance to human health and disease. This self-eating process was initially viewed as a non-selective mechanism used by eukaryotic cells to degrade and recycle macromolecules in response to stress; we now know that various cellular constituents, as well as pathogens, can also undergo selective autophagy. In contrast to non-selective autophagy, selective autophagy pathways rely on a plethora of selective autophagy receptors (SARs) that recognize and direct intracellular protein aggregates, organelles and pathogens for specific degradation. Although SARs themselves are not highly conserved, their modes of action and the signalling cascades that activate and regulate them are. Recent yeast studies have provided novel mechanistic insights into selective autophagy pathways, revealing principles of how various cargoes can be marked and targeted for selective degradation. PMID:27381245

  8. Circadian rhythms and addiction: Mechanistic insights and future directions

    PubMed Central

    Logan, Ryan W.; Williams, Wilbur P.; McClung, Colleen A.

    2014-01-01

    Circadian rhythms are prominent in many physiological and behavioral functions. Circadian disruptions either by environmental or molecular perturbation can have profound health consequences, including the development and progression of addiction. Both animal and humans studies indicate extensive bidirectional relationships between the circadian system and drugs of abuse. Addicted individuals display disrupted rhythms, and chronic disruption or particular chronotypes, may increase the risk for substance abuse and relapse. Moreover, polymorphisms in circadian genes and an evening chronotype have been linked to mood and addiction disorders, and recent efforts suggest an association with the function of reward neurocircuitry. Animal studies are beginning to determine how altered circadian gene function results in drug induced neuroplasticity and behaviors. Many studies suggest a critical role for circadian rhythms in reward-related pathways in the brain and indicate that drugs of abuse directly affect the central circadian pacemaker. In this review, we highlight key findings demonstrating the importance of circadian rhythms in addiction, and how future studies will reveal important mechanistic insights into the involvement of circadian rhythms in drug addiction. PMID:24731209

  9. Mechanistic insight into mycobacterial MmpL protein function.

    PubMed

    Székely, R; Cole, S T

    2016-03-01

    Mycobacterial cell walls are complex structures containing a broad range of unusual lipids, glycolipids and other polymers, some of which act as immunomodulators or virulence determinants. Better understanding of the enzymes involved in export processes would enlighten cell wall biogenesis. Bernut et al. () present the findings of a structural and functional investigation of one of the most important transporter families, the MmpL proteins, members of the resistance-nodulation-cell division (RND) superfamily. A Tyr842His missense mutation in the mmpL4a gene was shown to be responsible for the smooth-to-rough morphotype change of the near untreatable opportunistic pathogen Mycobacterium bolletii due to its failure to export a glycopeptidolipid (GPL). This mutation was pleiotropic and markedly increased virulence in infection models. Tyr842 is well conserved in all actinobacterial MmpL proteins suggesting that it is functionally important and this was confirmed by several approaches including replacing the corresponding residue in MmpL3 of Mycobacterium tuberculosis. Structural modelling combined with experimental results showed Tyr842 to be a critical residue for mediating the proton motive force required for GPL export. This mechanistic insight applies to all MmpL proteins and probably to all RND transporters. PMID:26710752

  10. Microneedle/nanoencapsulation-mediated transdermal delivery: Mechanistic insights

    PubMed Central

    Gomaa, Yasmine A.; Garland, Martin J.; McInnes, Fiona J.; Donnelly, Ryan F.; El-Khordagui, Labiba K.; Wilson, Clive G.

    2014-01-01

    A systematic study was undertaken to gain more insight into the mechanism of transdermal delivery of nanoencapsulated model dyes across microneedle (MN)-treated skin, a complex process not yet explored. Rhodamine B (Rh B) and fluorescein isothiocyanate (FITC) as model hydrophilic and hydrophobic small/medium-size molecules, respectively, were encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and delivered through full thickness porcine skin pretreated with MN array. Permeation through MN-treated skin was affected by physicochemical characteristics of NPs and the encapsulated dyes. Dye flux was enhanced by smaller particle size, hydrophilicity, and negative zeta potential of NPs. Regarding encapsulated dyes, solubility at physiological pH and potential interaction with skin proteins proved to outweigh molecular weight as determinants of skin permeation. Data were verified using confocal laser scanning microscopy imaging. Findings coupled with the literature data are supportive of a mechanism involving influx of NPs, particularly of smaller size, deep into MN-created channels, generating depot dye-rich reservoirs. Molecular diffusion of the released dye across viable skin layers proceeds at a rate determined by its molecular characteristics. Data obtained provide mechanistic information of importance to the development of formulation strategies for more effective intradermal and transdermal MN-mediated delivery of nanoencapsulated therapeutic agents. PMID:23461860

  11. Mechanistic insight into gramicidin-based detection of protein-ligand interactions via sensitized photoinactivation

    NASA Astrophysics Data System (ADS)

    Rokitskaya, Tatyana I.; Macrae, Michael X.; Blake, Steven; Egorova, Natalya S.; Kotova, Elena A.; Yang, Jerry; Antonenko, Yuri N.

    2010-11-01

    Among the many challenges for the development of ion channel-based sensors is the poor understanding of how to engineer modified transmembrane pores with tailored functionality that can respond to external stimuli. Here, we use the method of sensitized photoinactivation of gramicidin A (gA) channels in planar bilayer lipid membranes to help elucidate the underlying mechanistic details for changes in macroscopic transmembrane ionic current observed upon interaction of C-terminally attached gA ligands with specific proteins in solution. Three different systems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95 protein (belonging to the 'PDZ domain-containing protein') and a gA analog carrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) an anti-biotin antibody and gA-biotin. The results challenge a previously proposed mechanistic hypothesis suggesting that protein-induced current suppression is due to steric blockage of the ion passage through gA channels, while they reveal new insight for consideration in alternative mechanistic models. Additionally, we demonstrate that the length of a linker between the ligand and the gA channel may be less important for gramicidin-based detection of monovalent compared to multivalent protein-ligand interactions. These studies collectively shed new light on the mechanism of protein-induced current alterations in bilayer recordings of gA derivatives, which may be important in the design of new gramicidin-based sensors.

  12. Mechanistic insight into gramicidin-based detection of protein-ligand interactions via sensitized photoinactivation.

    PubMed

    Rokitskaya, Tatyana I; Macrae, Michael X; Blake, Steven; Egorova, Natalya S; Kotova, Elena A; Yang, Jerry; Antonenko, Yuri N

    2010-11-17

    Among the many challenges for the development of ion channel-based sensors is the poor understanding of how to engineer modified transmembrane pores with tailored functionality that can respond to external stimuli. Here, we use the method of sensitized photoinactivation of gramicidin A (gA) channels in planar bilayer lipid membranes to help elucidate the underlying mechanistic details for changes in macroscopic transmembrane ionic current observed upon interaction of C-terminally attached gA ligands with specific proteins in solution. Three different systems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95 protein (belonging to the 'PDZ domain-containing protein') and a gA analog carrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) an anti-biotin antibody and gA-biotin. The results challenge a previously proposed mechanistic hypothesis suggesting that protein-induced current suppression is due to steric blockage of the ion passage through gA channels, while they reveal new insight for consideration in alternative mechanistic models. Additionally, we demonstrate that the length of a linker between the ligand and the gA channel may be less important for gramicidin-based detection of monovalent compared to multivalent protein-ligand interactions. These studies collectively shed new light on the mechanism of protein-induced current alterations in bilayer recordings of gA derivatives, which may be important in the design of new gramicidin-based sensors. PMID:21339605

  13. Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights

    PubMed Central

    Harrison, Nicholas R.; Laroche, Fabrice J.F.; Gutierrez, Alejandro

    2016-01-01

    Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients. PMID:27165361

  14. Mechanistic Insight in the Function of Phosphite Additives for Protection of LiNi0.5Co0.2Mn0.3O2 Cathode in High Voltage Li-Ion Cells.

    PubMed

    He, Meinan; Su, Chi-Cheung; Peebles, Cameron; Feng, Zhenxing; Connell, Justin G; Liao, Chen; Wang, Yan; Shkrob, Ilya A; Zhang, Zhengcheng

    2016-05-11

    Triethlylphosphite (TEP) and tris(2,2,2-trifluoroethyl) phosphite (TTFP) have been evaluated as electrolyte additives for high-voltage Li-ion battery cells using a Ni-rich layered cathode material LiNi0.5Co0.2Mn0.3O2 (NCM523) and the conventional carbonate electrolyte. The repeated charge/discharge cycling for cells containing 1 wt % of these additives was performed using an NCM523/graphite full cell operated at the voltage window from 3.0-4.6 V. During the initial charge process, these additives decompose on the cathode surface at a lower oxidation potential than the baseline electrolyte. Impedance spectroscopy and post-test analyses indicate the formation of protective coatings by both additives on the cathode surface that prevent oxidative breakdown of the electrolyte. However, only TTFP containing cells demonstrate the improved capacity retention and Coulombic efficiency. For TEP, the protective coating is also formed, but low Li(+) ion mobility through the interphase layer results in inferior performance. These observations are rationalized through the inhibition of electrocatalytic centers present on the cathode surface and the formation of organophosphate deposits isolating the cathode surface from the electrolyte. The difference between the two phosphites clearly originates in the different properties of the resulting phosphate coatings, which may be in Li(+) ion conductivity through such materials. PMID:27090502

  15. Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems

    PubMed Central

    2015-01-01

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75–106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain–domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology. PMID:24779441

  16. Mechanistic insight into sonochemical biodiesel synthesis using heterogeneous base catalyst.

    PubMed

    Choudhury, Hanif A; Chakma, Sankar; Moholkar, Vijayanand S

    2014-01-01

    The beneficial effect of ultrasound on transesterification reaction is well known. Heterogeneous (or solid) catalysts for biodiesel synthesis have merit that they do not contaminate the byproduct of glycerol. In this paper, we have attempted to identify the mechanistic features of ultrasound-enhanced biodiesel synthesis with the base-catalyst of CaO. A statistical design of experiments (Box-Behnken) was used to identify the influence of temperature, alcohol to oil molar ratio and catalyst loading on transesterification yield. The optimum values of these parameters for the highest yield were identified through Response Surface Method (with a quadratic model) and ANOVA. These values are: temperature=62 °C, molar ratio=10:1 and catalyst loading=6 wt.%. The activation energy was determined as 82.3 kJ/mol, which is higher than that for homogeneous catalyzed system (for both acidic and basic catalyst). The experimental results have been analyzed vis-à-vis simulations of cavitation bubble dynamics. Due to 3-phase heterogeneity of the system, the yield was dominated by intrinsic kinetics, and the optimum temperature for the highest yield was close to boiling point of methanol. At this temperature, the influence of cavitation bubbles (in terms of both sonochemical and sonophysical effect) is negligible, and ultrasonic micro-streaming provided necessary convection in the system. The influence of all parameters on the reaction system was found to be strongly inter-dependent. PMID:23742888

  17. Environmental perception and epigenetic memory: mechanistic insight through FLC

    PubMed Central

    Berry, Scott; Dean, Caroline

    2015-01-01

    Chromatin plays a central role in orchestrating gene regulation at the transcriptional level. However, our understanding of how chromatin states are altered in response to environmental and developmental cues, and then maintained epigenetically over many cell divisions, remains poor. The floral repressor gene FLOWERING LOCUS C (FLC) in Arabidopsis thaliana is a useful system to address these questions. FLC is transcriptionally repressed during exposure to cold temperatures, allowing studies of how environmental conditions alter expression states at the chromatin level. FLC repression is also epigenetically maintained during subsequent development in warm conditions, so that exposure to cold may be remembered. This memory depends on molecular complexes that are highly conserved among eukaryotes, making FLC not only interesting as a paradigm for understanding biological decision-making in plants, but also an important system for elucidating chromatin-based gene regulation more generally. In this review, we summarize our understanding of how cold temperature induces a switch in the FLC chromatin state, and how this state is epigenetically remembered. We also discuss how the epigenetic state of FLC is reprogrammed in the seed to ensure a requirement for cold exposure in the next generation. Significance Statement FLOWERING LOCUS C (FLC) regulation provides a paradigm for understanding how chromatin can be modulated to determine gene expression in a developmental context. This review describes our current mechanistic understanding of how FLC expression is genetically specified and epigenetically regulated throughout the plant life cycle, and how this determines plant life-history strategy. PMID:25929799

  18. Mechanistic Insights into Homogeneous and Heterogeneous Asymmetric Iron Catalysis

    NASA Astrophysics Data System (ADS)

    Sonnenberg, Jessica

    Our group has been focused on replacing toxic and expensive precious metal catalysts with iron for the synthesis of enantiopure compounds for industrial applications. During an investigation into the mechanism of asymmetric transfer hydrogenation with our first generation iron-(P-N-N-P) catalysts we found substantial evidence for zero-valent iron nanoparticles coated in chiral ligand acting as the active site. Extensive experimental and computational experiments were undertaken which included NMR, DFT, reaction profile analysis, substoichiometric poisoning, electron microscope imaging, XPS and multiphasic analysis, all of which supported the fact that NPs were the active species in catalysis. Reversibility of this asymmetric reaction on the nanoparticle surface was then probed using oxidative kinetic resolution of racemic alcohols, yielding modest enantiopurity and high turnover frequencies (TOF) for a range of aromatic alcohols. Efficient dehydrogenation of ammonia-borane for hydrogen evolution and the formation of B-N oligomers was also shown using the NP system, yielding highly active systems, with a maximum TOF of 3.66 H2/s-1 . We have also begun to focus on the development of iron catalysts for asymmetric direct hydrogenation of ketones using hydrogen gas. New chiral iron-(P-N-P) catalysts were developed and shown to be quite active and selective for a wide range of substrates. Mechanistic investigations primarily using NMR and DFT indicated that a highly active trans-dihydride species was being formed during catalyst activation. Lastly, a new library of chiral P-N-P and P-NH-P ligands were developed, as well as their corresponding iron complexes, some of which show promise for the development of future generations of active asymmetric direct hydrogenation catalysts.

  19. Emerging mechanistic insights into AAA complexes regulating proteasomal degradation.

    PubMed

    Förster, Friedrich; Schuller, Jan M; Unverdorben, Pia; Aufderheide, Antje

    2014-01-01

    The 26S proteasome is an integral element of the ubiquitin-proteasome system(UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells.It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates.The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases,shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship.Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS). PMID:25102382

  20. Emerging Mechanistic Insights into AAA Complexes Regulating Proteasomal Degradation

    PubMed Central

    Förster, Friedrich; Schuller, Jan M.; Unverdorben, Pia; Aufderheide, Antje

    2014-01-01

    The 26S proteasome is an integral element of the ubiquitin-proteasome system (UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells. It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates. The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases, shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship. Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS). PMID:25102382

  1. The structural biology of HIV-1: mechanistic and therapeutic insights

    PubMed Central

    Engelman, Alan; Cherepanov, Peter

    2013-01-01

    Three-dimensional molecular structures can provide detailed information on biological mechanisms and, in cases where molecular function impacts on human health, significantly aid in the development of therapeutic interventions. Over the past 23 years, key components of the lentivirus HIV-1, including its envelope glycoproteins and capsid, and the replication enzymes reverse transcriptase, integrase and protease, have accordingly been scrutinized to near atomic scale resolution. Structural analyses of the interactions between viral and host cell components have moreover yielded key insights into the mechanisms of virus entry, chromosomal integration, transcription and egress from cells. Here, we review recent advances in HIV-1 structural biology, focusing on the impact these results have had on our understanding of virus replication and the development of new therapeutics. PMID:22421880

  2. Simple Sugar Intake and Hepatocellular Carcinoma: Epidemiological and Mechanistic Insight

    PubMed Central

    Laguna, Juan Carlos; Alegret, Marta; Roglans, Núria

    2014-01-01

    Sugar intake has dramatically increased during the last few decades. Specifically, there has been a clear trend towards higher consumption of fructose and high fructose corn syrup, which are the most common added sugars in processed food, soft drinks and other sweetened beverages. Although still controversial, this rising trend in simple sugar consumption has been positively associated with weight gain and obesity, insulin resistance and type 2 diabetes mellitus and non-alcoholic fatty liver disease. Interestingly, all of these metabolic alterations have also been related to the development of hepatocellular carcinoma. The purpose of this review is to discuss the evidence coming from epidemiological studies and data from animal models relating the consumption of simple sugars, and specifically fructose, with an increased risk of hepatocellular carcinoma and to gain insight into the putative molecular mechanisms involved. PMID:25533006

  3. Obesity and cancer: mechanistic insights from transdisciplinary studies.

    PubMed

    Allott, Emma H; Hursting, Stephen D

    2015-12-01

    Obesity is associated with a range of health outcomes that are of clinical and public health significance, including cancer. Herein, we summarize epidemiologic and preclinical evidence for an association between obesity and increased risk of breast and prostate cancer incidence and mortality. Moreover, we describe data from observational studies of weight change in humans and from calorie-restriction studies in mouse models that support a potential role for weight loss in counteracting tumor-promoting properties of obesity in breast and prostate cancers. Given that weight loss is challenging to achieve and maintain, we also consider evidence linking treatments for obesity-associated co-morbidities, including metformin, statins and non-steroidal anti-inflammatory drugs, with reduced breast and prostate cancer incidence and mortality. Finally, we highlight several challenges that should be considered when conducting epidemiologic and preclinical research in the area of obesity and cancer, including the measurement of obesity in population-based studies, the timing of obesity and weight change in relation to tumor latency and cancer diagnosis, and the heterogeneous nature of obesity and its associated co-morbidities. Given that obesity is a complex trait, comprised of behavioral, epidemiologic and molecular/metabolic factors, we argue that a transdisciplinary approach is the key to understanding the mechanisms linking obesity and cancer. As such, this review highlights the critical need to integrate evidence from both epidemiologic and preclinical studies to gain insight into both biologic and non-biologic mechanisms contributing to the obesity-cancer link. PMID:26373570

  4. Transformation of heavy metal speciation during sludge drying: mechanistic insights

    SciTech Connect

    Weng, Huanxin; Ma, Xue-Wen; Fu, Feng-Xia; Zhang, Jin-Jun; Liu, Zan; Tian, Li-Xun; Liu, Chongxuan

    2014-01-30

    Speciation can fundamentally affect on the stability and toxicity of heavy metals in sludge from wastewater treatment plants. This research investigated the speciation of heavy metals in sludge from both municipal and industrial sources, and metal speciation change as a result of drying process to reduce sludge volume. The changes in sludge properties including sludge moisture content, temperature, density, and electrical conductivity were also monitored to provide insights into the mechanisms causing the change in heavy metal speciation. The results show that the drying process generally stabilized the Cr, Cu, Cd and Pb in sludge by transforming acid-soluble, reducible and oxidizable species into structurally stable forms. Such transformation and stabilization occurred regardless of the sludge source and type, and were primarily caused by the changes in sludge properties associated with decomposition of organic matter and sulfide. The results enhanced our understanding of the geochemical behavior of heavy metals in municipal sludge, and are useful for designing a treatment system for environment-friendly disposal of sludge.

  5. Mechanistic insights into the mode of action of anticandidal sesamol.

    PubMed

    Ansari, Moiz A; Fatima, Zeeshan; Hameed, Saif

    2016-09-01

    Previously we have deciphered the antifungal effect of sesamol (Ses), a phenolic compound obtained from sesame oil, against human fungal pathogen Candida albicans. To gain deeper insights into the possible mechanisms involved, transcription profiling was done in presence of Ses which revealed various targets through which Ses was barricading the growth of C. albicans. We observed that Ses perturbs membrane integrity confirming our previous observations and displayed disrupted plasma membrane ATPase activity. We further investigated that Ses leads to inhibited morphological transition, biofilm formation and epithelial cell adhesion which are significant virulence attributes required for pathogenesis. Interestingly, Ses also causes amendment in iron homeostasis as revealed by hypersensitivity under iron deprivation, ferroxidase assay to estimate iron levels and concomitant upregulation of FTR2, a high affinity iron transporter. Finally we assessed that Ses causes defect in mitochondrial functioning and DNA repair mechanism. Together, being source of consumable natural product, further studies on Ses are warranted so that it can be exploited as effective antifungal agent. PMID:27392701

  6. Cobalt-Catalyzed C(sp(2))-H Borylation: Mechanistic Insights Inspire Catalyst Design.

    PubMed

    Obligacion, Jennifer V; Semproni, Scott P; Pappas, Iraklis; Chirik, Paul J

    2016-08-24

    A comprehensive study into the mechanism of bis(phosphino)pyridine (PNP) cobalt-catalyzed C-H borylation of 2,6-lutidine using B2Pin2 (Pin = pinacolate) has been conducted. The experimentally observed rate law, deuterium kinetic isotope effects, and identification of the catalyst resting state support turnover limiting C-H activation from a fully characterized cobalt(I) boryl intermediate. Monitoring the catalytic reaction as a function of time revealed that borylation of the 4-position of the pincer in the cobalt catalyst was faster than arene borylation. Cyclic voltammetry established the electron withdrawing influence of 4-BPin, which slows the rate of C-H oxidative addition and hence overall catalytic turnover. This mechanistic insight inspired the next generation of 4-substituted PNP cobalt catalysts with electron donating and sterically blocking methyl and pyrrolidinyl substituents that exhibited increased activity for the C-H borylation of unactivated arenes. The rationally designed catalysts promote effective turnover with stoichiometric quantities of arene substrate and B2Pin2. Kinetic studies on the improved catalyst, 4-(H)2BPin, established a change in turnover limiting step from C-H oxidative addition to C-B reductive elimination. The iridium congener of the optimized cobalt catalyst, 6-(H)2BPin, was prepared and crystallographically characterized and proved inactive for C-H borylation, a result of the high kinetic barrier for reductive elimination from octahedral Ir(III) complexes. PMID:27476954

  7. Chemoselective Transformation of Diarylethanones to Arylmethanoic Acids and Diarylmethanones and Mechanistic Insights.

    PubMed

    Wang, Xing; Chen, Rui-Xi; Wei, Zeng-Feng; Zhang, Chen-Yang; Tu, Hai-Yang; Zhang, Ai-Dong

    2016-01-01

    The chemoselective transformation of diarylethanones via either aerobic oxidative cleavage to give arylmethanoic acids or tandem aerobic oxidation/benzilic acid rearrangement/decarboxylation to give diarylmethanones has been developed. The transformation is controllable and applicable to a broad spectrum of substrates and affords the desired products in good to excellent yields. Mechanistic insights with control reactions, (1)H NMR tracking, and single-crystal X-ray diffraction reveal a complex mechanistic network in which two common intermediates, α-ketohydroperoxide and diarylethanedione, and three plausible pathways are proposed and verified. These pathways are interlinked and can be switched reasonably by changing the reaction conditions. This method enables scalable synthesis and access to a number of valuable compounds, including vitamin B3, diphenic acid, and the nonsteroidal anti-inflammatory drug ketoprofen. The present protocol represents a step forward in exploiting complex mechanistic networks to control reaction pathways, achieving divergent syntheses from the same class of starting materials. PMID:26618373

  8. Mechanistic Insights into PTS2-mediated Peroxisomal Protein Import

    PubMed Central

    Kunze, Markus; Malkani, Naila; Maurer-Stroh, Sebastian; Wiesinger, Christoph; Schmid, Johannes A.; Berger, Johannes

    2015-01-01

    The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have been characterized as peroxisomal targeting signals (PTS) residing either at the C terminus (PTS1) or close to the N terminus (PTS2). PTS2-carrying proteins interact with their cognate receptor protein PEX7 that mediates their transport to peroxisomes by a concerted action with a co-receptor protein, which in mammals is the PTS1 receptor PEX5L. Using a modified version of the mammalian two-hybrid assay, we demonstrate that the interaction strength between cargo and PEX7 is drastically increased in the presence of the co-receptor PEX5L. In addition, cargo binding is a prerequisite for the interaction between PEX7 and PEX5L and ectopic overexpression of PTS2-carrying cargo protein drastically increases the formation of PEX7-PEX5L complexes in this assay. Consistently, we find that the peroxisomal transfer of PEX7 depends on cargo binding and that ectopic overexpression of cargo protein stimulates this process. Thus, the sequential formation of a highly stable trimeric complex involving cargo protein, PEX7 and PEX5L stabilizes cargo binding and is a prerequisite for PTS2-mediated peroxisomal import. PMID:25538232

  9. The Cycloaddition of the Benzimidazolium Ylides with Alkynes: New Mechanistic Insights

    PubMed Central

    Moldoveanu, Costel; Zbancioc, Gheorghita; Mantu, Dorina; Maftei, Dan; Mangalagiu, Ionel

    2016-01-01

    New insights concerning the reaction mechanism in the cycloaddition reaction of benzimidazolium ylides to activated alkynes are presented. The proposed pathway leading both to 2-(1H-pyrrol-1-yl)anilines and to pyrrolo[1,2-a]quinoxalin-4(5H)-ones involves an opening of the imidazole ring from the cycloaddition product, followed by a nucleophilic attack of the aminic nitrogen to a proximal carbonyl group and the elimination of a leaving group. The mechanistic considerations are fully supported by experimental data, including the XRD resolved structure of the key reaction intermediate. PMID:27224656

  10. Cross-study and cross-omics comparisons of three nephrotoxic compounds reveal mechanistic insights and new candidate biomarkers

    SciTech Connect

    Matheis, Katja A.; Com, Emmanuelle; Gautier, Jean-Charles; Guerreiro, Nelson; Brandenburg, Arnd; Gmuender, Hans; Sposny, Alexandra; Hewitt, Philip; Amberg, Alexander; Boernsen, Olaf; Riefke, Bjoern; Hoffmann, Dana; Mally, Angela; Kalkuhl, Arno; Suter, Laura; Dieterle, Frank; Staedtler, Frank

    2011-04-15

    The European InnoMed-PredTox project was a collaborative effort between 15 pharmaceutical companies, 2 small and mid-sized enterprises, and 3 universities with the goal of delivering deeper insights into the molecular mechanisms of kidney and liver toxicity and to identify mechanism-linked diagnostic or prognostic safety biomarker candidates by combining conventional toxicological parameters with 'omics' data. Mechanistic toxicity studies with 16 different compounds, 2 dose levels, and 3 time points were performed in male Crl: WI(Han) rats. Three of the 16 investigated compounds, BI-3 (FP007SE), Gentamicin (FP009SF), and IMM125 (FP013NO), induced kidney proximal tubule damage (PTD). In addition to histopathology and clinical chemistry, transcriptomics microarray and proteomics 2D-DIGE analysis were performed. Data from the three PTD studies were combined for a cross-study and cross-omics meta-analysis of the target organ. The mechanistic interpretation of kidney PTD-associated deregulated transcripts revealed, in addition to previously described kidney damage transcript biomarkers such as KIM-1, CLU and TIMP-1, a number of additional deregulated pathways congruent with histopathology observations on a single animal basis, including a specific effect on the complement system. The identification of new, more specific biomarker candidates for PTD was most successful when transcriptomics data were used. Combining transcriptomics data with proteomics data added extra value.

  11. Pathways to neurodegeneration: mechanistic insights from GWAS in Alzheimer's disease, Parkinson's disease, and related disorders.

    PubMed

    Ramanan, Vijay K; Saykin, Andrew J

    2013-01-01

    The discovery of causative genetic mutations in affected family members has historically dominated our understanding of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). Nevertheless, most cases of neurodegenerative disease are not explained by Mendelian inheritance of known genetic variants, but instead are thought to have a complex etiology with numerous genetic and environmental factors contributing to susceptibility. Although unbiased genome-wide association studies (GWAS) have identified novel associations to neurodegenerative diseases, most of these hits explain only modest fractions of disease heritability. In addition, despite the substantial overlap of clinical and pathologic features among major neurodegenerative diseases, surprisingly few GWAS-implicated variants appear to exhibit cross-disease association. These realities suggest limitations of the focus on individual genetic variants and create challenges for the development of diagnostic and therapeutic strategies, which traditionally target an isolated molecule or mechanistic step. Recently, GWAS of complex diseases and traits have focused less on individual susceptibility variants and instead have emphasized the biological pathways and networks revealed by genetic associations. This new paradigm draws on the hypothesis that fundamental disease processes may be influenced on a personalized basis by a combination of variants - some common and others rare, some protective and others deleterious - in key genes and pathways. Here, we review and synthesize the major pathways implicated in neurodegeneration, focusing on GWAS from the most prevalent neurodegenerative disorders, AD and PD. Using literature mining, we also discover a novel regulatory network that is enriched with AD- and PD-associated genes and centered on the SP1 and AP-1 (Jun/Fos) transcription factors. Overall, this pathway- and

  12. New Mechanistic Insights on the Selectivity of Transition-Metal-Catalyzed Organic Reactions: The Role of Computational Chemistry.

    PubMed

    Zhang, Xinhao; Chung, Lung Wa; Wu, Yun-Dong

    2016-06-21

    With new advances in theoretical methods and increased computational power, applications of computational chemistry are becoming practical and routine in many fields of chemistry. In organic chemistry, computational chemistry plays an indispensable role in elucidating reaction mechanisms and the origins of various selectivities, such as chemo-, regio-, and stereoselectivities. Consequently, mechanistic understanding improves synthesis and assists in the rational design of new catalysts. In this Account, we present some of our recent works to illustrate how computational chemistry provides new mechanistic insights for improvement of the selectivities of several organic reactions. These examples include not only explanations for the existing experimental observations, but also predictions which were subsequently verified experimentally. This Account consists of three sections discuss three different kinds of selectivities. The first section discusses the regio- and stereoselectivities of hydrosilylations of alkynes, mainly catalyzed by [Cp*Ru(MeCN)3](+) or [CpRu(MeCN)3](+). Calculations suggest a new mechanism that involves a key ruthenacyclopropene intermediate. This mechanism not only explains the unusual Markovnikov regio-selectivity and anti-addition stereoselectivity observed by Trost and co-workers, but also motivated further experimental investigations. New intriguing experimental observations and further theoretical studies led to an extension of the reaction mechanism. The second section includes three cases of meta-selective C-H activation of aryl compounds. In the case of Cu-catalyzed selective meta-C-H activation of aniline, a new mechanism that involves a Cu(III)-Ar-mediated Heck-like transition state, in which the Ar group acts as an electrophile, was proposed. This mechanism predicted a higher reactivity for more electron-deficient Ar groups, which was supported by experiments. For two template-mediated, meta-selective C-H bond activations catalyzed by

  13. Mechanistic insights on the Dicer-independent AGO2-mediated processing of AgoshRNAs.

    PubMed

    Liu, Ying Poi; Karg, Margarete; Harwig, Alex; Herrera-Carrillo, Elena; Jongejan, Aldo; van Kampen, Antoine; Berkhout, Ben

    2015-01-01

    Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both for research and therapeutic purposes. The shRNA precursor is processed by the RNase III-like enzyme Dicer into biologically active small interfering RNA (siRNA). This effector molecule subsequently targets a complementary mRNA for destruction via the Argonaute 2 (AGO2) complex. The cellular role of Dicer concerns the processing of pre-miRNAs into mature microRNA (miRNA). Recently, a non-canonical pathway was reported for the biogenesis of miR-451, which bypasses Dicer and is processed instead by the slicer activity of AGO2, followed by the regular AGO2-mediated mRNA targeting step. Interestingly, shRNA designs that are characterized by a relatively short basepaired stem also bypass Dicer to be processed by AGO2. We named this design AgoshRNA as these molecules depend on AGO2 both for processing and silencing activity. In this study, we investigated diverse mechanistic aspects of this new class of AgoshRNA molecules. We probed the requirements for AGO2-mediated processing of AgoshRNAs by modification of the proposed cleavage site in the hairpin. We demonstrate by deep sequencing that AGO2-processed AgoshRNAs produce RNA effector molecules with more discrete ends than the products of the regular shRNA design. Furthermore, we tested whether trimming and tailing occurs upon AGO2-mediated processing of AgoshRNAs, similar to what has been described for miR-451. Finally, we tested the prediction that AgoshRNA activity, unlike that of regular shRNAs, is maintained in Dicer-deficient cell types. These mechanistic insights could aid in the design of optimised AgoshRNA tools and therapeutics. PMID:25826416

  14. Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila

    PubMed Central

    Pang, Yuxuan; Bai, Xiao-chen; Yan, Chuangye; Hao, Qi; Chen, Zheqin; Wang, Jia-Wei

    2015-01-01

    Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome. PMID:25644603

  15. Mechanistic insights into the first Lygus-active β-pore forming protein.

    PubMed

    Jerga, Agoston; Chen, Danqi; Zhang, Chunfen; Fu, Jinping; Kouadio, Jean-Louis K; Wang, Yanfei; Duff, Stephen M G; Howard, Jennifer E; Rydel, Timothy J; Evdokimov, Artem G; Ramaseshadri, Parthasarathy; Evans, Adam; Bolognesi, Renata; Park, Yoonseong; Haas, Jeffrey A

    2016-06-15

    The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active β-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the β-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered β-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality. PMID:27001423

  16. Pterostilbene-mediated Nrf2 activation: Mechanistic insights on Keap1:Nrf2 interface.

    PubMed

    Bhakkiyalakshmi, Elango; Dineshkumar, Kesavan; Karthik, Suresh; Sireesh, Dornadula; Hopper, Waheeta; Paulmurugan, Ramasamy; Ramkumar, Kunka Mohanram

    2016-08-15

    The discovery of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1-Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1-Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1-Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1-Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1-Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS. PMID:27312421

  17. Exploring novel mechanistic insights in Alzheimer's disease by assessing reliability of protein interactions.

    PubMed

    Malhotra, Ashutosh; Younesi, Erfan; Sahadevan, Sudeep; Zimmermann, Joerg; Hofmann-Apitius, Martin

    2015-01-01

    Protein interaction networks are widely used in computational biology as a graphical means of representing higher-level systemic functions in a computable form. Although, many algorithms exist that seamlessly collect and measure protein interaction information in network models, they often do not provide novel mechanistic insights using quantitative criteria. Measuring information content and knowledge representation in network models about disease mechanisms becomes crucial particularly when exploring new target candidates in a well-defined functional context of a potential disease mechanism. To this end, we have developed a knowledge-based scoring approach that uses literature-derived protein interaction features to quantify protein interaction confidence. Thereby, we introduce the novel concept of knowledge cliffs, regions of the interaction network where a significant gap between high scoring and low scoring interactions is observed, representing a divide between established and emerging knowledge on disease mechanism. To show the application of this approach, we constructed and assessed reliability of a protein-protein interaction model specific to Alzheimer's disease, which led to screening, and prioritization of four novel protein candidates. Evaluation of the identified candidates showed that two of them are already followed in clinical trials for testing potential AD drugs. PMID:26346705

  18. Mechanistic Insights into Reversible Photoactivation in Proteins of the GFP Family

    PubMed Central

    Gayda, Susan; Nienhaus, Karin; Nienhaus, G. Ulrich

    2012-01-01

    Light-controlled modification of the fluorescence emission properties of proteins of the GFP family is of crucial importance for many imaging applications including superresolution microscopy. Here, we have studied the reversibly photoswitchable fluorescent protein mIrisGFP using optical spectroscopy. By analyzing the pH dependence of isomerization and protonation equilibria and the isomerization kinetics, we have obtained insight into the coupling of the chromophore to the surrounding protein moiety and a better understanding of the photoswitching mechanism. A different acid-base environment of the chromophore’s protonating group in its two isomeric forms, which can be inferred from the x-ray structures of IrisFP, is key to the photoswitching function and ensures that isomerization and protonation are correlated. Amino acids near the chromophore, especially Glu212, rearrange upon isomerization, and Glu212 protonation modulates the chromophore pKa. In mIrisGFP, the cis chromophore protonates in two steps, with pKcis of 5.3 and 6, which is much lower than pKtrans (>10). Based on these results, we have put forward a mechanistic scheme that explains how the combination of isomeric and acid-base properties of the chromophore in its protein environment can produce negative and positive photoswitching modes. PMID:23260054

  19. Exploring novel mechanistic insights in Alzheimer’s disease by assessing reliability of protein interactions

    PubMed Central

    Malhotra, Ashutosh; Younesi, Erfan; Sahadevan, Sudeep; Zimmermann, Joerg; Hofmann-Apitius, Martin

    2015-01-01

    Protein interaction networks are widely used in computational biology as a graphical means of representing higher-level systemic functions in a computable form. Although, many algorithms exist that seamlessly collect and measure protein interaction information in network models, they often do not provide novel mechanistic insights using quantitative criteria. Measuring information content and knowledge representation in network models about disease mechanisms becomes crucial particularly when exploring new target candidates in a well-defined functional context of a potential disease mechanism. To this end, we have developed a knowledge-based scoring approach that uses literature-derived protein interaction features to quantify protein interaction confidence. Thereby, we introduce the novel concept of knowledge cliffs, regions of the interaction network where a significant gap between high scoring and low scoring interactions is observed, representing a divide between established and emerging knowledge on disease mechanism. To show the application of this approach, we constructed and assessed reliability of a protein-protein interaction model specific to Alzheimer’s disease, which led to screening, and prioritization of four novel protein candidates. Evaluation of the identified candidates showed that two of them are already followed in clinical trials for testing potential AD drugs. PMID:26346705

  20. Base-free non-noble-metal-catalyzed hydrogen generation from formic acid: scope and mechanistic insights.

    PubMed

    Mellmann, Dörthe; Barsch, Enrico; Bauer, Matthias; Grabow, Kathleen; Boddien, Albert; Kammer, Anja; Sponholz, Peter; Bentrup, Ursula; Jackstell, Ralf; Junge, Henrik; Laurenczy, Gábor; Ludwig, Ralf; Beller, Matthias

    2014-10-13

    The iron-catalyzed dehydrogenation of formic acid has been studied both experimentally and mechanistically. The most active catalysts were generated in situ from cationic Fe(II) /Fe(III) precursors and tris[2-(diphenylphosphino)ethyl]phosphine (1, PP3 ). In contrast to most known noble-metal catalysts used for this transformation, no additional base was necessary. The activity of the iron catalyst depended highly on the solvent used, the presence of halide ions, the water content, and the ligand-to-metal ratio. The optimal catalytic performance was achieved by using [FeH(PP3 )]BF4 /PP3 in propylene carbonate in the presence of traces of water. With the exception of fluoride, the presence of halide ions in solution inhibited the catalytic activity. IR, Raman, UV/Vis, and EXAFS/XANES analyses gave detailed insights into the mechanism of hydrogen generation from formic acid at low temperature, supported by DFT calculations. In situ transmission FTIR measurements revealed the formation of an active iron formate species by the band observed at 1543 cm(-1) , which could be correlated with the evolution of gas. This active species was deactivated in the presence of chloride ions due to the formation of a chloro species (UV/Vis, Raman, IR, and XAS). In addition, XAS measurements demonstrated the importance of the solvent for the coordination of the PP3 ligand. PMID:25196789

  1. Iron Trichloride and Air Mediated Guanylation of Acylthioureas. An Ecological Route to Acylguanidines: Scope and Mechanistic Insights.

    PubMed

    Pape, Simon; Wessig, Pablo; Brunner, Heiko

    2016-06-01

    Recently we introduced iron trichloride as an environmentally benign and cost-efficient reagent for the synthesis of N-benzoylguanidines. This highly attractive synthetic approach grants access to a broad spectrum of N-benzoylguanidines under mild conditions in short reaction times. In this work we present an extended scope of our methodology along with the results obtained from mechanistic studies via in situ IR spectroscopy in combination with LC (liquid chromatography)-MS analyses. On the basis of these new mechanistic insights we were able to optimize the synthetic protocol and to develop an alternative mechanistic proposal. In this context the symbiotic roles of iron trichloride and oxygen in the guanylation process are highlighted. PMID:27181741

  2. All Bariatric Surgeries Are Not Created Equal: Insights from Mechanistic Comparisons

    PubMed Central

    Stefater, Margaret A.; Wilson-Pérez, Hilary E.; Chambers, Adam P.; Sandoval, Darleen A.

    2012-01-01

    Despite considerable scientific progress on the biological systems that regulate energy balance, we have made precious little headway in providing new treatments to curb the obesity epidemic. Diet and exercise are the most popular treatment options for obesity, but rarely are they sufficient to produce long-term weight loss. Bariatric surgery, on the other hand, results in dramatic, sustained weight loss and for this reason has gained increasing popularity as a treatment modality for obesity. At least some surgical approaches also reduce obesity-related comorbidities including type 2 diabetes and hyperlipidemia. This success puts a premium on understanding how these surgeries exert their effects. This review focuses on the growing human and animal model literature addressing the underlying mechanisms. We compare three common procedures: Roux-en-Y Gastric Bypass (RYGB), vertical sleeve gastrectomy (VSG), and adjustable gastric banding (AGB). Although many would group together VSG and AGB as restrictive procedures of the stomach, VSG is more like RYGB than AGB in its effects on a host of endpoints including intake, food choice, glucose regulation, lipids and gut hormone secretion. Our strong belief is that to advance our understanding of these procedures, it is necessary to group bariatric procedures not on the basis of surgical similarity but rather on how they affect key physiological variables. This will allow for greater mechanistic insight into how bariatric surgery works, making it possible to help patients better choose the best possible procedure and to develop new therapeutic strategies that can help a larger portion of the obese population. PMID:22550271

  3. Structural and Mechanistic Insights into the Tropism of Epstein-Barr Virus

    PubMed Central

    Möhl, Britta S.; Chen, Jia; Sathiyamoorthy, Karthik; Jardetzky, Theodore S.; Longnecker, Richard

    2016-01-01

    Epstein-Barr virus (EBV) is the prototypical γ-herpesvirus and an obligate human pathogen that infects mainly epithelial cells and B cells, which can result in malignancies. EBV infects these target cells by fusing with the viral and cellular lipid bilayer membranes using multiple viral factors and host receptor(s) thus exhibiting a unique complexity in its entry machinery. To enter epithelial cells, EBV requires minimally the conserved core fusion machinery comprised of the glycoproteins gH/gL acting as the receptor-binding complex and gB as the fusogen. EBV can enter B cells using gp42, which binds tightly to gH/gL and interacts with host HLA class II, activating fusion. Previously, we published the individual crystal structures of EBV entry factors, such as gH/gL and gp42, the EBV/host receptor complex, gp42/HLA-DR1, and the fusion protein EBV gB in a postfusion conformation, which allowed us to identify structural determinants and regions critical for receptor-binding and membrane fusion. Recently, we reported different low resolution models of the EBV B cell entry triggering complex (gHgL/gp42/HLA class II) in “open” and “closed” states based on negative-stain single particle electron microscopy, which provide further mechanistic insights. This review summarizes the current knowledge of these key players in EBV entry and how their structures impact receptor-binding and the triggering of gB-mediated fusion. PMID:27094060

  4. Exploring soil organic matter-mineral interactions: mechanistic insights at the nanometer and molecular length scales

    NASA Astrophysics Data System (ADS)

    Newcomb, C.; Qafoku, N. P.; Grate, J. W.; Hufschmid, R.; Browning, N.; De Yoreo, J. J.

    2015-12-01

    With elevated levels of carbon dioxide in the atmosphere due to anthropogenic emissions and disruption to the carbon cycle, the effects of climate change are being accelerated. Approximately 80% of Earth's terrestrial organic carbon is stored in soil, and the residence time of this carbon can range from hours to millenia. Understanding the dynamics of this carbon pool in the carbon cycle is crucial to both predicting climate and sustaining ecosystem services. Soil organic carbon is known to be strongly associated with high surface area clay minerals. The nature of these interactions is not well understood primarily due to the heterogeneity of soil, as much of the current knowledge relies on experiments that take a top-down approach using bulk experimental measurements. Our work seeks to probe physical, chemical, and molecular-level interactions at the organic-mineral interface using a bottom-up approach that establishes a model system where complexity can be built in systematically. By performing in situ techniques such as dynamic force spectroscopy, a technique where organic molecules can be brought into contact with mineral surfaces in a controlled manner using an atomic force microscope, we demonstrate the ability to mechanistically probe the energy landscape of individual organic molecules with mineral surfaces. We demonstrate the ability to measure the binding energies of soil-inspired organic functional groups (including carboxylic acid, amine, methyl, and phosphate) with clay and mineral surfaces as a function of solution chemistry. This effort can provide researchers with both guiding principles about carbon dynamics at the sub-nanometer length scale and insights into early aggregation events, where organic-mineral interactions play a significant role.

  5. Three steps forward, two steps back: mechanistic insights into the assembly and disassembly of the SNARE complex.

    PubMed

    Bombardier, Jeffrey P; Munson, Mary

    2015-12-01

    Membrane fusion is a tightly controlled process in all eukaryotic cell types. The SNARE family of proteins is required for fusion throughout the exocytic and endocytic trafficking pathways. SNAREs on a transport vesicle interact with the cognate SNAREs on the target membrane, forming an incredibly stable SNARE complex that provides energy for the membranes to fuse, although many aspects of the mechanism remain elusive. Recent advances in single-molecule and high-resolution structural methods provide exciting new insights into how SNARE complexes assemble, including measurements of assembly energetics and identification of intermediates in the assembly pathway. These techniques were also key in elucidating mechanistic details into how the SNARE complex is disassembled, including details of the energetics required for ATP-dependent α-SNAP/NSF-mediated SNARE complex disassembly, and the structural changes that accompany ATP hydrolysis by the disassembly machinery. Additionally, SNARE complex formation and disassembly are tightly regulated processes; innovative biochemical and biophysical characterization has deepened our understanding of how these regulators work to control membrane fusion and exocytosis. PMID:26498108

  6. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    SciTech Connect

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2011-12-22

    Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 {angstrom} resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

  7. Toward a mechanistic understanding of the effect of biochar addition on soil water retention

    NASA Astrophysics Data System (ADS)

    Yi, S.; Chang, N.; Guo, M.; Imhoff, P. T.

    2014-12-01

    Biochar (BC) is a carbon-rich product produced by thermal degradation of biomass in an oxygen-free environment, whose application to sediment is said to improve water retention. However, BC produced from different feedstocks and pyrolyzed at different temperatures have distinct properties, which may alter water retention in ways difficult to predict a priori. Our goal is to develop a mechanistic understanding of BC addition on water retention by examining the impact of BC from two feedstocks, poultry litter (PL) and hardwood (HW), on the soil-water retention curves (SWRC) of a uniform sand and a sandy loam (SL). For experiments with sand, BC and sand were sieved to the same particle size (~ 0.547 mm) to minimize effects of BC addition on particle size distribution. Experiments with SL contained the same sieved BC. PL and HW bicohars were added at 2 and 7% (w/w), and water retention was measured from 0 to -4.38 × 106 cm-H2O. Both BCs increased porosities for sand and SL, up to 39 and 13% for sand and SL, respectively, with 7% HW BC addition. The primary cause for these increases was the internal porosity of BC particles. While the matric potential for air-entry was unchanged with BC addition, BC amendment increased water retention for sand and SL in the capillary region (0 to -15,000 cm-H2O) by an average of 26 and 33 % for 7% PL and HW BC in sand, respectively, but only 7 and 14 % for 7% PL and HW BC in SL. The most dramatic influence of BC amendment on water retention occurred in the adsorption region (< -15,000 cm-H2O), where water retention increased by a factor of 11 and 22 for 7% PL and HW BC in sand, respectively, but by 140 and 190 % for 7% PL and HW BC in SL, respectively. The impact of BC on water retention in these sediments is explained primarily by the additional surface area and internal porosity of PL and HW BC particles. van Genuchten (VG) models were fitted to the water retention data. For SL where the impact of BC addition on water retention was

  8. Dynamic Kinetic Resolution Enabled by Intramolecular Benzoin Reaction: Synthetic Applications and Mechanistic Insights.

    PubMed

    Zhang, Guoxiang; Yang, Shuang; Zhang, Xiaoyan; Lin, Qiqiao; Das, Deb K; Liu, Jian; Fang, Xinqiang

    2016-06-29

    The highly enantio-, diastereo-, and regioselective dynamic kinetic resolution of β-ketoesters and 1,3-diketones was achieved via a chiral N-heterocyclic carbene catalyzed intramolecular cross-benzoin reaction. A variety of tetralone derivatives bearing two contiguous stereocenters and multiple functionalities were liberated in moderate to excellent yields and with high levels of stereoselectivity (>95% ee and >20:1 dr in most cases). In addition, the excellent regioselectivity control for aryl/alkyl 1,3-diketones, and the superior electronic differentiation of 1,3-diarylketones were highlighted. Moreover, a set of new mechanistic rationale that differs with the currently widely accepted understanding of intramolecular benzoin reactions was established to demonstrate the superior preference of benzoin over aldol transformation: (1) A coexistence of competitive aldol and benzoin reactions was detected, but a retro-aldol-irreversible benzoin process performs a vital role in the generation of predominant benzoin products. (2) The most essential role of an N-electron-withdrawing substituent in triazolium catalysts was revealed to be accelerating the rate of the benzoin transformation, rather than suppressing the aldol process through reducing the inherent basicity of the catalyst. PMID:27270409

  9. Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

    PubMed Central

    Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.

    2014-01-01

    Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally. PMID:24459184

  10. Mechanistic insight into the nucleation and growth of oleic acid capped lead sulphide quantum dots.

    PubMed

    Shrestha, Aabhash; Spooner, Nigel A; Qiao, Shi Zhang; Dai, Sheng

    2016-05-18

    The quantum dots (QDs) of lead sulphide (PbS) are attractive near-infrared (NIR) active materials and have promising applications in a wide variety of applications. Till date many efforts have been made on optimizing its synthesis; however, current mechanistic understanding involving the nucleation and growth of these QDs has not reached the same level as that for other QDs. In this study, we present a detailed understanding on synthesis mechanism of PbS QDs so as to provide guidance for future QDs synthesis. The synthesis of PbS QDs is largely independent of classical nucleation process and the hot-injection of precursors may not be necessary for the successful synthesis of PbS QDs. The synthesis is basically a growth dominated process and is controlled by the Ostwald ripening of PbS QDs. In addition, reaction temperature and ligand are the key parameters for controlling QD growth. Temperature provides energy for overcoming activation barrier of QD growth while the ligands enhance QD growth via altering the environment for QD growth. Following the mechanism governing the synthesis of PbS QDs, we demonstrate that the size tuning of PbS QDs in ultra-small (<2 nm) can be achieved, which has been typically challenging following the hot injection synthesis. PMID:27156571

  11. Mechanistic Insight into the Photoredox Catalysis of Anti-Markovnikov Alkene Hydrofunctionalization Reactions

    PubMed Central

    2015-01-01

    We describe our efforts to understand the key mechanistic aspects of the previously reported alkene hydrofunctionalization reactions using 9-mesityl-10-methylacridinium (Mes-Acr+) as a photoredox catalyst. Importantly, we are able to detect alkene cation radical intermediates, and confirm that phenylthiyl radical is capable of oxidizing the persistent acridinyl radical in a fast process that unites the catalytic activity of the photoredox and hydrogen atom transfer (HAT) manifolds. Additionally, we present evidence that diphenyl disulfide ((PhS)2) operates on a common catalytic cycle with thiophenol (PhSH) by way of photolytic cleaveage of the disulfide bond. Transition structure analysis of the HAT step using DFT reveals that the activation barrier for H atom donation from PhSH is significantly lower than 2-phenylmalononitrile (PMN) due to structural reorganization. In the early stages of the reaction, Mes-Acr+ is observed to engage in off-cycle adduct formation, presumably as buildup of PhS− becomes significant. The kinetic differences between PhSH and (PhS)2 as HAT catalysts indicate that the proton transfer step may have significant rate limiting influence. PMID:25390821

  12. Mechanistic insights into the allosteric modulation of opioid receptors by sodium ions.

    PubMed

    Shang, Yi; LeRouzic, Valerie; Schneider, Sebastian; Bisignano, Paola; Pasternak, Gavril W; Filizola, Marta

    2014-08-12

    The idea of sodium ions altering G-protein-coupled receptor (GPCR) ligand binding and signaling was first suggested for opioid receptors (ORs) in the 1970s and subsequently extended to other GPCRs. Recently published ultra-high-resolution crystal structures of GPCRs, including that of the δ-OR subtype, have started to shed light on the mechanism underlying sodium control in GPCR signaling by revealing details of the sodium binding site. Whether sodium accesses different receptor subtypes from the extra- or intracellular sides, following similar or different pathways, is still an open question. Earlier experiments in brain homogenates suggested a differential sodium regulation of ligand binding to the three major OR subtypes, in spite of their high degree of sequence similarity. Intrigued by this possibility, we explored the dynamic nature of sodium binding to δ-OR, μ-OR, and κ-OR by means of microsecond-scale, all-atom molecular dynamics (MD) simulations. Rapid sodium permeation was observed exclusively from the extracellular milieu, and following similar binding pathways in all three ligand-free OR systems, notwithstanding extra densities of sodium observed near nonconserved residues of κ-OR and δ-OR, but not in μ-OR. We speculate that these differences may be responsible for the differential increase in antagonist binding affinity of μ-OR by sodium resulting from specific ligand binding experiments in transfected cells. On the other hand, sodium reduced the level of binding of subtype-specific agonists to all OR subtypes. Additional biased and unbiased MD simulations were conducted using the δ-OR ultra-high-resolution crystal structure as a model system to provide a mechanistic explanation for this experimental observation. PMID:25073009

  13. Mechanistic insights into folate supplementation from Crooked tail and other NTD-prone mutant mice.

    PubMed

    Gray, Jason D; Ross, M Elizabeth

    2009-04-01

    Despite two decades of research since Smithells and colleagues began exploring its benefits, the mechanisms through which folic acid supplementation supports neural tube closure and early embryonic development are still unclear. The greatest progress toward a molecular-genetic understanding of folate effects on neural tube defect (NTD) pathogenesis has come from animal models. The number of NTD-associated mouse mutants accumulated and studied over the past decade has illuminated the complexity of both genetic factors contributing to NTDs and also NTD-gene interactions with folate metabolism. This article discusses insights gained from mouse models into how folate supplementation impacts neurulation. A case is made for renewed efforts to systematically screen the folate responsiveness of the scores of NTD-associated mouse mutations now identified. Designed after Crooked tail, supplementation studies of additional mouse mutants could build the molecular network maps that will ultimately enable tailoring of therapeutic regimens to individual families. PMID:19067399

  14. Aortic Binding of AZD5248: Mechanistic Insight and Reactivity Assays To Support Lead Optimzation.

    PubMed

    Bragg, Ryan A; Brocklehurst, Simon; Gustafsson, Frida; Goodman, James; Hickling, Kevin; MacFaul, Philip A; Swallow, Steve; Tugwood, Jonathan

    2015-10-19

    The oral dipeptidyl peptidase 1 (DPP1) inhibitor AZD5248 showed aortic binding in a rat quantitative whole-body autoradiography (QWBA) study, and its development was terminated prior to human dosing. A mechanistic hypothesis for this finding was established invoking reactivity with aldehydes involved in the cross-linking of elastin, a major component of aortic tissue. This was tested by developing a simple aldehyde chemical reactivity assay and a novel in vitro competitive covalent binding assay. Results obtained with AZD5248, literature compounds, and close analogues of AZD5248 support the mechanistic hypothesis and provide validation for the use of these assays in a two tier screening approach to support lead optimization. The strengths and limitations of these assays are discussed. PMID:26351880

  15. Mechanistic insights into the potassium tert-butoxide-mediated synthesis of N-heterobiaryls.

    PubMed

    Stephens, David E; Lakey-Beitia, Johant; Burch, Jessica E; Arman, Hadi D; Larionov, Oleg V

    2016-08-01

    We report herein that symmetrical and non-symmetrical N-heterobiaryls are produced by a potassium tert-butoxide-mediated dimerization of heterocyclic N-oxides. The reaction is scalable and transition metal-free, and can be carried out under thermal and microwave conditions. Preliminary mechanistic studies point to the involvement of radical anionic intermediates arising from the N-oxide substrates and potassium tert-butoxide. PMID:27440397

  16. Mechanistic Insights into Molecular Targeting and Combined Modality Therapy for Aggressive, Localized Prostate Cancer

    PubMed Central

    Dal Pra, Alan; Locke, Jennifer A.; Borst, Gerben; Supiot, Stephane; Bristow, Robert G.

    2016-01-01

    Radiation therapy (RT) is one of the mainstay treatments for prostate cancer (PCa). The potentially curative approaches can provide satisfactory results for many patients with non-metastatic PCa; however, a considerable number of individuals may present disease recurrence and die from the disease. Exploiting the rich molecular biology of PCa will provide insights into how the most resistant tumor cells can be eradicated to improve treatment outcomes. Important for this biology-driven individualized treatment is a robust selection procedure. The development of predictive biomarkers for RT efficacy is therefore of utmost importance for a clinically exploitable strategy to achieve tumor-specific radiosensitization. This review highlights the current status and possible opportunities in the modulation of four key processes to enhance radiation response in PCa by targeting the: (1) androgen signaling pathway; (2) hypoxic tumor cells and regions; (3) DNA damage response (DDR) pathway; and (4) abnormal extra-/intracell signaling pathways. In addition, we discuss how and which patients should be selected for biomarker-based clinical trials exploiting and validating these targeted treatment strategies with precision RT to improve cure rates in non-indolent, localized PCa. PMID:26909338

  17. Mechanistic Insights into Molecular Targeting and Combined Modality Therapy for Aggressive, Localized Prostate Cancer.

    PubMed

    Dal Pra, Alan; Locke, Jennifer A; Borst, Gerben; Supiot, Stephane; Bristow, Robert G

    2016-01-01

    Radiation therapy (RT) is one of the mainstay treatments for prostate cancer (PCa). The potentially curative approaches can provide satisfactory results for many patients with non-metastatic PCa; however, a considerable number of individuals may present disease recurrence and die from the disease. Exploiting the rich molecular biology of PCa will provide insights into how the most resistant tumor cells can be eradicated to improve treatment outcomes. Important for this biology-driven individualized treatment is a robust selection procedure. The development of predictive biomarkers for RT efficacy is therefore of utmost importance for a clinically exploitable strategy to achieve tumor-specific radiosensitization. This review highlights the current status and possible opportunities in the modulation of four key processes to enhance radiation response in PCa by targeting the: (1) androgen signaling pathway; (2) hypoxic tumor cells and regions; (3) DNA damage response (DDR) pathway; and (4) abnormal extra-/intracell signaling pathways. In addition, we discuss how and which patients should be selected for biomarker-based clinical trials exploiting and validating these targeted treatment strategies with precision RT to improve cure rates in non-indolent, localized PCa. PMID:26909338

  18. Mechanistic insights related to the design and construction of lithium single ion conductors

    NASA Astrophysics Data System (ADS)

    Spahlinger, Gregory

    Lithium single ion conductors are a class of electrolytes, typically designed for lithium ion batteries, with the potential to improve the performance of these batteries. The benefits of single ion conductors arise out of the fact that their immobile anions are not capable of concentrating near the anode of the battery, causing an increase in resistance as the battery is discharged. Unfortunately lithium single ion conductors suffer severe drawbacks in their conductivity which have been attributed to diverse causes. Because of the low success rate of single ion conductors in the literature and previous work in the Baker group, I have chosen to investigate mechanistic questions related to the design and construction of these materials, without engineering new materials. An attractive design strategy for the screening of immobile anion moieties for single ion conductors would be the use of the copper catalyzed alkyne azide (CUAAC) "click" reaction in order to efficiently introduce anions onto a polymer or nanoparticle support in a way that is efficient and tunable. A variable added by this strategy would be the presence of a 1,2,3-triazole moiety which is without any significant precedent in the lithium ion electrolyte literature. In order to assess the impact of the triazole in on the conductivity of an electrolyte a series of model compounds were synthesized containing a variable number of triazoles in an otherwise poly(ethylene glycol) like oligomer chain. The model compounds were subjected to differential scanning calorimetry, electrochemical impedance spectroscopy, and in one case single crystal X-ray diffraction, and solvent shells were modeled for lithium with and without triazoles using ab initio quantum chemistry calculations. It was concluded that the triazole is not significantly stronger than an ether oxygen as a ligand in the electrolytes, however the triazole has a substantial dipole which exerts some deleterious effects on the conductivity, leading to

  19. New mechanistic insights in the NH3-SCR reactions at low temperature

    DOE PAGESBeta

    Ruggeri, Maria Pia; Selleri, Tomasso; Nova, Isabella; Tronconi, Enrico; Pihl, Josh A.; Toops, Todd J.; Partridge, Jr., William P.

    2016-05-06

    The present study is focused on the investigation of the low temperature Standard SCR reaction mechanism over Fe- and Cu-promoted zeolites. Different techniques are employed, including in situ DRIFTS, transient reaction analysis and chemical trapping techniques. The results present strong evidence of nitrite formation in the oxidative activation of NO and of their role in SCR reactions. These elements lead to a deeper understanding of the standard SCR chemistry at low temperature and can potentially improve the consistency of mechanistic mathematical models. Furthermore, comprehension of the mechanism on a fundamental level can contribute to the development of improved SCR catalysts.

  20. Pathways to neurodegeneration: mechanistic insights from GWAS in Alzheimer’s disease, Parkinson’s disease, and related disorders

    PubMed Central

    Ramanan, Vijay K; Saykin, Andrew J

    2013-01-01

    The discovery of causative genetic mutations in affected family members has historically dominated our understanding of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). Nevertheless, most cases of neurodegenerative disease are not explained by Mendelian inheritance of known genetic variants, but instead are thought to have a complex etiology with numerous genetic and environmental factors contributing to susceptibility. Although unbiased genome-wide association studies (GWAS) have identified novel associations to neurodegenerative diseases, most of these hits explain only modest fractions of disease heritability. In addition, despite the substantial overlap of clinical and pathologic features among major neurodegenerative diseases, surprisingly few GWAS-implicated variants appear to exhibit cross-disease association. These realities suggest limitations of the focus on individual genetic variants and create challenges for the development of diagnostic and therapeutic strategies, which traditionally target an isolated molecule or mechanistic step. Recently, GWAS of complex diseases and traits have focused less on individual susceptibility variants and instead have emphasized the biological pathways and networks revealed by genetic associations. This new paradigm draws on the hypothesis that fundamental disease processes may be influenced on a personalized basis by a combination of variants – some common and others rare, some protective and others deleterious – in key genes and pathways. Here, we review and synthesize the major pathways implicated in neurodegeneration, focusing on GWAS from the most prevalent neurodegenerative disorders, AD and PD. Using literature mining, we also discover a novel regulatory network that is enriched with AD- and PD-associated genes and centered on the SP1 and AP-1 (Jun/Fos) transcription factors. Overall, this pathway

  1. Phase Stability of t;#8242;-Zirconia-Based Thermal Barrier Coatings: Mechanistic Insights

    SciTech Connect

    Krogstad, Jessica A.; Krämer, Stephan; Lipkin, Don M.; Johnson, Curtis A.; Mitchell, David R.G.; Cairney, Julie M.; Levi, Carlos G.

    2011-11-07

    The temperature capability of yttria-stabilized zirconia thermal barrier coatings (TBCs) is ultimately tied to the rate of evolution of the 'nontransformable' t' phase into a depleted tetragonal form predisposed to the monoclinic transformation on cooling. The t' phase, however, has been shown to decompose in a small fraction of the time necessary to form the monoclinic phase. Instead, a modulated microstructure consisting of a coherent array of Y-rich and Y-lean lamellar phases develops early in the process, with mechanistic features suggestive of spinodal decomposition. Coarsening of this microstructure leads to loss of coherency and ultimately transformation into the monoclinic form, making the kinetics of this process, and not the initial decomposition, the critical factor in determining the phase stability of TBCs. Transmission electron microscopy is shown to be essential not only for characterizing the microstructure but also for proper interpretation of X-ray diffraction analysis.

  2. Glucosamine condensation catalyzed by 1-ethyl-3-methylimidazolium acetate: mechanistic insight from NMR spectroscopy.

    PubMed

    Jia, Lingyu; Pedersen, Christian Marcus; Qiao, Yan; Deng, Tiansheng; Zuo, Pingping; Ge, Wenzhi; Qin, Zhangfeng; Hou, Xianglin; Wang, Yingxiong

    2015-09-21

    The basic ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) could efficiently catalyze the conversion of 2-amino-2-deoxy-d-glucose (GlcNH2) into deoxyfructosazine (DOF) and fructosazine (FZ). Mechanistic investigation by NMR studies disclosed that [C2C1Im][OAc], exhibiting strong hydrogen bonding basicity, could coordinate with the hydroxyl and amino groups of GlcNH2via the promotion of hydrogen bonding in bifunctional activation of substrates and further catalyzing product formation, based on which a plausible reaction pathway involved in this homogeneous base-catalyzed reaction was proposed. Hydrogen bonding as an activation force, therefore, is responsible for the remarkable selectivity and rate enhancement observed. PMID:26278065

  3. Direct-acting antiviral agents for hepatitis C: structural and mechanistic insights.

    PubMed

    Götte, Matthias; Feld, Jordan J

    2016-06-01

    The treatment of HCV infection has evolved at an extremely rapid pace over the past few years. The development of direct-acting antiviral agents, which potently inhibit different stages in the viral life cycle, has led to the replacement of interferon with well-tolerated oral therapies with cure rates of >90% in most patient populations. Understanding the mechanisms of action of the various agents as well as related issues, including the molecular basis for resistance, helps to guide drug development and clinical use. In this Review, we provide a mechanistic description of NS3/4A protease inhibitors, nucleotide and non-nucleotide inhibitors of the NS5B viral polymerase and inhibitors of the NS5A protein, followed by a summary of clinical data from studies of each drug class alone and in combination. Remaining challenges in drug development efforts are also discussed. PMID:27147491

  4. Reactions of Co(III)-nitrosyl complexes with superoxide and their mechanistic insights.

    PubMed

    Kumar, Pankaj; Lee, Yong-Min; Park, Young Jun; Siegler, Maxime A; Karlin, Kenneth D; Nam, Wonwoo

    2015-04-01

    New Co(III)-nitrosyl complexes bearing N-tetramethylated cyclam (TMC) ligands, [(12-TMC)Co(III)(NO)](2+) (1) and [(13-TMC)Co(III)(NO)](2+) (2), were synthesized via [(TMC)Co(II)(CH3CN)](2+) + NO(g) reactions. Spectroscopic and structural characterization showed that these compounds bind the nitrosyl moiety in a bent end-on fashion. Complexes 1 and 2 reacted with KO2/2.2.2-cryptand to produce [(12-TMC)Co(II)(NO2)](+) (3) and [(13-TMC)Co(II)(NO2)](+) (4), respectively; these possess O,O'-chelated nitrito ligands. Mechanistic studies using (18)O-labeled superoxide ((18)O2(•-)) showed that one O atom in the nitrito ligand is derived from superoxide and the O2 produced comes from the other superoxide O atom. Evidence supporting the formation of a Co-peroxynitrite intermediate is also presented. PMID:25793706

  5. Mechanistic insights on immunosenescence and chronic immune activation in HIV-tuberculosis co-infection.

    PubMed

    Shankar, Esaki M; Velu, Vijayakumar; Kamarulzaman, Adeeba; Larsson, Marie

    2015-02-12

    Immunosenescence is marked by accelerated degradation of host immune responses leading to the onset of opportunistic infections, where senescent T cells show remarkably higher ontogenic defects as compared to healthy T cells. The mechanistic association between T-cell immunosenescence and human immunodeficiency virus (HIV) disease progression, and functional T-cell responses in HIV-tuberculosis (HIV-TB) co-infection remains to be elaborately discussed. Here, we discussed the association of immunosenescence and chronic immune activation in HIV-TB co-infection and reviewed the role played by mediators of immune deterioration in HIV-TB co-infection necessitating the importance of designing therapeutic strategies against HIV disease progression and pathogenesis. PMID:25674514

  6. Rhodium-Catalyzed Synthesis of Chiral Spiro-9-silabifluorenes by Dehydrogenative Silylation: Mechanistic Insights into the Construction of Tetraorganosilicon Stereocenters.

    PubMed

    Murai, Masahito; Takeuchi, Yutaro; Yamauchi, Kanae; Kuninobu, Yoichiro; Takai, Kazuhiko

    2016-04-18

    Mechanistic insight into the construction of quaternary silicon chiral centers by rhodium-catalyzed synthesis of spiro-9-silabifluorenes through dehydrogenative silylation is reported. The C2 -symmetric bisphosphine ligand, BINAP, was effective in controlling enantioselectivity, and axially chiral spiro-9-silabifluorenes were obtained in excellent yields with high enantiomeric excess. Monitoring of the reaction revealed the presence of a monohydrosilane intermediate as a mixture of two constitutional isomers. The reaction proceeded through two consecutive dehydrogenative silylations, and the absolute configuration was determined in the first silylative cyclization. Competitive reactions with electron-rich and electron-deficient dihydrosilanes indicated that the rate of silylative cyclization increased with decreasing electron density on the silicon atom of the starting dihydrosilane. Further investigation disclosed a rare interconversion between the two constitutional isomers of the monohydrosilane intermediate with retention of the absolute configuration. PMID:26970095

  7. Mechanistic Insights into the Oxidation of Substituted Phenols via Hydrogen Atom Abstraction by a Cupric–Superoxo Complex

    PubMed Central

    2015-01-01

    To obtain mechanistic insights into the inherent reactivity patterns for copper(I)–O2 adducts, a new cupric–superoxo complex [(DMM-tmpa)CuII(O2•–)]+ (2) [DMM-tmpa = tris((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)amine] has been synthesized and studied in phenol oxidation–oxygenation reactions. Compound 2 is characterized by UV–vis, resonance Raman, and EPR spectroscopies. Its reactions with a series of para-substituted 2,6-di-tert-butylphenols (p-X-DTBPs) afford 2,6-di-tert-butyl-1,4-benzoquinone (DTBQ) in up to 50% yields. Significant deuterium kinetic isotope effects and a positive correlation of second-order rate constants (k2) compared to rate constants for p-X-DTBPs plus cumylperoxyl radical reactions indicate a mechanism that involves rate-limiting hydrogen atom transfer (HAT). A weak correlation of (kBT/e) ln k2 versus Eox of p-X-DTBP indicates that the HAT reactions proceed via a partial transfer of charge rather than a complete transfer of charge in the electron transfer/proton transfer pathway. Product analyses, 18O-labeling experiments, and separate reactivity employing the 2,4,6-tri-tert-butylphenoxyl radical provide further mechanistic insights. After initial HAT, a second molar equiv of 2 couples to the phenoxyl radical initially formed, giving a CuII–OO–(ArO′) intermediate, which proceeds in the case of p-OR-DTBP substrates via a two-electron oxidation reaction involving hydrolysis steps which liberate H2O2 and the corresponding alcohol. By contrast, four-electron oxygenation (O–O cleavage) mainly occurs for p-R-DTBP which gives 18O-labeled DTBQ and elimination of the R group. PMID:24953129

  8. Mechanistic Insights into the Formation of Dodecanethiolate-Stabilized Magnetic Iridium Nanoparticles: Thiosulfate vs Thiol Ligands

    PubMed Central

    2015-01-01

    The synthesis of stable and isolable iridium nanoparticles with an average core size of ∼1.2 ± 0.3 nm was achieved by employing sodium S-dodecylthiosulfate as a ligand precursor during the modified Brust–Schiffrin reaction. Transmission electron microscopy (TEM) of the isolated Ir nanoparticles revealed a high degree of monodispersity. Further characterizations with 1H NMR, FT-IR, UV–vis spectroscopy, thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS) confirmed that the synthesized Ir nanoparticles are stabilized by dodecanethiolate ligands produced upon the adsorption/cleavage of S-dodecylthiosulfate on the growing Ir nanoparticle surface. By comparison, synthetic attempts employing dodecanethiol as a stabilizing ligand led to the formation of Ir-thiolate species (Ir(SR)3) as an intermediate and Ir-hydroxide species at the completion of reaction. Mechanistic investigations of these two reactions using S-dodecylthiosulfate and dodecanethiol provided deeper understandings on the novelty of thiosulfate ligands, which allow the successful formation of stable thiolate-capped Ir nanoparticles. Moreover, these Ir nanoparticles were shown to have strong magnetic properties. PMID:25018790

  9. Structure-based Mechanistic Insights into Terminal Amide Synthase in Nosiheptide-Represented Thiopeptides Biosynthesis

    PubMed Central

    Liu, Shanshan; Guo, Heng; Zhang, Tianlong; Han, Li; Yao, Pengfei; Zhang, Yan; Rong, Naiyan; Yu, Yi; Lan, Wenxian; Wang, Chunxi; Ding, Jianping; Wang, Renxiao; Liu, Wen; Cao, Chunyang

    2015-01-01

    Nosiheptide is a parent compound of thiopeptide family that exhibit potent activities against various bacterial pathogens. Its C-terminal amide formation is catalyzed by NosA, which is an unusual strategy for maturating certain thiopeptides by processing their precursor peptides featuring a serine extension. We here report the crystal structure of truncated NosA1-111 variant, revealing three key elements, including basic lysine 49 (K49), acidic glutamic acid 101 (E101) and flexible C-terminal loop NosA112-151, are crucial to the catalytic terminal amide formation in nosiheptide biosynthesis. The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions. The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation. The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI, and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis. PMID:26244829

  10. Mechanistic Insights into the Rate-Limiting Step in Purine-Specific Nucleoside Hydrolase.

    PubMed

    Chen, Nanhao; Zhao, Yuan; Lu, Jianing; Wu, Ruibo; Cao, Zexing

    2015-07-14

    A full enzymatic catalysis cycle in the inosine-adenosine-guanosine specific nucleoside hydrolase (IAG-NH) was assumed to be comprised of four steps: substrate binding, chemical reaction, base release, and ribose release. Nevertheless, the mechanistic details for the rate-limiting step of the entire enzymatic reaction are still unknown, even though the ribose release was likely to be the most difficult stage. Based on state-of-the-art quantum mechanics and molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the ribose release process can be divided into two steps: "ribose dissociation" and "ribose release". The "ribose dissociation" includes "cleavage" and "exchange" stages, in which a metastable 6-fold intermediate will recover to an 8-fold coordination shell of Ca(2+) as observed in apo- IAG-NH. Extensive random acceleration molecular dynamics and MD simulations have been employed to verify plausible release channels, and the estimated barrier for the rate-determining step of the entire reaction is 13.0 kcal/mol, which is comparable to the experimental value of 16.7 kcal/mol. Moreover, the gating mechanism arising from loop1 and loop2, as well as key residues around the active pocket, has been found to play an important role in manipulating the ribose release. PMID:26575755

  11. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design

    PubMed Central

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M.

    2016-01-01

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared – non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents. PMID:27147293

  12. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design

    NASA Astrophysics Data System (ADS)

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M.

    2016-05-01

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared – non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

  13. Carcinogenicity and mechanistic insights on the behavior of epoxides and epoxide-forming chemicals.

    PubMed

    Melnick, Ronald L

    2002-12-01

    Many epoxides and their precursors are high production volume chemicals that have major uses in the polymer industry and as intermediates in the manufacture of other chemicals. Several of these chemicals were demonstrated to be carcinogenic in laboratory animal studies conducted by the Ramazzini Foundation (e.g., vinyl chloride, acrylonitrile, styrene, styrene oxide, and benzene) and by the National Toxicology Program (e.g., ethylene oxide, 1,3-butadiene, isoprene, chloroprene, acrylonitrile, glycidol, and benzene). The most common sites of tumor induction were lung, liver, harderian gland, and circulatory system in mice; Zymbal's gland and brain in rats; and mammary gland and forestomach in both species. Differences in cancer outcome among studies of epoxide chemicals may be related to differences in study design (e.g., dose, duration, and route of exposure; observation period; animal strains), as well as biological factors affecting target organ dosimetry of the DNA-reactive epoxide (toxicokinetics) and tissue response (toxicodynamics). N7-Alkylguanine, N1-alkyladenine, and cyclic etheno adducts, as well as K-ras and p53 mutations, have been detected in animals and/or workers exposed to several of these chemicals. The classifications of these chemical carcinogens by IARC and NTP are based on animal and human data and results of mechanistic studies. Reducing occupational and environmental exposures to these chemicals will certainly reduce human cancer risks. PMID:12562636

  14. Crystal structure of the sodium–proton antiporter NhaA dimer and new mechanistic insights

    PubMed Central

    Lee, Chiara; Yashiro, Shoko; Dotson, David L.; Uzdavinys, Povilas; Iwata, So; Sansom, Mark S.P.; von Ballmoos, Christoph

    2014-01-01

    Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA. PMID:25422503

  15. Mechanistic insights into the effects of climate change on larval cod.

    PubMed

    Kristiansen, Trond; Stock, Charles; Drinkwater, Kenneth F; Curchitser, Enrique N

    2014-05-01

    Understanding the biophysical mechanisms that shape variability in fisheries recruitment is critical for estimating the effects of climate change on fisheries. In this study, we used an Earth System Model (ESM) and a mechanistic individual-based model (IBM) for larval fish to analyze how climate change may impact the growth and survival of larval cod in the North Atlantic. We focused our analysis on five regions that span the current geographical range of cod and are known to contain important spawning populations. Under the SRES A2 (high emissions) scenario, the ESM-projected surface ocean temperatures are expected to increase by >1 °C for 3 of the 5 regions, and stratification is expected to increase at all sites between 1950-1999 and 2050-2099. This enhanced stratification is projected to decrease large (>5 μm ESD) phytoplankton productivity and mesozooplankton biomass at all 5 sites. Higher temperatures are projected to increase larval metabolic costs, which combined with decreased food resources will reduce larval weight, increase the probability of larvae dying from starvation and increase larval exposure to visual and invertebrate predators at most sites. If current concentrations of piscivore and invertebrate predators are maintained, larval survival is projected to decrease at all five sites by 2050-2099. In contrast to past observed responses to climate variability in which warm anomalies led to better recruitment in cold-water stocks, our simulations indicated that reduced prey availability under climate change may cause a reduction in larval survival despite higher temperatures in these regions. In the lower prey environment projected under climate change, higher metabolic costs due to higher temperatures outweigh the advantages of higher growth potential, leading to negative effects on northern cod stocks. Our results provide an important first large-scale assessment of the impacts of climate change on larval cod in the North Atlantic. PMID

  16. Understanding impacts of climatic extremes on diarrheal disease epidemics: Insights from mechanistic disease propagation models

    NASA Astrophysics Data System (ADS)

    Jutla, A.; Akanda, A. S.; Colwell, R. R.

    2013-12-01

    An epidemic outbreak of diarrheal diseases (primarily cholera) in Haiti in 2010 is a reminder that our understanding on disease triggers, transmission and spreading mechanisms is incomplete. Cholera can occur in two forms - epidemic (defined as sudden outbreak in a historically disease free region) and endemic (recurrence and persistence of the disease for several consecutive years). Examples of countries with epidemic cholera include Pakistan (2008), Congo (2008), and most recently Haiti (2010). A significant difference between endemic and epidemic regions is the mortality rate, i.e., 1% or lower in an endemic regions versus 3-7% during recent epidemic outbreaks. A fundamentally transformational approach - a warning system with several months prediction lead time - is needed to prevent disease outbreak and minimize its impact on population. Lack of information on spatial and temporal variability of disease incidence as well as transmission in human population continues to be significant challenge in the development of early-warning systems for cholera. Using satellite data on regional hydroclimatic processes, water and sanitation infrastructure indices, and biological pathogen growth information, here we present a Simple, Mechanistic, Adaptive, Remote sensing based Regional Transmission or SMART model to (i) identify regions of potential cholera outbreaks and (ii) quantify mechanism of spread of the disease in previously disease free region. Our results indicate that epidemic regions are located near regional rivers and are characterized by sporadic outbreaks, which are likely to be initiated during episodes of prevailing warm air temperature with low river flows, creating favorable environmental conditions for the growth of cholera bacteria. Heavy rainfall, through inundation or breakdown of sanitary infrastructure, accelerates interaction between contaminated water and human activities, resulting in an epidemic. We discuss the above findings in light of

  17. Mechanistic Insight into the Chemical Exfoliation and Functionalization of Ti3C2 MXene.

    PubMed

    Srivastava, Pooja; Mishra, Avanish; Mizuseki, Hiroshi; Lee, Kwang-Ryeol; Singh, Abhishek K

    2016-09-14

    MXene, a two-dimensional layer of transition metal carbides/nitrides, showed great promise for energy storage, sensing, and electronic applications. MXene are chemically exfoliated from the bulk MAX phase; however, mechanistic understanding of exfoliation and subsequent functionalization of these technologically important materials is still lacking. Here, using density-functional theory we show that exfoliation of Ti3C2 MXene proceeds via HF insertion through edges of Ti3AlC2 MAX phase. Spontaneous dissociation of HF and subsequent termination of edge Ti atoms by H/F weakens Al-MXene bonds. Consequent opening of the interlayer gap allows further insertion of HF that leads to the formation of AlF3 and H2, which eventually come out of the MAX, leaving fluorinated MXene behind. Density of state and electron localization function shows robust binding between F/OH and Ti, which makes it very difficult to obtain controlled functionalized or pristine MXene. Analysis of the calculated Gibbs free energy (ΔG) shows fully fluorinated MXene to be lowest in energy, whereas the formation of pristine MXene is thermodynamically least favorable. In the presence of water, mixed functionalized Ti3C2Fx(OH)1-x (x ranges from 0 to 1) MXene can be obtained. The ΔG values for the mixed functionalized MXenes are very close in energy, indicating the random and nonuniform functionalization of MXene. The microscopic understanding gained here unveils the challenges in exfoliation and controlling the functionalization of MXene, which is essential for its practical application. PMID:27537784

  18. A genetic cause of Alzheimer disease: mechanistic insights from Down syndrome

    PubMed Central

    Wiseman, Frances K.; Al-Janabi, Tamara; Hardy, John; Karmiloff-Smith, Annette; Nizetic, Dean; Tybulewicz, Victor L. J.; Fisher, Elizabeth M. C.; Strydom, André

    2015-01-01

    Down syndrome, which arises in individuals carrying an extra copy of chromosome 21, is associated with a greatly increased risk of early-onset Alzheimer disease. It is thought that this risk is conferred by the presence of three copies of the gene encoding amyloid precursor protein (APP) — an Alzheimer disease risk factor — although the possession of extra copies of other chromosome 21 genes may also play a part. Further study of the mechanisms underlying the development of Alzheimer disease in people with Down syndrome could provide insights into the mechanisms that cause dementia in the general population. PMID:26243569

  19. Structural and mechanistic insights into methane oxidation by particulate methane monooxygenase.

    PubMed

    Balasubramanian, Ramakrishnan; Rosenzweig, Amy C

    2007-07-01

    Particulate methane monooxygense (pMMO) is an integral membrane copper-containing enzyme that converts methane to methanol. Knowledge of how pMMO selectively oxidizes methane under ambient conditions could impact the development of new catalysts. The crystal structure of Methylococcus capsulatus (Bath) pMMO reveals the composition and location of three metal centers. Spectroscopic data provide insight into the coordination environments and oxidation states of these metal centers. These results, combined with computational studies and comparisons to relevant systems, are discussed in the context of identifying the most likely site for O 2 activation. PMID:17444606

  20. Stereochemistry and Mechanistic Insight in the [2(k)+2(i)+2(i)] Annulations of Ketenes and Imines.

    PubMed

    Yang, Zhanhui; He, Wei; Cheng, Baoxiang; Xu, Jiaxi

    2016-06-01

    The stereochemistry and mechanistic insight in the annulations of one ketene molecule with two imine molecules ([2(k)+2(i)+2(i)] annulation) are studied by using six-membered 3,4-dihydroisoquinoline as an imine probe. A concerted hetero-Diels-Alder cycloaddition mechanism is proposed to explain the stereochemical outcomes. In most cases, the zwitterionic 2-aza-1,3-butadiene-type intermediates, generated from ketenes and imines, undergo endo hetero-Diels-Alder cycloaddition with the second imine molecule. For ketenes with electron-donating substituents, (2,4)-cis-(4,5)-cis-[2(k)+2(i)+2(i)] annuladducts formed stereospecifically, while, for ketenes with electron-accepting substituents, (2,4)-cis-(4,5)-trans-[2(k)+2(i)+2(i)] annuladducts are generated stereospecifically. The [2(k)+2(i)+2(i)] annulations of aryloxyketenes and 3,4-dihydroisoquinoline give stereodivergent products due to the occurrence of the stepwise nucleophilic annulation. However, in the [2(k)+2(i)+2(i)] annulations of seven-membered cyclic imine dibenzo[b,f][1,4]oxazepine, the zwitterionic aza-butadiene-type intermediates exclusively undergo exo hetero-Diels-Alder cycloadditions with another molecule of imine to yield (2,4)-trans-(4,5)-trans-[2(k)+2(i)+2(i)] annuladducts stereospecifically, regardless of the ketene substituents. The mechanistic model not only discloses the nature of the [2(k)+2(i)+2(i)] annulations, but also can be used to explain and predict the stereochemistry of the [2(k)+2(i)+2(i)] annuladducts from different ketenes and imines. PMID:27166848

  1. C-H Bond Oxidation Catalyzed by an Imine-Based Iron Complex: A Mechanistic Insight.

    PubMed

    Olivo, Giorgio; Nardi, Martina; Vìdal, Diego; Barbieri, Alessia; Lapi, Andrea; Gómez, Laura; Lanzalunga, Osvaldo; Costas, Miquel; Di Stefano, Stefano

    2015-11-01

    A family of imine-based nonheme iron(II) complexes (LX)2Fe(OTf)2 has been prepared, characterized, and employed as C-H oxidation catalysts. Ligands LX (X = 1, 2, 3, and 4) stand for tridentate imine ligands resulting from spontaneous condensation of 2-pycolyl-amine and 4-substituted-2-picolyl aldehydes. Fast and quantitative formation of the complex occurs just upon mixing aldehyde, amine, and Fe(OTf)2 in a 2:2:1 ratio in acetonitrile solution. The solid-state structures of (L1)2Fe(OTf)(ClO4) and (L3)2Fe(OTf)2 are reported, showing a low-spin octahedral iron center, with the ligands arranged in a meridional fashion. (1)H NMR analyses indicate that the solid-state structure and spin state is retained in solution. These analyses also show the presence of an amine-imine tautomeric equilibrium. (LX)2Fe(OTf)2 efficiently catalyze the oxidation of alkyl C-H bonds employing H2O2 as a terminal oxidant. Manipulation of the electronic properties of the imine ligand has only a minor impact on efficiency and selectivity of the oxidative process. A mechanistic study is presented, providing evidence that C-H oxidations are metal-based. Reactions occur with stereoretention at the hydroxylated carbon and selectively at tertiary over secondary C-H bonds. Isotopic labeling analyses show that H2O2 is the dominant origin of the oxygen atoms inserted in the oxygenated product. Experimental evidence is provided that reactions involve initial oxidation of the complexes to the ferric state, and it is proposed that a ligand arm dissociates to enable hydrogen peroxide binding and activation. Selectivity patterns and isotopic labeling studies strongly suggest that activation of hydrogen peroxide occurs by heterolytic O-O cleavage, without the assistance of a cis-binding water or alkyl carboxylic acid. The sum of these observations provides sound evidence that controlled activation of H2O2 at (LX)2Fe(OTf)2 differs from that occurring in biomimetic iron catalysts described to date. PMID

  2. Mechanistic insights into chemical and photochemical transformations of bismuth vanadate photoanodes

    PubMed Central

    Toma, Francesca M.; Cooper, Jason K.; Kunzelmann, Viktoria; McDowell, Matthew T.; Yu, Jie; Larson, David M.; Borys, Nicholas J.; Abelyan, Christine; Beeman, Jeffrey W.; Yu, Kin Man; Yang, Jinhui; Chen, Le; Shaner, Matthew R.; Spurgeon, Joshua; Houle, Frances A.; Persson, Kristin A.; Sharp, Ian D.

    2016-01-01

    Artificial photosynthesis relies on the availability of semiconductors that are chemically stable and can efficiently capture solar energy. Although metal oxide semiconductors have been investigated for their promise to resist oxidative attack, materials in this class can suffer from chemical and photochemical instability. Here we present a methodology for evaluating corrosion mechanisms and apply it to bismuth vanadate, a state-of-the-art photoanode. Analysis of changing morphology and composition under solar water splitting conditions reveals chemical instabilities that are not predicted from thermodynamic considerations of stable solid oxide phases, as represented by the Pourbaix diagram for the system. Computational modelling indicates that photoexcited charge carriers accumulated at the surface destabilize the lattice, and that self-passivation by formation of a chemically stable surface phase is kinetically hindered. Although chemical stability of metal oxides cannot be assumed, insight into corrosion mechanisms aids development of protection strategies and discovery of semiconductors with improved stability. PMID:27377305

  3. Mechanistic insights into chemical and photochemical transformations of bismuth vanadate photoanodes.

    PubMed

    Toma, Francesca M; Cooper, Jason K; Kunzelmann, Viktoria; McDowell, Matthew T; Yu, Jie; Larson, David M; Borys, Nicholas J; Abelyan, Christine; Beeman, Jeffrey W; Yu, Kin Man; Yang, Jinhui; Chen, Le; Shaner, Matthew R; Spurgeon, Joshua; Houle, Frances A; Persson, Kristin A; Sharp, Ian D

    2016-01-01

    Artificial photosynthesis relies on the availability of semiconductors that are chemically stable and can efficiently capture solar energy. Although metal oxide semiconductors have been investigated for their promise to resist oxidative attack, materials in this class can suffer from chemical and photochemical instability. Here we present a methodology for evaluating corrosion mechanisms and apply it to bismuth vanadate, a state-of-the-art photoanode. Analysis of changing morphology and composition under solar water splitting conditions reveals chemical instabilities that are not predicted from thermodynamic considerations of stable solid oxide phases, as represented by the Pourbaix diagram for the system. Computational modelling indicates that photoexcited charge carriers accumulated at the surface destabilize the lattice, and that self-passivation by formation of a chemically stable surface phase is kinetically hindered. Although chemical stability of metal oxides cannot be assumed, insight into corrosion mechanisms aids development of protection strategies and discovery of semiconductors with improved stability. PMID:27377305

  4. Mechanistic insights into chemical and photochemical transformations of bismuth vanadate photoanodes

    NASA Astrophysics Data System (ADS)

    Toma, Francesca M.; Cooper, Jason K.; Kunzelmann, Viktoria; McDowell, Matthew T.; Yu, Jie; Larson, David M.; Borys, Nicholas J.; Abelyan, Christine; Beeman, Jeffrey W.; Yu, Kin Man; Yang, Jinhui; Chen, Le; Shaner, Matthew R.; Spurgeon, Joshua; Houle, Frances A.; Persson, Kristin A.; Sharp, Ian D.

    2016-07-01

    Artificial photosynthesis relies on the availability of semiconductors that are chemically stable and can efficiently capture solar energy. Although metal oxide semiconductors have been investigated for their promise to resist oxidative attack, materials in this class can suffer from chemical and photochemical instability. Here we present a methodology for evaluating corrosion mechanisms and apply it to bismuth vanadate, a state-of-the-art photoanode. Analysis of changing morphology and composition under solar water splitting conditions reveals chemical instabilities that are not predicted from thermodynamic considerations of stable solid oxide phases, as represented by the Pourbaix diagram for the system. Computational modelling indicates that photoexcited charge carriers accumulated at the surface destabilize the lattice, and that self-passivation by formation of a chemically stable surface phase is kinetically hindered. Although chemical stability of metal oxides cannot be assumed, insight into corrosion mechanisms aids development of protection strategies and discovery of semiconductors with improved stability.

  5. New developments providing mechanistic insight into the impact of the microbiota on allergic disease.

    PubMed

    McCoy, Kathy D; Köller, Yasmin

    2015-08-01

    The increase in allergic diseases over the past several decades is correlated with changes in the composition and diversity of the intestinal microbiota. Microbial-derived signals are critical for instructing the developing immune system and conversely, immune regulation can impact the microbiota. Perturbations in the microbiota composition may be especially important during early-life when the immune system is still developing, resulting in a critical window of opportunity for instructing the immune system. This review highlights recent studies investigating the role of the microbiome in susceptibility or development of allergic diseases with a focus on animal models that provide insight into the mechanisms and pathways involved. Identification of a causal link between reduced microbial diversity or altered microbial composition and increased susceptibility to immune-mediated diseases will hopefully pave the way for better preventive therapies. PMID:25988860

  6. Iron-Mediated Oxidation of Methoxyhydroquinone under Dark Conditions: Kinetic and Mechanistic Insights.

    PubMed

    Yuan, Xiu; Davis, James A; Nico, Peter S

    2016-02-16

    Despite the biogeochemical significance of the interactions between natural organic matter (NOM) and iron species, considerable uncertainty still remains as to the exact processes contributing to the rates and extents of complexation and redox reactions between these important and complex environmental components. Investigations on the reactivity of low-molecular-weight quinones, which are believed to be key redox active compounds within NOM, toward iron species, could provide considerable insight into the kinetics and mechanisms of reactions involving NOM and iron. In this study, the oxidation of 2-methoxyhydroquinone (MH2Q) by ferric iron (Fe(III)) under dark conditions in the absence and presence of oxygen was investigated within a pH range of 4-6. Although Fe(III) was capable of stoichiometrically oxidizing MH2Q under anaerobic conditions, catalytic oxidation of MH2Q was observed in the presence of O2 due to further cycling between oxygen, semiquinone radicals, and iron species. A detailed kinetic model was developed to describe the predominant mechanisms, which indicated that both the undissociated and monodissociated anions of MH2Q were kinetically active species toward Fe(III) reduction, with the monodissociated anion being the key species accounting for the pH dependence of the oxidation. The generated radical intermediates, namely semiquinone and superoxide, are of great importance in reaction-chain propagation. The kinetic model may provide critical insight into the underlying mechanisms of the thermodynamic and kinetic characteristics of metal-organic interactions and assist in understanding and predicting the factors controlling iron and organic matter transformation and bioavailability in aquatic systems. PMID:26789138

  7. Turbulent Flow Enhancement by Polyelectrolyte Additives: Mechanistic Implications for Drag Reduction.

    NASA Astrophysics Data System (ADS)

    Wagger, David Leonard

    1992-01-01

    The drag reduction phenomenon was experimentally studied in two pipes, of diameters 1.46 and 1.02 cm, using seven polyelectrolytic HPAM additives, with molecular weights from 1 to 20 times 10^6 g/mole and degree of backbone hydrolysis from 8 to 60%, at concentrations from 1 to 1000 wppm, in saline solutions containing from 0.3 to 0.00001 N NaCl. Both laminar and turbulent flow behavior were greatly influenced by salinity-induced changes in the initial conformation of the HPAM additives. Initially collapsed, random-coiling conformations exhibited Newtonian laminar flow and Type-A turbulent drag reduction, while initially extended conformations exhibited shear-thinning in laminar flow and Type-B turbulent drag reduction. The gross-flow physics of Type-B drag reduction were delineated. A characteristic "ladder" structure prevailed, with polymeric regime segments that were roughly parallel to, but shifted upward from, the Prandtl-Karman line. In the polymeric regime, both Type-A fan and Type -B ladder structures were essentially independent of pipe diameter, and were scaled by the wall shear stress. The wall shear stress also scaled degradation during drag reduction. New onset and slope increment correlations were presented for Type-A drag reduction by HPAM additives. In Type-B drag reduction, flow enhancement was found proportional to additive concentration, and the intrinsic slip, Sigma = S^'/(c/M _{rm w}), varied roughly as the third power of backbone chain links N_ {rm bb}. New intrinsic slip and retro-onset correlations were presented for Type-B drag reduction by HPAM additives. Analysis of Type-B literature revealed a wide range of additive efficacies, with specific slips S^'/c from 0.0001 to 4. For the most effective additives, HPAM and asbestos fibers, the additive-pervaded volume fraction per unit flow enhancement, X_{rm v} /S^' ~ 3000, implied that these additives align during drag reduction. The slip ratio R_{rm sc}, which is the relative flow enhancement

  8. Antimicrobial combinations: Bliss independence and Loewe additivity derived from mechanistic multi-hit models.

    PubMed

    Baeder, Desiree Y; Yu, Guozhi; Hozé, Nathanaël; Rolff, Jens; Regoes, Roland R

    2016-05-26

    Antimicrobial peptides (AMPs) and antibiotics reduce the net growth rate of bacterial populations they target. It is relevant to understand if effects of multiple antimicrobials are synergistic or antagonistic, in particular for AMP responses, because naturally occurring responses involve multiple AMPs. There are several competing proposals describing how multiple types of antimicrobials add up when applied in combination, such as Loewe additivity or Bliss independence. These additivity terms are defined ad hoc from abstract principles explaining the supposed interaction between the antimicrobials. Here, we link these ad hoc combination terms to a mathematical model that represents the dynamics of antimicrobial molecules hitting targets on bacterial cells. In this multi-hit model, bacteria are killed when a certain number of targets are hit by antimicrobials. Using this bottom-up approach reveals that Bliss independence should be the model of choice if no interaction between antimicrobial molecules is expected. Loewe additivity, on the other hand, describes scenarios in which antimicrobials affect the same components of the cell, i.e. are not acting independently. While our approach idealizes the dynamics of antimicrobials, it provides a conceptual underpinning of the additivity terms. The choice of the additivity term is essential to determine synergy or antagonism of antimicrobials.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'. PMID:27160596

  9. Mechanistic insight into the ability of American ginseng to suppress colon cancer associated with colitis

    PubMed Central

    Cui, Xiangli; Jin, Yu; Poudyal, Deepak; Chumanevich, Alexander A.; Davis, Tia; Windust, Anthony; Hofseth, Anne; Wu, Wensong; Habiger, Joshua; Pena, Edsel; Wood, Patricia; Nagarkatti, Mitzi; Nagarkatti, Prakash S.; Hofseth, Lorne

    2010-01-01

    We have recently shown that American ginseng (AG) prevents and treats mouse colitis. Because both mice and humans with chronic colitis have a high colon cancer risk, we tested the hypothesis that AG can be used to prevent colitis-driven colon cancer. Using the azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of ulcerative colitis, we show that AG can suppress colon cancer associated with colitis. To explore the molecular mechanisms of the anticancer effects of AG, we also carried out antibody array experiments on colon cells isolated at a precancerous stage. We found there were 82 protein end points that were either significantly higher (41 proteins) or significantly lower (41 proteins) in the AOM + DSS group compared with the AOM-alone (control) group. In contrast, there were only 19 protein end points that were either significantly higher (10 proteins) or significantly lower (9 proteins) in the AOM + DSS + AG group compared with the AOM-alone (control) group. Overall, these results suggest that AG keeps the colon environment in metabolic equilibrium when mice are treated with AOM + DSS and gives insight into the mechanisms by which AG protects from colon cancer associated with colitis. PMID:20729391

  10. Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

    NASA Astrophysics Data System (ADS)

    Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

    2015-07-01

    Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

  11. New mechanistic insight in the thermal helix inversion of second-generation molecular motors.

    PubMed

    Klok, Martin; Walko, Martin; Geertsema, Edzard M; Ruangsupapichat, Nopporn; Kistemaker, Jos C M; Meetsma, Auke; Feringa, Ben L

    2008-01-01

    The introduction of dibenzocyclohepten-5-ylidene as part of a unidirectional light-driven molecular motor allows a more complete picture of the pathway of thermal helix inversion to be developed. The most stable conformation is similar to that found in related motors in that it has, overall, an anti-folded structure with the substituent at the stereogenic centre adopting an axial orientation. Photochemical cis/trans isomerisation at -40 degrees C results in the formation of an isomer in a syn-folded conformation with the methyl group in an axial orientation. This contrasts with previous studies on related molecular rotary motors. The conformation of the higher energy intermediate typically observed for this class of compound is the anti-folded conformation, in which the methyl group is in an equatorial orientation. This conformation is available through an energetically uphill upper half ring inversion of the observed photochemical product. However, this pathway competes with a second process that leads to the more stable anti-folded conformation in which the methyl group is oriented axially. It has been shown that the conformations and pathways available for second-generation molecular motors can be described by using similar overall geometries. Differences in the metastable high-energy species are attributable to the relative energy and position on the reaction coordinate of the transition states. Kinetic studies on these new molecular motors thus provide important insights into the conformational dynamics of the rotation cycle. PMID:18979464

  12. Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

    PubMed Central

    Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, XinMei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

    2015-01-01

    Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur–DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur–feoAB1 operator complex and the Fur–Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs. PMID:26134419

  13. Structure of the Vacuolar H+-ATPase Rotary Motor Reveals New Mechanistic Insights

    PubMed Central

    Rawson, Shaun; Phillips, Clair; Huss, Markus; Tiburcy, Felix; Wieczorek, Helmut; Trinick, John; Harrison, Michael A.; Muench, Stephen P.

    2015-01-01

    Summary Vacuolar H+-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases. PMID:25661654

  14. Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

    PubMed

    Matsuura, Yoshiyuki

    2016-05-22

    Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza. PMID:26519791

  15. Mechanistic insight into the Staudinger reaction catalyzed by N-heterocyclic carbenes.

    PubMed

    Hans, Morgan; Wouters, Johan; Demonceau, Albert; Delaude, Lionel

    2013-07-15

    Four zwitterions were prepared by treating 1,3-dimesitylimidazolin-2-ylidene (SIMes) or 1,3-dimesitylimidazol-2-ylidene (IMes) with either N-tosyl benzaldimine or diphenylketene. They were isolated in high yields and characterized by IR and NMR spectroscopy. The molecular structures of three of them were determined by using X-ray crystallography and their thermal stability was monitored by using thermogravimetric analysis. The imidazol(in)ium-2-amides were rather labile white solids that did not show any tendency to tautomerize into the corresponding 1,2,2-triaminoethene derivatives. They displayed a mediocre catalytic activity in the Staudinger reaction of N-tosyl benzaldimine with diphenylketene. In contrast, the imidazol(in)ium-2-enolates were orange-red crystalline materials that remained stable over extended periods of time. Despite their greater stability, these zwitterions turned out to be efficient promoters for the model cycloaddition under scrutiny. As a matter of fact, their catalytic activity matched those recorded with the free carbenes. Altogether, these results provide strong experimental insight into the mechanism of the Staudinger reaction catalyzed by N-heterocyclic carbenes. They also highlight the superior catalytic activity of the imidazole-based carbene IMes compared with its saturated analogue SIMes in the reaction under consideration. PMID:23754585

  16. Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function

    DOE PAGESBeta

    Sun, Jingchuan; Li, Huilin; Fernandez-Cid, Alejandra; Riera, Alberto; Tognetti, Sivia; Yuan, Zuanning; Stillman, Bruce; Speck, Christian

    2014-10-15

    Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex andmore » an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.« less

  17. The Microbial Signature Provides Insight into the Mechanistic Basis of Coral Success across Reef Habitats

    PubMed Central

    Leggat, William; Bongaerts, Pim

    2016-01-01

    ABSTRACT For ecosystems vulnerable to environmental change, understanding the spatiotemporal stability of functionally crucial symbioses is fundamental to determining the mechanisms by which these ecosystems may persist. The coral Pachyseris speciosa is a successful environmental generalist that succeeds in diverse reef habitats. The generalist nature of this coral suggests it may have the capacity to form functionally significant microbial partnerships to facilitate access to a range of nutritional sources within different habitats. Here, we propose that coral is a metaorganism hosting three functionally distinct microbial interactions: a ubiquitous core microbiome of very few symbiotic host-selected bacteria, a microbiome of spatially and/or regionally explicit core microbes filling functional niches (<100 phylotypes), and a highly variable bacterial community that is responsive to biotic and abiotic processes across spatial and temporal scales (>100,000 phylotypes). We find that this coral hosts upwards of 170,000 distinct phylotypes and provide evidence for the persistence of a select group of bacteria in corals across environmental habitats of the Great Barrier Reef and Coral Sea. We further show that a higher number of bacteria are consistently associated with corals on mesophotic reefs than on shallow reefs. An increase in microbial diversity with depth suggests reliance by this coral on bacteria for nutrient acquisition on reefs exposed to nutrient upwelling. Understanding the complex microbial communities of host organisms across broad biotic and abiotic environments as functionally distinct microbiomes can provide insight into those interactions that are ubiquitous niche symbioses and those that provide competitive advantage within the hosts’ environment. PMID:27460792

  18. Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

    PubMed Central

    Sun, Jingchuan; Fernandez-Cid, Alejandra; Riera, Alberto; Tognetti, Silvia; Yuan, Zuanning

    2014-01-01

    Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability. PMID:25319829

  19. High pressure thermal inactivation of Clostridium botulinum type E endospores – kinetic modeling and mechanistic insights

    PubMed Central

    Lenz, Christian A.; Reineke, Kai; Knorr, Dietrich; Vogel, Rudi F.

    2015-01-01

    Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT) processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores. We investigated the inactivation of C. botulinum type E spores by (near) isothermal HPT treatments at 300–1200 MPa at 30–75°C for 1 s to 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone), large heat susceptible (HPT-induced germinated) or lysozyme-dependently germinable (damaged coat layer) spore fractions were not detected. Inactivation followed first order kinetics. Dipicolinic acid release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective) physiologic-like (similar to nutrient-induced) germination at ≤450 MPa/≤45°C and non-physiological germination at >500 MPa/>60–70°C. Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores compared with the resistance of spores from other C. botulinum types could allow for the implementation of

  20. Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function

    SciTech Connect

    Sun, Jingchuan; Li, Huilin; Fernandez-Cid, Alejandra; Riera, Alberto; Tognetti, Sivia; Yuan, Zuanning; Stillman, Bruce; Speck, Christian

    2014-10-15

    Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

  1. Mechanistic Insights into the Role of C-Type Lectin Receptor/CARD9 Signaling in Human Antifungal Immunity

    PubMed Central

    Drummond, Rebecca A.; Lionakis, Michail S.

    2016-01-01

    Human CARD9 deficiency is an autosomal recessive primary immunodeficiency disorder caused by biallelic mutations in the gene CARD9, which encodes a signaling protein that is found downstream of many C-type lectin receptors (CLRs). CLRs encompass a large family of innate recognition receptors, expressed predominantly by myeloid and epithelial cells, which bind fungal carbohydrates and initiate antifungal immune responses. Accordingly, human CARD9 deficiency is associated with the spontaneous development of persistent and severe fungal infections that primarily localize to the skin and subcutaneous tissue, mucosal surfaces and/or central nervous system (CNS). In the last 3 years, more than 15 missense and nonsense CARD9 mutations have been reported which associate with the development of a wide spectrum of fungal infections caused by a variety of fungal organisms. The mechanisms by which CARD9 provides organ-specific protection against these fungal infections are now emerging. In this review, we summarize recent immunological and clinical advances that have provided significant mechanistic insights into the pathogenesis of human CARD9 deficiency. We also discuss how genetic mutations in CARD9-coupled receptors (Dectin-1, Dectin-2) and CARD9-binding partners (MALT1, BCL10) affect human antifungal immunity relative to CARD9 deficiency, and we highlight major understudied research questions which merit future investigation. PMID:27092298

  2. Fluid Mechanics in Dentinal Microtubules Provides Mechanistic Insights into the Difference between Hot and Cold Dental Pain

    PubMed Central

    Lin, Min; Luo, Zheng Yuan; Bai, Bo Feng; Xu, Feng; Lu, Tian Jian

    2011-01-01

    Dental thermal pain is a significant health problem in daily life and dentistry. There is a long-standing question regarding the phenomenon that cold stimulation evokes sharper and more shooting pain sensations than hot stimulation. This phenomenon, however, outlives the well-known hydrodynamic theory used to explain dental thermal pain mechanism. Here, we present a mathematical model based on the hypothesis that hot or cold stimulation-induced different directions of dentinal fluid flow and the corresponding odontoblast movements in dentinal microtubules contribute to different dental pain responses. We coupled a computational fluid dynamics model, describing the fluid mechanics in dentinal microtubules, with a modified Hodgkin-Huxley model, describing the discharge behavior of intradental neuron. The simulated results agreed well with existing experimental measurements. We thence demonstrated theoretically that intradental mechano-sensitive nociceptors are not “equally sensitive” to inward (into the pulp) and outward (away from the pulp) fluid flows, providing mechanistic insights into the difference between hot and cold dental pain. The model developed here could enable better diagnosis in endodontics which requires an understanding of pulpal histology, neurology and physiology, as well as their dynamic response to the thermal stimulation used in dental practices. PMID:21448459

  3. Mechanistic Insights into the Role of C-Type Lectin Receptor/CARD9 Signaling in Human Antifungal Immunity.

    PubMed

    Drummond, Rebecca A; Lionakis, Michail S

    2016-01-01

    Human CARD9 deficiency is an autosomal recessive primary immunodeficiency disorder caused by biallelic mutations in the gene CARD9, which encodes a signaling protein that is found downstream of many C-type lectin receptors (CLRs). CLRs encompass a large family of innate recognition receptors, expressed predominantly by myeloid and epithelial cells, which bind fungal carbohydrates and initiate antifungal immune responses. Accordingly, human CARD9 deficiency is associated with the spontaneous development of persistent and severe fungal infections that primarily localize to the skin and subcutaneous tissue, mucosal surfaces and/or central nervous system (CNS). In the last 3 years, more than 15 missense and nonsense CARD9 mutations have been reported which associate with the development of a wide spectrum of fungal infections caused by a variety of fungal organisms. The mechanisms by which CARD9 provides organ-specific protection against these fungal infections are now emerging. In this review, we summarize recent immunological and clinical advances that have provided significant mechanistic insights into the pathogenesis of human CARD9 deficiency. We also discuss how genetic mutations in CARD9-coupled receptors (Dectin-1, Dectin-2) and CARD9-binding partners (MALT1, BCL10) affect human antifungal immunity relative to CARD9 deficiency, and we highlight major understudied research questions which merit future investigation. PMID:27092298

  4. High Content Analysis Provides Mechanistic Insights on the Pathways of Toxicity Induced by Amine-Modified Polystyrene Nanoparticles

    PubMed Central

    Anguissola, Sergio; Garry, David; Salvati, Anna; O'Brien, Peter J.; Dawson, Kenneth A.

    2014-01-01

    The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH2) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH2 NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH2 NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for in vitro screening of nanomaterials in highthroughput format. PMID:25238162

  5. Molecular interaction between natural IgG and ficolin - mechanistic insights on adaptive-innate immune crosstalk

    NASA Astrophysics Data System (ADS)

    Panda, Saswati; Zhang, Jing; Yang, Lifeng; Anand, Ganesh S.; Ding, Jeak L.

    2014-01-01

    Recently, we found that natural IgG (nIgG; a non-specific immunoglobulin of adaptive immunity) is not quiescent, but plays a crucial role in immediate immune defense by collaborating with ficolin (an innate immune protein). However, how the nIgG and ficolin interplay and what factors control the complex formation during infection is unknown. Here, we found that mild acidosis and hypocalcaemia induced by infection- inflammation condition increased the nIgG:ficolin complex formation. Hydrogen-deuterium exchange mass spectrometry delineated the binding interfaces to the CH2-CH3 region of nIgG Fc and P-subdomain of ficolin FBG domain. Infection condition exposes novel binding sites. Site-directed mutagenesis and surface plasmon resonance analyses of peptides, derived from nIgG and ficolin, defined the interacting residues between the proteins. These results provide mechanistic insights on the interaction between two molecules representing the adaptive and innate immune pathways, prompting potential development of immunomodulatory/prophylactic peptides tunable to prevailing infection conditions.

  6. Mechanistic insights into 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Li, Heng; Tian, Jie; Sun, Wei; Qin, Wei; Gao, Wen-Yun

    2013-11-01

    The binding mode of 1-deoxy-D-xylulose 5-phosphate (DXP) to 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) (EC 1.1.1.267) from Escherichia coli was investigated via (18) O isotope exchange experiments and determination of the kinetic parameters of the reaction. The results support a C3-C4 substrate binding mode in which DXP chelates a DXR-bound divalent cation via its hydroxyl groups at C3 and C4. Based on this binding mode and the early results, a catalytic cycle for the conversion of DXP to 2-methyl-D-erythritol 4-phosphate mediated by DXR including a pseudo-single molecule transition state of the retro-aldol intermediates is proposed. Taking into account the binding mode of DXP and the catalytic cycle of DXR, the mechanistic insights of DXR are disclosed and the current discrepancies concerning the catalysis of this enzyme are interpreted within the accepted retro-aldol/aldol sequence. PMID:24010408

  7. A Mechanistic Study of Halogen Addition and Photoelimination from π-Conjugated Tellurophenes.

    PubMed

    Carrera, Elisa I; Lanterna, Anabel E; Lough, Alan J; Scaiano, Juan C; Seferos, Dwight S

    2016-03-01

    The ability to drive reactivity using visible light is of importance for many disciplines of chemistry and has significant implications for sustainable chemistry. Identifying photochemically active compounds and understanding photochemical mechanisms is important for the development of useful materials for synthesis and catalysis. Here we report a series of photoactive diphenyltellurophene compounds bearing electron-withdrawing and electron-donating substituents synthesized by alkyne coupling/ring closing or palladium-catalyzed ipso-arylation chemistry. The redox chemistry of these compounds was studied with respect to oxidative addition and photoelimination of bromine, which is of importance for energy storage reactions involving X2. The oxidative addition reaction mechanism was studied using density functional theory, the results of which support a three-step mechanism involving the formation of an initial η(1) association complex, a monobrominated intermediate, and finally the dibrominated product. All of the tellurophene derivatives undergo photoreduction using 430, 447, or 617 nm light depending on the absorption properties of the compound. Compounds bearing electron-withdrawing substituents have the highest photochemical quantum efficiencies in the presence of an alkene trap, with efficiencies of up to 42.4% for a pentafluorophenyl-functionalized tellurophene. The photoelimination reaction was studied in detail through bromine trapping experiments and laser flash photolysis, and a mechanism is proposed. The photoreaction, which occurs by release of bromine radicals, is competitive with intersystem crossing to the triplet state of the brominated species, as evidenced by the formation of singlet oxygen. These findings should be useful for the design of new photochemically active compounds supported by main-group elements. PMID:26853739

  8. Mechanistic Insight into the Facilitation of β-Lactam Fragmentation through Metal Assistance.

    PubMed

    Casarrubios, Luis; Esteruelas, Miguel A; Larramona, Carmen; Lledós, Agustí; Muntaner, Jaime G; Oñate, Enrique; Ortuño, Manuel A; Sierra, Miguel A

    2015-11-16

    The mechanism of OsH6(PiPr3)2-mediated fragmentation of a 4-(2 pyridyl)-2-azetidinone has been investigated by DFT calculations. The addition of the C4-H bond of the substrate to OsH2(PiPr3)2 allows the active participation of an osmium lone pair in the B-type β-lactam fragmentation process. This new mechanism makes the N1-C4/C2-C3 fragmentation of the lactamic core thermally accessible through a stepwise process. PMID:26437692

  9. Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact.

    PubMed

    Karousis, Evangelos D; Nasif, Sofia; Mühlemann, Oliver

    2016-09-01

    Nonsense-mediated mRNA decay (NMD) was originally coined to define a quality control mechanism that targets mRNAs with truncated open reading frames due to the presence of a premature termination codon. Meanwhile, it became clear that NMD has a much broader impact on gene expression and additional biological functions beyond quality control are continuously being discovered. We review here the current views regarding the molecular mechanisms of NMD, according to which NMD ensues on mRNAs that fail to terminate translation properly, and point out the gaps in our understanding. We further summarize the recent literature on an ever-rising spectrum of biological processes in which NMD appears to be involved, including homeostatic control of gene expression, development and differentiation, as well as viral defense. WIREs RNA 2016, 7:661-682. doi: 10.1002/wrna.1357 For further resources related to this article, please visit the WIREs website. PMID:27173476

  10. Mechanistic Insights into the Mode of Action of Bifunctional Pyrrolidine-Squaramide-Derived Organocatalysts.

    PubMed

    Roca-López, David; Uria, Uxue; Reyes, Efraim; Carrillo, Luisa; Jørgensen, Karl Anker; Vicario, Jose L; Merino, Pedro

    2016-01-18

    The catalytic modes of action of three squaramide-derived bifunctional organocatalysts have been investigated using DFT methods. The [5+2] cycloaddition between oxidopyrylium ylides and enals was used as the model reaction. Two primary modes were possible for the different catalysts studied. The preference for one mode over the other was due to the possibility of additional favorable π-π interactions between the hydrogen-bond activated pyrylium ylide and an electron-deficient aromatic ring bonded to the squaramide NH group. The model can be extended to other reactions catalyzed by the same catalysts, such as formal [2+2] cycloadditions between nitroalkenes and α,β-unsaturated aldehydes. The computational results were in excellent concurrence with the available experimental reports on the observed total enantioselectivity and differences in diastereoselectivity depending on the substrate and the reaction. PMID:26612630

  11. Degradation of transparent conductive oxides: mechanistic insights across configurations and exposures

    NASA Astrophysics Data System (ADS)

    Lemire, Heather M.; Peterson, Kelly A.; Sprawls, Samuel; Singer, Kenneth; Martin, Ina T.; French, Roger H.

    2013-09-01

    Understanding transparent conductive oxide (TCO) degradation is critical to improving stability and lifetime of both organic and inorganic thin lm PV modules, which utilize TCOs, like indium tin oxide (ITO), aluminumdoped zinc oxide (AZO) and uorine-doped tin oxide (FTO) as electrodes. These TCOs must retain their long-term functionality in diverse outdoor environments. In addition to bulk material degradation, interfacial degradation, a frequent avenue for failure in PV systems, is promoted by exposure to environmental stressors such as irradiance, heat and humidity. ITO, AZO and FTO samples in an open-faced con guration were exposed to damp heat and ASTM G154 for up to 1000 hours. The e ect of exposure on electrical and optical properties and surface energies of cleaned samples was measured. Yellowness, haze, water contact angle and resistivity of the di erent materials trended di erently with exposure time and type, indicating the activation of distinct degradation mechanisms. An encapsulated con guration study was conducted on ITO and AZO, exposing samples to the above accelerated exposures and two outdoor exposures (1x suns and 5x suns on a dual axis trackers), with and without PEDOT:PSS layers. PEDOT:PSS increases the yellowness and haze of ITO and AZO, but does not accelerate the increase in resistivity, suggesting that the optical and electrical degradation mechanisms are not coupled. Additionally, the hazing/roughening mechanism of PEDOT:PSS on AZO appears to be photo-sensitive; 5x outdoor exposure samples demonstrated distinctly higher haze than damp heat exposed samples.

  12. HCl uptake by volcanic ash in the high temperature eruption plume: Mechanistic insights

    NASA Astrophysics Data System (ADS)

    Ayris, Paul M.; Delmelle, Pierre; Cimarelli, Corrado; Maters, Elena C.; Suzuki, Yujiro J.; Dingwell, Donald B.

    2014-11-01

    The injection of HCl into the stratosphere by large volcanic eruptions has long been considered to be of minor importance. This is due to the widespread assumption that any HCl will be efficiently taken up by hydrometeors in the cooling plume. However, this assumption does not account for the possibility that prior scavenging processes can act within the high temperature core of the eruption plume. The adsorption of HCl onto ash surfaces to form soluble -Cl salts is a hitherto unconstrained scavenging mechanism, and their fate within the atmosphere and environment is uncertain. Here, we investigate the capacity of volcanic glasses of tephrite, phonolite, dacite and rhyolite composition to adsorb HCl. The experiments are conducted in the presence of He-SO2-CO2 mixtures at temperatures of 200-800 °C. Our experimental findings show that only the tephrite and phonolite glasses exhibit significant reactivity to HCl, which show optimal efficiency of uptake at 400-600 °C. The primary reaction product formed during adsorption is halite (NaCl), in addition to minor quantities of Ca-, K-, Al- and Fe-bearing chlorides. Uptake of HCl by glass surfaces is sustained by the outward diffusion of Na+ and other Cl-reactive cations via exchange with H+. Simple mathematical models can be used to yield Na diffusion coefficients for the four experimental glasses, and suggest that a varying structural role for Na within the glass network governs the capacity for HCl retention. The uptake of HCl under experimental conditions is limited above 500 °C by a Cl-induced dehydroxylation process, but the presence of H2O in the hydrous plume may sustain or even enhance adsorption. The present experimental data, combined with cooling gradients obtained from established plume evolution models, lead us to conclude that HCl adsorption within the eruption plume core can be a more significant scavenging mechanism in large explosive eruptions than previously considered. We additionally highlight the

  13. Mechanistic insights into the gold(I)-catalyzed activation of glycosyl ortho-alkynylbenzoates for glycosidation.

    PubMed

    Tang, Yu; Li, Jiakun; Zhu, Yugen; Li, Yao; Yu, Biao

    2013-12-11

    Anomerization, which involves cleavage and formation of the anomeric C-O bond, is of fundamental importance in the carbohydrate chemistry. Herein, the unexpected gold(I)-catalyzed anomerization of glycosyl ortho-alkynylbenzoates has been studied in detail. Especially, crossover experiments in the presence of an exogenous isochromen-4-yl gold(I) complex confirm that the anomerization proceeds via the exocleavage mechanism, involving (surprisingly) the addition of the isochromen-4-yl gold(I) complex onto a sugar oxocarbenium (or dioxolenium) and an elimination of LAu(+) from the vinyl gold(I) complex. The inhibitory effect of the exogenous isochromen-4-yl gold(I) complex when in stoichiometric amount on the anomerization has guided us to disclose an isochromen-4-yl gem-gold(I) complex, which is inactive in catalysis but in equilibrium with the monogold(I) complex and the LAu(+) catalyst. The proposed key intermediate in the anomerization, a transient glycosyloxypyrylium species, is successfully trapped via a cycloaddition reaction with n-butyl vinyl ether as a dienophile. SN2-like substitution of the initially formed glycosyloxypyrylium intermediate has then been achieved to a large extent via charging with acceptors in an excess amount to lead to the corresponding glycosides in a stereoselective manner. PMID:24252170

  14. Post-infectious irritable bowel syndrome: mechanistic insights into chronic disturbances following enteric infection.

    PubMed

    Beatty, Jennifer K; Bhargava, Amol; Buret, Andre G

    2014-04-14

    Irritable bowel syndrome (IBS) is a commonly encountered chronic functional gastrointestinal (GI) disorder. Approximately 10% of IBS patients can trace the onset of their symptoms to a previous a bout of infectious dysentery. The appearance of new IBS symptoms following an infectious event is defined as post-infectious-IBS. Indeed, with the World Health Organization estimating between 2 and 4 billion cases annually, infectious diarrheal disease represents an incredible international healthcare burden. Additionally, compounding evidence suggests many commonly encountered enteropathogens as unique triggers behind IBS symptom generation and underlying pathophysiological features. A growing body of work provides evidence supporting a role for pathogen-mediated modifications in the resident intestinal microbiota, epithelial barrier integrity, effector cell functions, and innate and adaptive immune features, all proposed physiological manifestations that can underlie GI abnormalities in IBS. Enteric pathogens must employ a vast array of machinery to evade host protective immune mechanisms, and illicit successful infections. Consequently, the impact of infectious events on host physiology can be multidimensional in terms of anatomical location, functional scope, and duration. This review offers a unique discussion of the mechanisms employed by many commonly encountered enteric pathogens that cause acute disease, but may also lead to the establishment of chronic GI dysfunction compatible with IBS. PMID:24744587

  15. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

    PubMed Central

    Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

    2016-01-01

    Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity. PMID:27112574

  16. Mechanistic insights into the hydrolysis of 2-chloroethyl ethyl sulfide: the expanded roles of sulfonium salts.

    PubMed

    Bae, Su Y; Winemiller, Mark D

    2013-07-01

    The hydrolysis of 2-chloroethyl ethyl sulfide has been examined in an effort to better understand its mechanism under more concentrated conditions. Two salts formed during hydrolysis were synthesized, and an emphasis was placed on determining their effect on the reaction as it proceeded. Unexpected changes in mechanism were seen when excess chloride was added to the reaction. By measuring rates and product distributions as the products were added back into the hydrolysis, a mechanism was developed. The formation of these sulfonium salts represents additional products in the disappearance of 2-chloroethyl ethyl sulfide with k3 in particular causing a deviation away from expected first-order behavior. Sulfonium salts 3 and 4 do not appear to interconvert, and the system as a whole had fewer pathways available than previously proposed. Initial conditions for studying the hydrolysis were very important and could lead to different conclusions depending on the conditions used. This work will aid in better understanding the hydrolysis of the very toxic chemical warfare agent mustard (bis(2-chloroethyl)sulfide) in the environment and during its decontamination. PMID:23767819

  17. PIK3CA somatic mutations in breast cancer: Mechanistic insights from Langevin dynamics simulations

    PubMed Central

    Mankoo, Parminder K.; Sukumar, Saraswati; Karchin, Rachel

    2011-01-01

    Somatic mutations in PIK3CA (phosphati-dylinositol-3 kinase, catalytic subunit, alpha isoform) are reported in breast and other human cancers to concentrate at hotspots within its kinase and helical domains. Most of these mutations cause kinase gain of function in vitro and are associated with oncogenicity in vivo. However, little is known about the mechanisms driving tumor development. We have performed computational structural studies on a homology model of wildtype PIK3CA plus recurrent H1047R, H1047L, and P539R mutations, located in the kinase and helical domains, respectively. The time evolution of the structures show that H1047R/L mutants exhibit a larger area of the catalytic cleft between the kinase N- and C-lobes compared with the wildtype that could facilitate the entrance of substrates. This larger area might yield enhanced substrate-to-product turnover associated with oncogenicity. In addition, the H1047R/L mutants display increased kinase activation loop mobility, compared with the wildtype. The P539R mutant forms more hydrogen bonds and salt-bridge interactions than the wildtype, properties that are associated with enhanced thermostability. Mutant-specific differences in the catalytic cleft and activation loop behavior suggest that structure-based mutant-specific inhibitors can be designed for PIK3CA-positive breast cancers. PMID:18951408

  18. Unraveling Comparative Anti-Amyloidogenic Behavior of Pyrazinamide and D-Cycloserine: A Mechanistic Biophysical Insight

    PubMed Central

    Chaturvedi, Sumit Kumar; Zaidi, Nida; Alam, Parvez; Khan, Javed Masood; Qadeer, Atiyatul; Siddique, Ibrar Ahmad; Asmat, Shamoon; Zaidi, Yusra; Khan, Rizwan Hasan

    2015-01-01

    Amyloid fibril formation by proteins leads to variety of degenerative disorders called amyloidosis. While these disorders are topic of extensive research, effective treatments are still unavailable. Thus in present study, two anti-tuberculosis drugs, i.e., pyrazinamide (PYZ) and D-cycloserine (DCS), also known for treatment for Alzheimer’s dementia, were checked for the anti-aggregation and anti-amyloidogenic ability on Aβ-42 peptide and hen egg white lysozyme. Results demonstrated that both drugs inhibit the heat induced aggregation; however, PYZ was more potent and decelerated the nucleation phase as observed from various spectroscopic and microscopic techniques. Furthermore, pre-formed amyloid fibrils incubated with these drugs also increased the PC12/SH-SY5Y cell viability as compare to the amyloid fibrils alone; however, the increase was more pronounced for PYZ as confirmed by MTT assay. Additionally, molecular docking study suggested that the greater inhibitory potential of PYZ as compare to DCS may be due to strong binding affinity and more occupancy of hydrophobic patches of HEWL, which is known to form the core of the protein fibrils. PMID:26312749

  19. Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

    NASA Astrophysics Data System (ADS)

    Kabadi, Pradeep G.; Sankaran, Praveen Kallamvalliillam; Palanivelu, Dinesh V.; Adhikary, Laxmi; Khedkar, Anand; Chatterjee, Amarnath

    2016-08-01

    We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation.

  20. Mechanistic insight into cadmium-induced inactivation of the Bloom protein

    PubMed Central

    Qin, Wei; Bazeille, Nicolas; Henry, Etienne; Zhang, Bo; Deprez, Eric; Xi, Xu-Guang

    2016-01-01

    Cadmium is a toxic metal that inactivates DNA-repair proteins via multiple mechanisms, including zinc substitution. In this study, we investigated the effect of Cd2+ on the Bloom protein (BLM), a DNA-repair helicase carrying a zinc-binding domain (ZBD) and playing a critical role to ensure genomic stability. One characteristics of BLM-deficient cells is the elevated rate of sister chromatid exchanges, a phenomenon that is also induced by Cd2+. Here, we show that Cd2+ strongly inhibits both ATPase and helicase activities of BLM. Cd2+ primarily prevents BLM-DNA interaction via its binding to sulfhydryl groups of solvent-exposed cysteine residues and, concomitantly, promotes the formation of large BLM multimers/aggregates. In contrast to previously described Cd2+ effects on other zinc-containing DNA-repair proteins, the ZBD appears to play a minor role in the Cd2+-mediated inhibition. While the Cd2+-dependent formation of inactive multimers and the defect of DNA-binding were fully reversible upon addition of EDTA, the inhibition of the DNA unwinding activity was not counteracted by EDTA, indicating another mechanism of inhibition by Cd2+ relative to the targeting of a catalytic residue. Altogether, our results provide new clues for understanding the mechanism behind the ZBD-independent inactivation of BLM by Cd2+ leading to accumulation of DNA double-strand breaks. PMID:27194376

  1. Mechanistic insights into the lipid interaction of an ancient saposin-like protein.

    PubMed

    Michalek, Matthias; Leippe, Matthias

    2015-03-10

    The members of the expanding family of saposin-like proteins (SAPLIPs) have various biological functions in plants, animals, and humans. In addition to a similar protein backbone, these proteins have in common the fact that they interact with lipid membranes. According to their phylogenetic position, it has long been thought that amoeboid protozoans produce archetypes of SAPLIPs and that these are lytic proteins that can perforate membranes of prokaryotic and eukaryotic target cells. Here, we show that an amoebic SAPLIP from Entamoeba invadens does not form lytic pores in membranes but displays several characteristics that are known from human saposins. The protein named invaposin changes the conformation from a closed to an open form in the presence of lipid membranes, acts in a pH-dependent manner, selectively binds anionic lipids, aggregates lipid vesicles of the preferred composition, and dimerizes upon acidification. Our data indicate that the principal features of the lipid-binding saposins evolved long before the appearance of the vertebrate lineage and push the origin of saposins even deeper down the phylogenetic tree to unicellular organisms. PMID:25715682

  2. Mechanistic Insights from Reaction of α-Oxiranyl-Aldehydes with Cyanobacterial Aldehyde Deformylating Oxygenase

    PubMed Central

    Das, Debasis; Ellington, Benjamin; Paul, Bishwajit; Marsh, E. Neil G.

    2014-01-01

    The biosynthesis of long-chain aliphatic hydrocarbons, which are derived from fatty acids, is widespread in Nature. The last step in this pathway involves the decarbonylation of fatty aldehydes to the corresponding alkanes or alkenes. In cyanobacteria this is catalyzed by an aldehyde deformylating oxygenase. We have investigated the mechanism of this enzyme using substrates bearing an oxirane ring adjacent to the aldehyde carbon. The enzyme catalyzed the deformylation of these substrates to produce the corresponding oxiranes. Performing the reaction in D2O allowed the facial selectivity of proton addition to be examined by 1H-NMR spectroscopy. The proton is delivered with equal probability to either face of the oxirane ring, indicating the formation of an oxiranyl radical intermediate that is free to rotate during the reaction. Unexpectedly, the enzyme also catalyzes a side reaction in which oxiranyl-aldehydes undergo tandem deformylation to furnish alkanes two carbons shorter. We present evidence that this involves the rearrangement of the intermediate oxiranyl radical formed in the first step, resulting an aldehyde that is further deformylated in a second step. These observations provide support for a radical mechanism for deformylation and, furthermore, allow the lifetime of the radical intermediate to be estimated based on prior measurements of rate constants for the rearrangement of oxiranyl radicals. PMID:24313866

  3. Attenuated exercise induced hyperaemia with age: mechanistic insight from passive limb movement

    PubMed Central

    McDaniel, John; Hayman, Melissa A; Ives, Steve; Fjeldstad, Anette S; Trinity, Joel D; Wray, D Walter; Richardson, Russell S

    2010-01-01

    The influence of age on the central and peripheral contributors to exercise-induced hyperaemia is unclear. Utilizing a reductionist approach, we compared the peripheral and central haemodynamic responses to passive limb movement (exercise without an increase in metabolism) in 11 old (71 ± 9 years of age s.d.) and 11 young (24 ± 2 years of age) healthy subjects. Cardiac output (CO), heart rate (HR), stroke volume (SV), mean arterial pressure (MAP), and femoral blood flow of the passively moved and control legs were evaluated second-by-second during 2 min of passive knee extension at a rate of 1 Hz. Compared to the young, the old group exhibited a significantly attenuated increase in HR (7 ± 4%vs. 13 ± 7%s.d.), CO (10 ± 6%vs. 18 ± 8%) and femoral blood flow in the passively moved (123 ± 55%vs. 194 ± 57%) and control legs (47 ± 43%vs. 77 ± 96%). In addition, the change in vascular conductance in the passively moving limb was also significantly attenuated in the old (2.4 ± 1.2 ml min−1 mmHg−1) compared to the young (4.3 ± 1.7 ml min−1 mmHg−1). In both groups all main central and peripheral changes that occurred at the onset of passive knee extension were transient, lasting only 45 s. In a paradigm where metabolism does not play a role, these data reveal that both central and peripheral haemodynamic mechanisms are likely to be responsible for the 30% reduction in exercise-induced hyperaemia with age. PMID:20876201

  4. HCl uptake by volcanic ash in the high temperature eruption plume: mechanistic insights

    NASA Astrophysics Data System (ADS)

    Ayris, P. M.; Delmelle, P.; Cimarelli, C.; Maters, E. C.; Suzuki, Y.; Dingwell, D. B.

    2014-12-01

    The injection of HCl into the stratosphere by large volcanic eruptions is considered to be little importance, due to the efficient incorporation of the former into hydrometeors within the cooling plume. However, HCl is also adsorbed onto ash surfaces to form soluble -Cl salts within the high temperature core of the eruption plume, and the atmospheric and environmental significance of this process is uncertain. We investigate the capacity of volcanic glasses with tephrite, phonolite, dacite, and rhyolite compositions to adsorb HCl at temperatures of 200-800°C in the presence of SO2, CO2 and He. Experiments show that only tephrite and phonolite glasses are significantly reactive to HCl, exhibiting optimal uptake at 400-600°C. The primary reaction product formed during adsorption is NaCl, but Ca-, K-, Al- and Fe- chlorides are also identified. Uptake of HCl by glass surfaces is sustained by interdiffusion of Na+ and other Cl-reactive cations with H+. Diffusion coefficient calculations yield Na diffusion coefficients for the four glasses, suggesting that the structural role for Na within the glass network governs the capacity for HCl retention. The uptake of HCl under experimental conditions is limited above 500°C by a Cl-induced dehydroxylation process, but the presence of H2O in the hydrous eruption plume may sustain or enhance adsorption. The experimental data, combined with simulated plume cooling profiles, suggest that HCl adsorption can be a significant scavenging mechanism in large explosive eruptions, particularly in peralkaline systems. The fate of adsorbed HCl is variable; some may be retained on ash surfaces within pyroclastic flows, while chloride-coated ash in the stratosphere could promote the formation of reactive Cl species associated with O3 destruction. Additionally, Fe- and Cl-bearing salts emplaced on ash surfaces by HCl adsorption within the plume cores of large explosive eruptions may increase the ocean fertilising potential of such events.

  5. Mechanistic insights into landscape genetic structure of two tropical amphibians using field-derived resistance surfaces.

    PubMed

    Nowakowski, A Justin; DeWoody, J Andrew; Fagan, Matthew E; Willoughby, Janna R; Donnelly, Maureen A

    2015-02-01

    Conversion of forests to agriculture often fragments distributions of forest species and can disrupt gene flow. We examined effects of prevalent land uses on genetic connectivity of two amphibian species in northeastern Costa Rica. We incorporated data from field surveys and experiments to develop resistance surfaces that represent local mechanisms hypothesized to modify dispersal success of amphibians, such as habitat-specific predation and desiccation risk. Because time lags can exist between forest conversion and genetic responses, we evaluated landscape effects using land-cover data from different time periods. Populations of both species were structured at similar spatial scales but exhibited differing responses to landscape features. Litter frog population differentiation was significantly related to landscape resistances estimated from abundance and experiment data. Model support was highest for experiment-derived surfaces that represented responses to microclimate variation. Litter frog genetic variation was best explained by contemporary landscape configuration, indicating rapid population response to land-use change. Poison frog genetic structure was strongly associated with geographic isolation, which explained up to 45% of genetic variation, and long-standing barriers, such as rivers and mountains. However, there was also partial support for abundance- and microclimate response-derived resistances. Differences in species responses to landscape features may be explained by overriding effects of population size on patterns of differentiation for poison frogs, but not litter frogs. In addition, pastures are likely semi-permeable to poison frog gene flow because the species is known to use pastures when remnant vegetation is present, but litter frogs do not. Ongoing reforestation efforts will probably increase connectivity in the region by increasing tree cover and reducing area of pastures. PMID:25533403

  6. Methane to acetic acid over Cu-exchanged zeolites: mechanistic insights from a site-specific carbonylation reaction.

    PubMed

    Narsimhan, Karthik; Michaelis, Vladimir K; Mathies, Guinevere; Gunther, William R; Griffin, Robert G; Román-Leshkov, Yuriy

    2015-02-11

    The selective low temperature oxidation of methane is an attractive yet challenging pathway to convert abundant natural gas into value added chemicals. Copper-exchanged ZSM-5 and mordenite (MOR) zeolites have received attention due to their ability to oxidize methane into methanol using molecular oxygen. In this work, the conversion of methane into acetic acid is demonstrated using Cu-MOR by coupling oxidation with carbonylation reactions. The carbonylation reaction, known to occur predominantly in the 8-membered ring (8MR) pockets of MOR, is used as a site-specific probe to gain insight into important mechanistic differences existing between Cu-MOR and Cu-ZSM-5 during methane oxidation. For the tandem reaction sequence, Cu-MOR generated drastically higher amounts of acetic acid when compared to Cu-ZSM-5 (22 vs 4 μmol/g). Preferential titration with sodium showed a direct correlation between the number of acid sites in the 8MR pockets in MOR and acetic acid yield, indicating that methoxy species present in the MOR side pockets undergo carbonylation. Coupled spectroscopic and reactivity measurements were used to identify the genesis of the oxidation sites and to validate the migration of methoxy species from the oxidation site to the carbonylation site. Our results indicate that the Cu(II)-O-Cu(II) sites previously associated with methane oxidation in both Cu-MOR and Cu-ZSM-5 are oxidation active but carbonylation inactive. In turn, combined UV-vis and EPR spectroscopic studies showed that a novel Cu(2+) site is formed at Cu/Al <0.2 in MOR. These sites oxidize methane and promote the migration of the product to a Brønsted acid site in the 8MR to undergo carbonylation. PMID:25562431

  7. Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions

    PubMed Central

    Chen, Haoyuan; Giese, Timothy J.; Huang, Ming; Wong, Kin-Yiu; Harris, Michael E.; York, Darrin M.

    2015-01-01

    Phosphoryl transfer reactions are ubiquitous in biology, and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate-controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition state structure and bonding optimally requires the use of theoretical models. In this work, we apply density-functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2’-O-transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well-studied series of phosphate and phosphorothioate mono-, di- and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non-enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate-KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of

  8. Mechanistic study of copper-catalyzed aerobic oxidative coupling of arylboronic esters and methanol: insights into an organometallic oxidase reaction.

    PubMed

    King, Amanda E; Brunold, Thomas C; Stahl, Shannon S

    2009-04-15

    Copper-catalyzed aerobic oxidative coupling of arylboronic acid derivatives and heteroatom nucleophiles is a highly useful method for the formation of aryl-heteroatom bonds. Mechanistic studies reveal that this reaction proceeds via an "oxidase"-style mechanism. Kinetic and spectroscopic studies establish that transmetalation of the aryl group from boron to Cu(II) is the turnover-limiting step and reoxidation of the reduced catalyst by O(2) is rapid. Further mechanistic analysis implicates the involvement of an aryl-copper(III) intermediate that undergoes facile C-O bond formation. PMID:19309072

  9. Mechanistic insight into the Brust-Schiffrin two-phase method for organochalcogenate-protected metal nanoparticles

    NASA Astrophysics Data System (ADS)

    Lopez Jimenez, Francisco

    The primary objective of this thesis research was to explore the mechanism of the Brust---Schiffrin two--phase method, which has been widely used for preparing the thiolate--protected metal nanoparticles with the core diameter less than 5nm; to synthesize and characterize the ligand--protected gold nanoparticles using dialkyl ditelluride with different alkyl chain length as ligand precursor; and to develop a new synthetic route for obtaining ultra--small (< 2 nm) gold nanoparticels covered by tellurium-anchoring ligands. Since the Brust---Schiffrin two--phase method was first reported in 1994, many groups have tried to identify the precursors of metal ions prior to the addition of NaBH4. The widely accepted assumption has been that the addition of thiol reduces Au(III) to Au(I) and forms [AuSR] n--like polymers. Recently Lennox and coworkers have demonstrated that the metal precursor before the addition of the reductant is TOA metal(I) halide complex [TOA][MX2], and not [MSR]n--like polymers. Our Raman spectroscopic investigations showed that, in a typical BSM synthesis, no metal(M)---sulfur(S) bonds were formed after thiol addition. These observations provide independent spectroscopic confirmation of the recent work by Lennox and co--workers. On the basis of Raman, NMR, and surface plasmon resonance characterizations, we found that before the formation of any metal-chalcogen bonds, metal nucleation centers/NPs were first formed inside the inverse micelles of the tetrabutylammonium bromide in the organic solvent, where the metal ions were reduced by NaBH4. The ensuing formation of the metal---chalcogen bonds between the naked metal NPs inside the micelles and the organochalcogen ligands in the organic solvent is the mechanism by which the further growth of the metal core can be controlled. This proposed mechanism has been further examined in the formation of Ag and Cu nanoparticles. This new mechanistic understanding enabled the design of some specifically targeted

  10. A Mechanistic Dichotomy Leading to a Ruthenium-Catalyzed cis-Addition for Stereoselective Formation of (Z)-Vinyl Bromides.

    PubMed

    Trost; Pinkerton

    2000-01-01

    Either trans- or cis-haloalkylation is possible through a three-component coupling [Eq. (1)]. The cis-bromoruthenation of an alkyne by lithium bromide and [CpRu(CH(3)CN)(3)]PF(6), catalyzed by SnBr(4), gives (Z)-vinyl bromides with high chemoselectivity. The degree of control over the sterochemistry raises intriguing mechanistic questions as well as offering practical synthetic utility. PMID:10649409

  11. Advantages of Crystallographic Fragment Screening: Functional and Mechanistic Insights from a Powerful Platform for Efficient Drug Discovery

    PubMed Central

    Patel, Disha; Bauman, Joseph D.; Arnold, Eddy

    2015-01-01

    X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this “chemical interrogation” of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility. PMID:25117499

  12. A mechanistic view of human mitochondrial DNA polymerase γ: providing insight into drug toxicity and mitochondrial disease

    PubMed Central

    Bailey, Christopher M.; Anderson, Karen S.

    2010-01-01

    Summary Mitochondrial DNA polymerase gamma (Pol γ) is the sole polymerase responsible for replication of the mitochondrial genome. The study of human Pol γ is of key importance to clinically relevant issues such as nucleoside analog toxicity and mitochondrial disorders such as progressive external ophthalmoplegia. The development of a recombinant form of the human Pol γ holoenzyme provided an essential tool in understanding the mechanism of these clinically relevant phenomena using kinetic methodologies. This review will provide a brief history on the discovery and characterization of human mitochondrial DNA polymerase γ, focusing on kinetic analyses of the polymerase and mechanistic data illustrating structure-function relationships to explain drug toxicity and mitochondrial disease. PMID:20083238

  13. Velocity map imaging as a tool for gaining mechanistic insight from closed-loop control studies of molecular fragmentation

    SciTech Connect

    Jochim, Bethany; Averin, R.; Gregerson, Neal; Wells, E.; McKenna, J.; De, S.; Ray, D.; Zohrabi, M.; Carnes, K. D.; Ben-Itzhak, I.; Bergues, B.; Kling, M. F.

    2011-04-15

    Strong-field closed-loop control schemes using shaped ultrafast laser pulses have been used to selectively fragment a variety of molecules in recent years. The resulting pulses are often complex and resist an easy mechanistic interpretation. We report on the use of velocity map imaging to study the dissociative ionization of CO molecules by optimally-shaped ultrafast laser pulses. Using this technique, a mechanism is identified for the optimized CO{sup +} {yields} C + O{sup +} dissociation, and some of the observed control over the CO{sup +} dissociation branching ratio is ascribed to an angular discrimination effect. Furthermore, we demonstrate that the acquisition of two-dimensional velocity map images is rapid enough to incorporate directly into the adaptive control loop.

  14. Mechanistic validation.

    PubMed

    Hartung, Thomas; Hoffmann, Sebastian; Stephens, Martin

    2013-01-01

    Validation of new approaches in regulatory toxicology is commonly defined as the independent assessment of the reproducibility and relevance (the scientific basis and predictive capacity) of a test for a particular purpose. In large ring trials, the emphasis to date has been mainly on reproducibility and predictive capacity (comparison to the traditional test) with less attention given to the scientific or mechanistic basis. Assessing predictive capacity is difficult for novel approaches (which are based on mechanism), such as pathways of toxicity or the complex networks within the organism (systems toxicology). This is highly relevant for implementing Toxicology for the 21st Century, either by high-throughput testing in the ToxCast/Tox21 project or omics-based testing in the Human Toxome Project. This article explores the mostly neglected assessment of a test's scientific basis, which moves mechanism and causality to the foreground when validating/qualifying tests. Such mechanistic validation faces the problem of establishing causality in complex systems. However, pragmatic adaptations of the Bradford Hill criteria, as well as bioinformatic tools, are emerging. As critical infrastructures of the organism are perturbed by a toxic mechanism we argue that by focusing on the target of toxicity and its vulnerability, in addition to the way it is perturbed, we can anchor the identification of the mechanism and its verification. PMID:23665802

  15. Structural and Mechanistic Insights into the Regulation of the Fundamental Rho Regulator RhoGDIα by Lysine Acetylation.

    PubMed

    Kuhlmann, Nora; Wroblowski, Sarah; Knyphausen, Philipp; de Boor, Susanne; Brenig, Julian; Zienert, Anke Y; Meyer-Teschendorf, Katrin; Praefcke, Gerrit J K; Nolte, Hendrik; Krüger, Marcus; Schacherl, Magdalena; Baumann, Ulrich; James, Leo C; Chin, Jason W; Lammers, Michael

    2016-03-11

    Rho proteins are small GTP/GDP-binding proteins primarily involved in cytoskeleton regulation. Their GTP/GDP cycle is often tightly connected to a membrane/cytosol cycle regulated by the Rho guanine nucleotide dissociation inhibitor α (RhoGDIα). RhoGDIα has been regarded as a housekeeping regulator essential to control homeostasis of Rho proteins. Recent proteomic screens showed that RhoGDIα is extensively lysine-acetylated. Here, we present the first comprehensive structural and mechanistic study to show how RhoGDIα function is regulated by lysine acetylation. We discover that lysine acetylation impairs Rho protein binding and increases guanine nucleotide exchange factor-catalyzed nucleotide exchange on RhoA, these two functions being prerequisites to constitute a bona fide GDI displacement factor. RhoGDIα acetylation interferes with Rho signaling, resulting in alteration of cellular filamentous actin. Finally, we discover that RhoGDIα is endogenously acetylated in mammalian cells, and we identify CBP, p300, and pCAF as RhoGDIα-acetyltransferases and Sirt2 and HDAC6 as specific deacetylases, showing the biological significance of this post-translational modification. PMID:26719334

  16. Mechanistic insight into ultrasound induced enhancement of simultaneous saccharification and fermentation of Parthenium hysterophorus for ethanol production.

    PubMed

    Singh, Shuchi; Agarwal, Mayank; Sarma, Shyamali; Goyal, Arun; Moholkar, Vijayanand S

    2015-09-01

    This paper presents investigations into mechanism of ultrasound assisted bioethanol synthesis using Parthenium hysterophorus biomass through simultaneous saccharification and fermentation (SSF) mode. Approach of coupling experimental results to mathematical model for SSF using Genetic Algorithm based optimization has been adopted. Comparison of model parameters for experiments with mechanical shaking and sonication (10% duty cycle) give an interesting mechanistic account of influence of ultrasound on SSF system. A 4-fold rise in ethanol and cell mass productivity is seen with ultrasound. The analysis reveals following facets of influence of ultrasound on SSF: increase in Monod constant for glucose for cell growth, maximal specific growth rate and inhibition constant of cell growth by glucose and reduction in specific cell death rate. Values of inhibition constant of cell growth by ethanol (K3E), and constants for growth associated (a) and non-growth associated (b) ethanol production remained unaltered with sonication. Beneficial effects of ultrasound are attributed to enhanced cellulose hydrolysis, enhanced trans-membrane transport of substrate and products as well as dilution of the toxic substances due to micro-convection induced by ultrasound. Intrinsic physiological functioning of cells remained unaffected by ultrasound as indicated by unaltered values of K3E, a and b. PMID:25813894

  17. Homogeneous catalytic O2 reduction to water by a cytochrome c oxidase model with trapping of intermediates and mechanistic insights

    PubMed Central

    Halime, Zakaria; Kotani, Hiroaki; Li, Yuqi; Fukuzumi, Shunichi; Karlin, Kenneth D.

    2011-01-01

    An efficient and selective four-electron plus four-proton (4e-/4H+) reduction of O2 to water by decamethylferrocene and trifluoroacetic acid can be catalyzed by a synthetic analog of the heme a3/CuB site in cytochrome c oxidase (6LFeCu) or its Cu-free version (6LFe) in acetone. A detailed mechanistic-kinetic study on the homogeneous catalytic system reveals spectroscopically detectable intermediates and that the rate-determining step changes from the O2-binding process at 25 °C room temperature (RT) to the O-O bond cleavage of a newly observed FeIII-OOH species at lower temperature (-60 °C). At RT, the rate of O2-binding to 6LFeCu is significantly faster than that for 6LFe, whereas the rates of the O-O bond cleavage of the FeIII-OOH species observed (-60 °C) with either the 6LFeCu or 6LFe catalyst are nearly the same. Thus, the role of the Cu ion is to assist the heme and lead to faster O2-binding at RT. However, the proximate Cu ion has no effect on the O-O bond cleavage of the FeIII-OOH species at low temperature. PMID:21808032

  18. Structural and Mechanistic Insights into Fast Lithium-Ion Conduction in Li4SiO4-Li3PO4 Solid Electrolytes.

    PubMed

    Deng, Yue; Eames, Christopher; Chotard, Jean-Noël; Lalère, Fabien; Seznec, Vincent; Emge, Steffen; Pecher, Oliver; Grey, Clare P; Masquelier, Christian; Islam, M Saiful

    2015-07-22

    Solid electrolytes that are chemically stable and have a high ionic conductivity would dramatically enhance the safety and operating lifespan of rechargeable lithium batteries. Here, we apply a multi-technique approach to the Li-ion conducting system (1-z)Li4SiO4-(z)Li3PO4 with the aim of developing a solid electrolyte with enhanced ionic conductivity. Previously unidentified superstructure and immiscibility features in high-purity samples are characterized by X-ray and neutron diffraction across a range of compositions (z = 0.0-1.0). Ionic conductivities from AC impedance measurements and large-scale molecular dynamics (MD) simulations are in good agreement, showing very low values in the parent phases (Li4SiO4 and Li3PO4) but orders of magnitude higher conductivities (10(-3) S/cm at 573 K) in the mixed compositions. The MD simulations reveal new mechanistic insights into the mixed Si/P compositions in which Li-ion conduction occurs through 3D pathways and a cooperative interstitial mechanism; such correlated motion is a key factor in promoting high ionic conductivity. Solid-state (6)Li, (7)Li, and (31)P NMR experiments reveal enhanced local Li-ion dynamics and atomic disorder in the solid solutions, which are correlated to the ionic diffusivity. These unique insights will be valuable in developing strategies to optimize the ionic conductivity in this system and to identify next-generation solid electrolytes. PMID:26118319

  19. Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-Assisted Catalysis in Family 85 Glycoside Hydrolases

    SciTech Connect

    Abbott, D.; Macauley, M; Vocadlo, D; Boraston, A

    2009-01-01

    Endo-?-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (Kd = 28 ?m) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

  20. Mechanistic insights into Lp(a)-induced IL-8 expression: a role for oxidized phospholipid modification of apo(a).

    PubMed

    Scipione, Corey A; Sayegh, Sera E; Romagnuolo, Rocco; Tsimikas, Sotirios; Marcovina, Santica M; Boffa, Michael B; Koschinsky, Marlys L

    2015-12-01

    Elevated lipoprotein (a) [Lp(a)] levels are a causal risk factor for coronary heart disease. Accumulating evidence suggests that Lp(a) can stimulate cellular inflammatory responses through the kringle-containing apolipoprotein (a) [apo(a)] component. Here, we report that recombinant apo(a) containing 17 kringle (17K) IV domains elicits a dose-dependent increase in interleukin (IL)-8 mRNA and protein expression in THP-1 and U937 macrophages. This effect was blunted by mutation of the lysine binding site in apo(a) kringle IV type 10, which resulted in the loss of oxidized phospholipid (oxPL) on apo(a). Trypsin-digested 17K had the same stimulatory effect on IL-8 expression as intact apo(a), while enzymatic removal of oxPL from apo(a) significantly blunted this effect. Using siRNA to assess candidate receptors, we found that CD36 and TLR2 may play roles in apo(a)-mediated IL-8 stimulation. Downstream of these receptors, inhibitors of MAPKs, Jun N-terminal kinase and ERK1/2, abolished the effect of apo(a) on IL-8 gene expression. To assess the roles of downstream transcription factors, luciferase reporter gene experiments were conducted using an IL-8 promoter fragment. The apo(a)-induced expression of this reporter construct was eliminated by mutation of IL-8 promoter binding sites for either NF-κB or AP-1. Our results provide a mechanistic link between oxPL modification of apo(a) and stimulation of proinflammatory intracellular signaling pathways. PMID:26474593

  1. Toward a mechanistic understanding of patterns in biomineralization and new insights for old dogmas in geological settings (Invited)

    NASA Astrophysics Data System (ADS)

    Dove, P. M.; Hamm, L.; Giuffre, A. J.; Han, N.; De Yoreo, J. J.

    2013-12-01

    The ability of organisms to mineralize tissues into skeletons and other functional structures is a remarkable achievement of biology. Yet, the physical basis for how macromolecules regulate the placement and onset of mineral formation is not well established. Efforts to understand nucleation onto organic substrates have produced two, seemingly contradictory, lines of thought: The biomineralization community widely assumes the organic matrix promotes nucleation through stereochemical matching to guide the organization of solute ions, while materials synthesis groups use simple binding assays to correlate high binding strength with good promoters of nucleation. This study reconciles the two views and provides a mechanistic explanation for template-directed nucleation by correlating heterogeneous nucleation barriers with crystal-substrate binding free energies. Using surface assembled monolayers (SAM) as simple model systems, we first measure the kinetics of calcite nucleation onto model substrates that present different functional group chemistries (carboxyl, thiol, phosphate, hydroxyl) and conformations (C11, C16 chain lengths). We find rates are substrate-specific and obey predictions of classical nucleation theory at supersaturations that extend above the solubility of amorphous calcium carbonate (ACC). Analysis of the kinetic data shows the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy of the system, γ. We then use dynamic force spectroscopy to independently measure calcite-substrate binding free energies, ΔGb. Moreover, we show that within the classical theory of nucleation, γ and ΔGb should be linearly related. The results bear out this prediction and demonstrate that low energy barriers to nucleation correlate with strong crystal-substrate binding. This relationship is general to all functional group chemistries and conformations. These findings reconcile the long-standing concept of templated nucleation through

  2. Mechanistic Photochemistry of Methyl-4-hydroxycinnamate Chromophore and Its One-Water Complexes: Insights from MS-CASPT2 Study.

    PubMed

    Xie, Xiao-Ying; Li, Chun-Xiang; Fang, Qiu; Cui, Ganglong

    2016-08-01

    Herein we computationally studied the excited-state properties and decay dynamics of methyl-4-hydroxycinnamate (OMpCA) in the lowest three electronic states, that is, (1)ππ*, (1)nπ*, and S0 using combined MS-CASPT2 and CASSCF electronic structure methods. We found that one-water hydration can significantly stabilize and destabilize the vertical excitation energies of the spectroscopically bright (1)ππ* and dark (1)nπ* excited singlet states, respectively; in contrast, it has a much smaller effect on the (1)ππ* and (1)nπ* adiabatic excitation energies. Mechanistically, we located two (1)ππ* excited-state relaxation channels. One is the internal conversion to the dark (1)nπ* state, and the other is the (1)ππ* photoisomerization that eventually leads the system to a (1)ππ*/S0 conical intersection region, near which the radiationless internal conversion to the S0 state occurs. These two (1)ππ* relaxation pathways play distinct roles in OMpCA and its two one-water complexes (OMpCA-W1 and OMpCA-W2). In OMpCA, the predominant (1)ππ* decay route is the state-switching to the dark (1)nπ* state, while in one-water complexes, the importance of the (1)ππ* photoisomerization is significantly enhanced because the internal conversion to the (1)nπ* state is heavily suppressed due to the one-water hydration. PMID:27398611

  3. Reduction of O2 slow component by priming exercise: novel mechanistic insights from time-resolved near-infrared spectroscopy

    PubMed Central

    Fukuoka, Yoshiyuki; Poole, David C; Barstow, Thomas J; Kondo, Narihiko; Nishiwaki, Masato; Okushima, Dai; Koga, Shunsaku

    2015-01-01

    Novel time-resolved near-infrared spectroscopy (TR-NIRS), with adipose tissue thickness correction, was used to test the hypotheses that heavy priming exercise reduces the V̇O2 slow component (V̇O2SC) (1) by elevating microvascular [Hb] volume at multiple sites within the quadriceps femoris (2) rather than reducing the heterogeneity of muscle deoxygenation kinetics. Twelve subjects completed two 6-min bouts of heavy work rate exercise, separated by 6 min of unloaded cycling. Priming exercise induced faster overall V̇O2 kinetics consequent to a substantial reduction in the V̇O2SC (0.27 ± 0.12 vs. 0.11 ± 0.09 L·min−1, P < 0.05) with an unchanged primary V̇O2 time constant. An increased baseline for the primed bout [total (Hb + Mb)] (197.5 ± 21.6 vs. 210.7 ± 22.5 μmol L−1, P < 0.01), reflecting increased microvascular [Hb] volume, correlated significantly with the V̇O2SC reduction. At multiple sites within the quadriceps femoris, priming exercise reduced the baseline and slowed the increase in [deoxy (Hb + Mb)]. Changes in the intersite coefficient of variation in the time delay and time constant of [deoxy (Hb + Mb)] during the second bout were not correlated with the V̇O2SC reduction. These results support a mechanistic link between priming exercise-induced increase in muscle [Hb] volume and the reduced V̇O2SC that serves to speed overall V̇O2 kinetics. However, reduction in the heterogeneity of muscle deoxygenation kinetics does not appear to be an obligatory feature of the priming response. PMID:26109190

  4. The pathogenicity of splicing defects: mechanistic insights into pre-mRNA processing inform novel therapeutic approaches.

    PubMed

    Daguenet, Elisabeth; Dujardin, Gwendal; Valcárcel, Juan

    2015-12-01

    Removal of introns from pre-mRNA precursors (pre-mRNA splicing) is a necessary step for the expression of most genes in multicellular organisms, and alternative patterns of intron removal diversify and regulate the output of genomic information. Mutation or natural variation in pre-mRNA sequences, as well as in spliceosomal components and regulatory factors, has been implicated in the etiology and progression of numerous pathologies. These range from monogenic to multifactorial genetic diseases, including metabolic syndromes, muscular dystrophies, neurodegenerative and cardiovascular diseases, and cancer. Understanding the molecular mechanisms associated with splicing-related pathologies can provide key insights into the normal function and physiological context of the complex splicing machinery and establish sound basis for novel therapeutic approaches. PMID:26566663

  5. Mechanistic insight into size-dependent activity and durability in Pt/CNT catalyzed hydrolytic dehydrogenation of ammonia borane.

    PubMed

    Chen, Wenyao; Ji, Jian; Feng, Xiang; Duan, Xuezhi; Qian, Gang; Li, Ping; Zhou, Xinggui; Chen, De; Yuan, Weikang

    2014-12-01

    We report a size-dependent activity in Pt/CNT catalyzed hydrolytic dehydrogenation of ammonia borane. Kinetic study and model calculations revealed that Pt(111) facet is the dominating catalytically active surface. There is an optimized Pt particle size of ca. 1.8 nm. Meanwhile, the catalyst durability was found to be highly sensitive to the Pt particle size. The smaller Pt particles appear to have lower durability, which could be related to more significant adsorption of B-containing species on Pt surfaces as well as easier changes in Pt particle size and shape. The insights reported here may pave the way for the rational design of highly active and durable Pt catalysts for hydrogen generation. PMID:25405630

  6. A Mechanistic Understanding of a Binary Additive System to Synergistically Boost Efficiency in All-Polymer Solar Cells

    PubMed Central

    Kim, Yu Jin; Ahn, Sunyong; Wang, Dong Hwan; Park, Chan Eon

    2015-01-01

    All-polymer solar cells are herein presented utilizing the PBDTTT-CT donor and the P(NDI2OD-T2) acceptor with 1,8-diiodooctane (DIO) and 1-chloronaphthalene (CN) binary solvent additives. A systematic study of the polymer/polymer bulk heterojunction photovoltaic cells processed from the binary additives revealed that the microstructures and photophysics were quite different from those of a pristine system. The combination of DIO and CN with a DIO/CN ratio of 3:1 (3 vol% DIO, 1 vol% CN and 96 vol% o-DCB) led to suitable penetrating polymer networks, efficient charge generation and balanced charge transport, which were all beneficial to improving the efficiency. This improvement is attributed to increase in power conversion efficiency from 2.81% for a device without additives to 4.39% for a device with the binary processing additives. A detailed investigation indicates that the changes in the polymer:polymer interactions resulted in the formation of a percolating nasnoscale morphology upon processing with the binary additives. Depth profile measurements with a two-dimensional grazing incidence wide-angle X-ray scattering confirm this optimum phase feature. Furthermore impedance spectroscopy also finds evidence for synergistically boosting the device performance. PMID:26658472

  7. Mechanistic insights on the responses of plant and ecosystem gas exchange to global environmental change: lessons from Biosphere 2.

    PubMed

    Gonzalez-Meler, Miquel A; Rucks, Jessica S; Aubanell, Gerard

    2014-09-01

    Scaling up leaf processes to canopy/ecosystem level fluxes is critical for examining feedbacks between vegetation and climate. Collectively, studies from Biosphere 2 Laboratory have provided important insight of leaf-to-ecosystem investigations of multiple environmental parameters that were not before possible in enclosed or field studies. B2L has been a testing lab for the applicability of new technologies such as spectral approaches to detect spatial and temporal changes in photosynthesis within canopies, or for the development of cavity ring-down isotope applications for ecosystem evapotranspiration. Short and long term changes in atmospheric CO2, drought or temperature allowed for intensive investigation of the interactions between photosynthesis and leaf, soil and ecosystem respiration. Experiments conducted in the rainforest biome have provided some of the most comprehensive dataset to date on the effects of climate change variables on tropical ecosystems. Results from these studies have been later corroborated in natural rainforest ecosystems and have improved the predictive capabilities of models that now show increased resilience of tropics to climate change. Studies of temperature and CO2 effects on ecosystem respiration and its leaf and soil components have helped reconsider the use of simple first-order kinetics for characterizing respiration in models. The B2L also provided opportunities to quantify the rhizosphere priming effect, or establish the relationships between net primary productivity, atmospheric CO2 and isoprene emissions. PMID:25113446

  8. How does α-actinin-3 deficiency alter muscle function? Mechanistic insights into ACTN3, the 'gene for speed'.

    PubMed

    Lee, Fiona X Z; Houweling, Peter J; North, Kathryn N; Quinlan, Kate G R

    2016-04-01

    An estimated 1.5 billion people worldwide are deficient in the skeletal muscle protein α-actinin-3 due to homozygosity for the common ACTN3 R577X polymorphism. α-Actinin-3 deficiency influences muscle performance in elite athletes and the general population. The sarcomeric α-actinins were originally characterised as scaffold proteins at the muscle Z-line. Through studying the Actn3 knockout mouse and α-actinin-3 deficient humans, significant progress has been made in understanding how ACTN3 genotype alters muscle function, leading to an appreciation of the diverse roles that α-actinins play in muscle. The α-actinins interact with a number of partner proteins, which broadly fall into three biological pathways-structural, metabolic and signalling. Differences in functioning of these pathways have been identified in α-actinin-3 deficient muscle that together contributes to altered muscle performance in mice and humans. Here we discuss new insights that have been made in understanding the molecular mechanisms that underlie the consequences of α-actinin-3 deficiency. PMID:26802899

  9. Mechanistic insights into a Ca2+-dependent family of α-mannosidases in a human gut symbiont

    PubMed Central

    Zhu, Yanping; Suits, Michael D. L.; Thompson, Andrew J.; Chavan, Sambhaji; Dinev, Zoran; Dumon, Claire; Smith, Nicola; Moremen, Kelley W.; Xiang, Yong; Siriwardena, Aloysius; Williams, Spencer J.; Gilbert, Harry J.; Davies, Gideon J.

    2014-01-01

    Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are α-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca2+-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca2+ plays a key catalytic role in helping distort the mannoside away from its ground-state 4C1 chair conformation toward the transition state. PMID:20081828

  10. Mechanistic insights into osmolyte action in protein stabilization under harsh conditions: N-methylacetamide in glycine betaine-urea mixture

    NASA Astrophysics Data System (ADS)

    Kumar, Narendra; Kishore, Nand

    2014-10-01

    Glycine betaine (GB), a small naturally occurring osmolyte, stabilizes proteins and counteracts harsh denaturing conditions such as extremes of temperature, cellular dehydration, and presence of high concentration of urea. In spite of several studies on understanding mechanism of protein stabilization and counteraction of these harsh conditions by osmolytes, studies centred on GB, one of the most important osmolyte, are scarce, hence, there is need for more investigations. To explore mechanism of protein stabilization and counteraction of denaturing property of urea by GB, molecular dynamics studies of N-methylacetamide (NMA), a model peptide representing denatured state of a protein, in the presence of GB, urea, and GB-urea mixture were carried out. The results show that GB and urea work such that the strength of GB as a protecting osmolyte is increased and the denaturing ability of urea is decreased in the GB-urea mixture. It can be inferred that GB counteracts urea by decreasing its hydrophobic interactions with proteins. The mutual interactions between GB and urea also play an important role in protein stabilization. This study provides insights on osmolyte induced counteraction of denaturing property of urea.

  11. Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

    PubMed

    Chen, Yao-Sheng; Luo, Wen-I; Yang, Chung-Ling; Tu, Yi-Jung; Chang, Chun-Wei; Chiang, Chih-Hsiang; Chang, Chi-Yao; Chan, Sunney I; Yu, Steve S-F

    2014-05-01

    The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions. PMID:24629413

  12. Mechanistic Insights into Validoxylamine A 7'-Phosphate Synthesis by VldE Using the Structure of the Entire Product Complex

    SciTech Connect

    Cavalier, Michael C.; Yim, Young-Sun; Asamizu, Shumpei; Neau, David; Almabruk, Khaled H.; Mahmud, Taifo; Lee, Yong-Hwan

    2013-09-09

    The pseudo-glycosyltransferase VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the stereochemical configuration of the substrates to form validoxylamine A 7'-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7'-phosphate, and in complex with GDP and trehalose. The structure of VldE with the catalytic site in both an “open” and “closed” conformation is also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the stereochemical configuration of the donated cyclitol.

  13. A mechanistic study of the addition of alcohol to a five-membered ring silene via a photochemical reaction.

    PubMed

    Su, Ming-Der

    2016-03-21

    The mechanism for the photochemical rearrangement of a cyclic divinyldisilane (1-Si) in its first excited state ((1)π → (1)π*) is determined using the CAS/6-311G(d) and MP2-CAS/6-311++G(3df,3pd) levels of theory. The photoproduct, a cyclic silene, reacts with various alcohols to yield a mixture of cis- and trans- adducts. The two reaction pathways are denoted as the cis- addition path (path A) and the trans-addition path (path B). These model studies demonstrate that conical intersections play a crucial role in the photo-rearrangements of cyclic divinyldisilanes. The theoretical evidence also demonstrates that the addition of alcohol to a cyclic divinyldisilane follows the reaction path: cyclic divinyldisilane → Franck-Condon region → conical intersection → photoproduct (cyclic silene) → local intermediate (with alcohol) → transition state → cis- or trans-adduct. The theoretical studies demonstrate that the steric effects as well as the concentrations of CH3OH must have a dominant role in determining the yields of the final adducts by stereochemistry. The same mechanism for the carbon derivative (1-C) is also considered in this work. However, the theoretical results indicate that 1-C does not undergo a methanol addition reaction via the photochemical reaction pathway, since its energy of conical intersection (S1/S0-CI-C) is more than that of its FC (FC-C). The reason for these phenomena could be that the atomic radius of carbon is much smaller than that of silicon (77 and 117 pm, respectively). As a result, the conformation for 1-C is more sterically congested than that for 1-Si, along the 1,3-silyl-migration pathway. PMID:26928893

  14. Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa

    PubMed Central

    Matsuyama, Bruno Y.; Krasteva, Petya V.; Baraquet, Claudine; Harwood, Caroline S.; Sondermann, Holger; Navarro, Marcos V. A. S.

    2016-01-01

    Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ54-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ’s AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP–complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs. PMID:26712005

  15. Olanzapine Activates Hepatic Mammalian Target of Rapamycin: New Mechanistic Insight into Metabolic Dysregulation with Atypical Antipsychotic Drugs

    PubMed Central

    Schmidt, Robin H.; Jokinen, Jenny D.; Massey, Veronica L.; Falkner, K. Cameron; Shi, Xue; Yin, Xinmin; Zhang, Xiang; Beier, Juliane I.

    2013-01-01

    Olanzapine (OLZ), an effective treatment of schizophrenia and other disorders, causes weight gain and metabolic syndrome. Most studies to date have focused on the potential effects of OLZ on the central nervous system’s mediation of weight; however, peripheral changes in liver or other key metabolic organs may also play a role in the systemic effects of OLZ. Thus, the purpose of this study was to investigate the effects of OLZ on hepatic metabolism in a mouse model of OLZ exposure. Female C57Bl/6J mice were administered OLZ (8 mg/kg per day) or vehicle subcutaneously by osmotic minipumps for 28 days. Liver and plasma were taken at sacrifice for biochemical analyses and for comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry metabolomics analysis. OLZ increased body weight, fat pad mass, and liver-to-body weight ratio without commensurate increase in food consumption, indicating that OLZ altered energy expenditure. Expression and biochemical analyses indicated that OLZ induced anaerobic glycolysis and caused a pseudo-fasted state, which depleted hepatic glycogen reserves. OLZ caused similar effects in cultured HepG2 cells, as determined by Seahorse analysis. Metabolomic analysis indicated that OLZ increased hepatic concentrations of amino acids that can alter metabolism via the mTOR pathway; indeed, hepatic mTOR signaling was robustly increased by OLZ. Interestingly, OLZ concomitantly activated AMP-activated protein kinase (AMPK) signaling. Taken together, these data suggest that disturbances in glucose and lipid metabolism caused by OLZ in liver may be mediated, at least in part, via simultaneous activation of both catabolic (AMPK) and anabolic (mammalian target of rapamycin) pathways, which yields new insight into the metabolic side effects of this drug. PMID:23926289

  16. Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa.

    PubMed

    Matsuyama, Bruno Y; Krasteva, Petya V; Baraquet, Claudine; Harwood, Caroline S; Sondermann, Holger; Navarro, Marcos V A S

    2016-01-12

    Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs. PMID:26712005

  17. Mechanistic insights into the Bazarov synthesis of urea from NH3 and CO2 using electronic structure calculation methods.

    PubMed

    Tsipis, Constantinos A; Karipidis, Paraskevas A

    2005-09-29

    The mechanism of the noncatalyzed and reagent-catalyzed Bazarov synthesis of urea has extensively been investigated in the gas phase by means of density functional (B3LYP/6-31G(d,p)) and high quality ab initio (CBS-QB3) computational techniques. It was found that the first step of urea formation from NH3(g) and CO2(g) corresponds to a simple addition reaction leading to the carbamic acid intermediate, a process being slightly endothermic. Among the three possible reaction mechanisms considered, the addition-elimination-addition (AEA) and the double addition-elimination (DAE) mechanisms are almost equally favored, while the concerted (C) one was predicted kinetically forbidden. The second step involves the formation of loose adducts between NH3 and carbamic acid corresponding to an ammonium carbamate intermediate, which subsequently dehydrates to urea. The formation of "ammonium carbamate" corresponds to an almost thermoneutral process, whereas its dehydration, which is the rate-determining step, is highly endothermic. The Bazarov synthesis of urea is strongly assisted by the active participation of extra NH3 or H2O molecules (autocatalysis). For all reaction pathways studied the entire geometric and energetic profiles were computed and thoroughly analyzed. The reaction scheme described herein can be related with the formation of both isocyanic acid, H-N=C=O, and carbamic acid, H2N-COOH, as key intermediates in the initial formation of organic molecules, such as urea, under prebiotic conditions. PMID:16834254

  18. Mechanistic Insights of Ethanol Steam Reforming over Ni–CeO x (111): The Importance of Hydroxyl Groups for Suppressing Coke Formation

    DOE PAGESBeta

    Liu, Zongyuan; Duchoň, Tomáš; Wang, Huanru; Peterson, Erik W.; Zhou, Yinghui; Luo, Si; Zhou, Jing; Matolín, Vladimir; Stacchiola, Dario J.; Rodriguez, José A.; et al

    2015-07-30

    We have studied the reaction of ethanol and water over Ni–CeO2-x(111) model surfaces to elucidate the mechanistic steps associated with the ethanol steam reforming (ESR) reaction. Our results provide insights about the importance of hydroxyl groups to the ESR reaction over Ni-based catalysts. Systematically, we have investigated the reaction of ethanol on Ni–CeO2-x(111) at varying Ce³⁺ concentrations (CeO1.8–2.0) with absence/presence of water using a combination of soft X-ray photoelectron spectroscopy (sXPS) and temperature-programmed desorption (TPD). Consistent with previous reports, upon annealing, metallic Ni formed on reduced ceria while NiO was the main component on fully oxidized ceria. Ni⁰ is themore » active phase leading to both the C–C and C–H cleavage of ethanol but is also responsible for carbon accumulation or coking. We have identified a Ni₃C phase that formed prior to the formation of coke. At temperatures above 600K, the lattice oxygen from ceria and the hydroxyl groups from water interact cooperatively in the removal of coke, likely through a strong metal–support interaction between nickel and ceria that facilitates oxygen transfer.« less

  19. Mechanistic Insights of Ethanol Steam Reforming over Ni–CeO x (111): The Importance of Hydroxyl Groups for Suppressing Coke Formation

    SciTech Connect

    Liu, Zongyuan; Duchoň, Tomáš; Wang, Huanru; Peterson, Erik W.; Zhou, Yinghui; Luo, Si; Zhou, Jing; Matolín, Vladimir; Stacchiola, Dario J.; Rodriguez, José A.; Senanayake, Sanjaya D.

    2015-07-30

    We have studied the reaction of ethanol and water over Ni–CeO2-x(111) model surfaces to elucidate the mechanistic steps associated with the ethanol steam reforming (ESR) reaction. Our results provide insights about the importance of hydroxyl groups to the ESR reaction over Ni-based catalysts. Systematically, we have investigated the reaction of ethanol on Ni–CeO2-x(111) at varying Ce³⁺ concentrations (CeO1.8–2.0) with absence/presence of water using a combination of soft X-ray photoelectron spectroscopy (sXPS) and temperature-programmed desorption (TPD). Consistent with previous reports, upon annealing, metallic Ni formed on reduced ceria while NiO was the main component on fully oxidized ceria. Ni⁰ is the active phase leading to both the C–C and C–H cleavage of ethanol but is also responsible for carbon accumulation or coking. We have identified a Ni₃C phase that formed prior to the formation of coke. At temperatures above 600K, the lattice oxygen from ceria and the hydroxyl groups from water interact cooperatively in the removal of coke, likely through a strong metal–support interaction between nickel and ceria that facilitates oxygen transfer.

  20. A mechanistic insight into the mechanical role of the stratum corneum during stretching and compression of the skin.

    PubMed

    Leyva-Mendivil, Maria F; Page, Anton; Bressloff, Neil W; Limbert, Georges

    2015-09-01

    The study of skin biophysics has largely been driven by consumer goods, biomedical and cosmetic industries which aim to design products that efficiently interact with the skin and/or modify its biophysical properties for health or cosmetic benefits. The skin is a hierarchical biological structure featuring several layers with their own distinct geometry and mechanical properties. Up to now, no computational models of the skin have simultaneously accounted for these geometrical and material characteristics to study their complex biomechanical interactions under particular macroscopic deformation modes. The goal of this study was, therefore, to develop a robust methodology combining histological sections of human skin, image-processing and finite element techniques to address fundamental questions about skin mechanics and, more particularly, about how macroscopic strains are transmitted and modulated through the epidermis and dermis. The work hypothesis was that, as skin deforms under macroscopic loads, the stratum corneum does not experience significant strains but rather folds/unfolds during skin extension/compression. A sample of fresh human mid-back skin was processed for wax histology. Sections were stained and photographed by optical microscopy. The multiple images were stitched together to produce a larger region of interest and segmented to extract the geometry of the stratum corneum, viable epidermis and dermis. From the segmented structures a 2D finite element mesh of the skin composite model was created and geometrically non-linear plane-strain finite element analyses were conducted to study the sensitivity of the model to variations in mechanical properties. The hybrid experimental-computational methodology has offered valuable insights into the simulated mechanics of the skin, and that of the stratum corneum in particular, by providing qualitative and quantitative information on strain magnitude and distribution. Through a complex non-linear interplay

  1. Physiological and transcriptomic analyses reveal mechanistic insight into the adaption of marine Bacillus subtilis C01 to alumina nanoparticles.

    PubMed

    Mu, Dashuai; Yu, Xiuxia; Xu, Zhenxing; Du, Zongjun; Chen, Guanjun

    2016-01-01

    An increasing number of studies have investigated the effects of nanoparticles (NPs) on microbial systems; however, few existing reports have focused on the defense mechanisms of bacteria against NPs. Whether secondary metabolism biosynthesis is a response to NP stress and contributes to the adaption of bacteria to NPs is unclear. Here, a significant induction in the surfactin production and biofilm formation were detected by adding Al2O3 NPs to the B. subtilis fermentation broth. Physiological analysis showed that Al2O3 NP stress could also affect the cell and colony morphogenesis and inhibit the motility and sporulation. Exogenously adding commercial surfactin restored the swarming motility. Additionally, a suite of toxicity assays analyzing membrane damage, cellular ROS generation, electron transport activity and membrane potential was used to determine the molecular mechanisms of toxicity of Al2O3 NPs. Furthermore, whole transcriptomic analysis was used to elucidate the mechanisms of B. subtilis adaption to Al2O3 NPs. These results revealed several mechanisms by which marine B. subtilis C01 adapt to Al2O3 NPs. Additionally, this study broadens the applications of nanomaterials and describes the important effects on secondary metabolism and multicellularity regulation by using Al2O3 NPs or other nano-products. PMID:27440502

  2. Physiological and transcriptomic analyses reveal mechanistic insight into the adaption of marine Bacillus subtilis C01 to alumina nanoparticles

    PubMed Central

    Mu, Dashuai; Yu, Xiuxia; Xu, Zhenxing; Du, Zongjun; Chen, Guanjun

    2016-01-01

    An increasing number of studies have investigated the effects of nanoparticles (NPs) on microbial systems; however, few existing reports have focused on the defense mechanisms of bacteria against NPs. Whether secondary metabolism biosynthesis is a response to NP stress and contributes to the adaption of bacteria to NPs is unclear. Here, a significant induction in the surfactin production and biofilm formation were detected by adding Al2O3 NPs to the B. subtilis fermentation broth. Physiological analysis showed that Al2O3 NP stress could also affect the cell and colony morphogenesis and inhibit the motility and sporulation. Exogenously adding commercial surfactin restored the swarming motility. Additionally, a suite of toxicity assays analyzing membrane damage, cellular ROS generation, electron transport activity and membrane potential was used to determine the molecular mechanisms of toxicity of Al2O3 NPs. Furthermore, whole transcriptomic analysis was used to elucidate the mechanisms of B. subtilis adaption to Al2O3 NPs. These results revealed several mechanisms by which marine B. subtilis C01 adapt to Al2O3 NPs. Additionally, this study broadens the applications of nanomaterials and describes the important effects on secondary metabolism and multicellularity regulation by using Al2O3 NPs or other nano-products. PMID:27440502

  3. Mechanistic insights into the distribution of carbohydrate clusters on cell membranes revealed by dSTORM imaging

    NASA Astrophysics Data System (ADS)

    Chen, Junling; Gao, Jing; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

    2016-07-01

    Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the carbohydrate-binding proteins (i.e., galectins) cross-linking their specific carbohydrate ligands. Our results clarify the organizational mechanism of carbohydrates on cell surfaces from their formation, stable existence and size-restriction, which promotes a better understanding of the relationship between the function and distribution of carbohydrates, as well as the structure of cell membranes.Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the

  4. Organically treated biochar increases plant production and reduces N2O emissions: mechanistic insights by 15N tracing

    NASA Astrophysics Data System (ADS)

    Kammann, Claudia; Messerschmidt, Nicole; Clough, Tim; Schmidt, Hans-Peter; Marhan, Sven; Koyro, Hans-Werner; Steffens, Diedrich; Müller, Christoph

    2014-05-01

    Pyrogenic carbon (biochar) offers considerable potential for carbon capture and soil storage (CCSS) compared to other, less recalcitrant soil-C additives. Recent meta-analysis demonstrated that it can significantly reduce agricultural N2O emissions. Freshly produced biochars, however, do not always have yield-improving effects, i.e. there is no immediate economic incentive for using it. Hence, combining biochar with organic nutrient-rich amendments may be a promising agricultural strategy to accelerate CCSS, but it is unclear if biochar still reduces N2O emissions, in particular when it may act as nutrient carrier. We explored the potential of biochar to improve the GHG-cost/yield ratio and thereby its socio-economic value as soil amendment in two subsequent studies under controlled conditions: (1) A proof-of-concept study where the effects of untreated biochar were compared to those of co-composted biochar combined with stepwise improved nutritional regimes (+/- compost; +/- mineral-N application), and (2) a 15N-labeling-tracing study to unravel N exchange on biochar particles and N2O production and reduction mechanisms. Both studies were carried out in nutrient-poor sandy soils, the most likely initial target soils for biochar-CCSS strategies. While the untreated biochar reduced plant growth under N-limiting conditions, or at best did not reduce it, the co-composted biochar always significantly stimulated plant growth. The relative stimulation was largest with the lowest nutrient additions (305% versus 61% of control with untreated biochar). Electro-ultra-filtration analyses revealed that the co-composted but not the untreated biochar carried considerable amounts of easily extractable as well as more strongly sorbed plant nutrients, in particular nitrate and phosphorus. The subsequent 15N labelling-tracing study revealed that the co-composted biochar still (i) acted as a mineral-N exchange site for nitrate and ammonium despite its N-preloading, (ii) reduced N2O

  5. Mechanistic insights on chaotropic interactions of liophilic ions with basic pharmaceuticals in polar ionic mode liquid chromatography.

    PubMed

    Sanganyado, Edmond; Lu, Zhijiang; Gan, Jay

    2014-11-14

    We report for the first time the effect of liophilic mobile phase additives on the mechanism of chiral recognition of basic chiral pharmaceutical on a vancomycin based chiral stationary phase (CSP). Using methanol as the mobile phase and 0.005% formic acid as pH modifier, we evaluated the effect of different concentrations of three types of liophilic anions, formate (HCOO(-)), nitrate (NO3(-)), and acetate (CH3COO(-)), on enantioresolution (Rs), enantioselectivity (α) and retention factor (k) of enantiomers of fluoxetine and atenolol. The effect of liophilic ion types on k followed the Hofmeister series: CH3COO(-)>HCOO(-)>NO3(-). Increasing concentration from 4 to 20mM resulted in decreases in Rs and k in accordance to hydrophobicity of the liophilic anion. The effect of temperature or mobile phase composition on enantioseparation was determined at 13-40°C. For both analytes, standard changes in enthalpy (ΔH°) and entropy (ΔS°) calculated using van't Hoff plots (lnk against 1/T) to varied from -4.99 to -0.63 kJ/mol and -11.82 to 9.47 J/mol, respectively. The van't Hoff plots showed elution order of the enantiomers of each analyte did not reverse in the temperature range studied. Chiral recognition of the enantiomers of atenolol and fluoxetine in the presence of liophilic ions was enthalpy driven. PMID:25311487

  6. Mechanistic insights into the distribution of carbohydrate clusters on cell membranes revealed by dSTORM imaging.

    PubMed

    Chen, Junling; Gao, Jing; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

    2016-07-14

    Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the carbohydrate-binding proteins (i.e., galectins) cross-linking their specific carbohydrate ligands. Our results clarify the organizational mechanism of carbohydrates on cell surfaces from their formation, stable existence and size-restriction, which promotes a better understanding of the relationship between the function and distribution of carbohydrates, as well as the structure of cell membranes. PMID:27362510

  7. Insights into Mechanistic Models for Evaporation of Organic Liquids in the Environment Obtained by Position-Specific Carbon Isotope Analysis.

    PubMed

    Julien, Maxime; Nun, Pierrick; Robins, Richard J; Remaud, Gérald S; Parinet, Julien; Höhener, Patrick

    2015-11-01

    Position-specific isotope effects (PSIEs) have been measured by isotope ratio monitoring (13)C nuclear magnetic resonance spectrometry during the evaporation of 10 liquids of different polarities under 4 evaporation modes (passive evaporation, air-vented evaporation, low pressure evaporation, distillation). The observed effects are used to assess the validity of the Craig-Gordon isotope model for organic liquids. For seven liquids the overall isotope effect (IE) includes a vapor-liquid contribution that is strongly position-specific in polar compounds but less so in apolar compounds and a diffusive IE that is not position-specific, except in the alcohols, ethanol and propan-1-ol. The diffusive IE is diminished under forced evaporation. The position-specific isotope pattern created by liquid-vapor IEs is manifest in five liquids, which have an air-side limitation for volatilization. For the alcohols, undefined processes in the liquid phase create additional PSIEs. Three other liquids with limitations on the liquid side have a lower, highly position-specific, bulk diffusive IE. It is concluded that evaporation of organic pollutants creates unique position-specific isotope patterns that may be used to assess the progress of remediation or natural attenuation of pollution and that the Craig-Gordon isotope model is valid for the volatilization of nonpolar organic liquids with air-side limitation of the volatilization rate. PMID:26443423

  8. Simple cathode design for Li–S batteries: cell performance and mechanistic insights by in operando X-ray diffraction.

    PubMed

    Kulisch, Jörn; Sommer, Heino; Brezesinski, Torsten; Janek, Jürgen

    2014-09-21

    Rechargeable batteries have been receiving increasing attention over the past several years, particularly with regard to the accelerated development of electric vehicles, but also for their potential in grid storage applications. Among the broad range of cathode active materials, elemental sulfur has the highest theoretical specific capacity, thereby making it one of the most promising positive electrode materials these days. In the present work, we show that already a simple cathode design (cathodes with a non-optimized composite microstructure) provides good electrochemical performance both in coin and pouch cells with sulfur loadings of 2 mg cm−2. Our research data demonstrate that (1) specific capacities of 1000 mA h g−1 can be achieved over 60 cycles at room temperature while the cyclability at elevated temperatures (here, θ > 40 °C) is poor, (2) the discharge is the kinetically rate-limiting process, (3) the major fraction of active sulfur in the electrode is lost during the formation cycle at C/50 and (4) the Li–S cells suffer from drying-out due to continuous electrolyte decomposition on the lithium metal anode. In addition, in operando X-ray diffraction shows Li2S formation (grain size of <10 nm) on discharge and the appearance of single phase β-sulfur in the sub-100 nm size range – rather than the thermodynamically stable orthorhombic polymorph (α-sulfur) – by the end of the charge cycle. PMID:25077958

  9. Mechanistic insight into CM18-Tat11 peptide membrane-perturbing action by whole-cell patch-clamp recording.

    PubMed

    Fasoli, Anna; Salomone, Fabrizio; Benedusi, Mascia; Boccardi, Claudia; Rispoli, Giorgio; Beltram, Fabio; Cardarelli, Francesco

    2014-01-01

    The membrane-destabilization properties of the recently-introduced endosomolytic CM18-Tat11 hybrid peptide (KWKLFKKIGAVLKVLTTG-YGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein) are investigated in CHO-K1 cells by using the whole-cell configuration of the patch-clamp technique. CM18-Tat11, CM18, and Tat11 peptides are administered to the cell membrane with a computer-controlled micro-perfusion system. CM18-Tat11 induces irreversible cell-membrane permeabilization at concentrations (≥4 µM) at which CM18 triggers transient pore formation, and Tat11 does not affect membrane integrity. We argue that the addition of the Tat11 module to CM18 is able to trigger a shift in the mechanism of membrane destabilization from "toroidal" to "carpet", promoting a detergent-like membrane disruption. Collectively, these results rationalize previous observations on CM18-Tat11 delivery properties that we believe can guide the engineering of new modular peptides tailored to specific cargo-delivery applications. PMID:24991756

  10. Mechanistic insight on (E)-methyl 3-(2-aminophenyl)acrylate cyclization reaction by multicatalysis of solvent and substrate.

    PubMed

    Liu, Jiahui; Zheng, Yiying; Liu, Ying; Yuan, Haiyan; Zhang, Jingping

    2016-10-01

    The reaction mechanism of (E)-methyl 3-(2-aminophenyl)acrylate (A) with phenylisothiocyanate (B) as well as the vital roles of substrate A and solvent water were investigated under unassisted, water-assisted, substrate A-assisted, and water-A-assisted conditions. The reaction proceeds with four processes via nucleophilic addition, deprotonation and protonation, intramolecular cyclization with hydrogen transfer, and keto-enol tautomerization. According to the different H-shift mode, two possible types of H-shift P1 and P2 are carefully investigated to identify the most preferred pathway, differing in the NH2 group deprotonation and CH group of A protonation processes. It is found that substrate A and water not only act as reactant and solvent, but also as catalyst, proton shuttle, and stabilizer in effectively lowering the energy barrier. Therefore, the results demonstrate that the strong donating and accepting ability of NH2 group on A and the presence of bulk water are the keys to the title reaction proceed. © 2016 Wiley Periodicals, Inc. PMID:27487815

  11. Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II).

    PubMed

    Wrobel, Katarzyna; Corrales Escobosa, Alma Rosa; Gonzalez Ibarra, Alan Alexander; Mendez Garcia, Manuel; Yanez Barrientos, Eunice; Wrobel, Kazimierz

    2015-12-30

    Over the past few decades, reduction of hexavalent chromium (Cr(VI)) has been studied in many physicochemical contexts. In this research, we reveal the mechanism underlying the favorable effect of Mn(II) observed during Cr(VI) reduction by oxalic acid using liquid chromatography with spectrophotometric diode array detector (HPLC-DAD), nitrogen microwave plasma atomic emission spectrometry (HPLC-MP-AES), and high resolution mass spectrometry (ESI-QTOFMS). Both reaction mixtures contained potassium dichromate (0.67 mM Cr(VI)) and oxalic acid (13.3mM), pH 3, one reaction mixture contained manganese sulfate (0.33 mM Mn(II)). In the absence of Mn(II) only trace amounts of reaction intermediates were generated, most likely in the following pathways: (1) Cr(VI)→ Cr(IV) and (2) Cr(VI)+Cr(IV)→ 2Cr(V). In the presence of Mn(II), the active reducing species appeared to be Mn(II) bis-oxalato complex (J); the proposed reaction mechanism involves a one-electron transfer from J to any chromium compound containing CrO bond, which is reduced to CrOH, and the generation of Mn(III) bis-oxalato complex (K). Conversion of K to J was observed, confirming the catalytic role of Mn(II). Since no additional acidification was required, the results obtained in this study may be helpful in designing a new, environmentally friendly strategy for the remediation of environments contaminated with Cr(VI). PMID:26177490

  12. Co-composted biochar can promote plant growth by serving as a nutrient carrier: first mechanistic insights

    NASA Astrophysics Data System (ADS)

    Kammann, Claudia; Haider, Ghulam; Messerschmidt, Nicole; Schmidt, Hans-Peter; Koyro, Hans-Werner; Steffens, Diedrich; Clough, Timothy; Müller, Christoph

    2014-05-01

    Pyrogenic carbon (biochar) offers considerable potential for carbon capture (CCSS) and soil storage and meta-analysis suggests that it can significantly reduce soil N2O emissions. Freshly produced biochars, however, do not always have yield-improving effects; pure, production-fresh biochar seems to 'claim' some nutrients initially from soil, particularly nitrogen, although the mechanisms are unclear to date. Hence, combining biochar with organic nutrient-rich materials and waste streams may be a promising strategy to enable CCSS by yield improvements, which may enable economically feasible biochar use in agriculture. We explored the potential of organically post-treated biochar to act as a nutrient carrier and thus to increase its socio-economic value as beneficial soil amendment with associated CCSS value. In a first proof-of-concept study the effects of untreated biochar were compared to those of co-composted biochar, combined with stepwise improved nutritional regimes (+/- compost; +/- mineral-N application). While the untreated biochar reduced plant growth under N-limiting conditions, or at best did not reduce it, the co-composted biochar always significantly stimulated plant growth. The relative stimulation was largest with the lowest nutrient additions (305% versus 61% of control with untreated biochar). Subsequent electro-ultra-filtration analyses revealed that the co-composted but not the untreated biochar carried considerable amounts of easily extractable as well as more strongly sorbed plant nutrients, in particular nitrate and phosphorus. Nevertheless the co-composted N-rich biochar still sorbed 15N labelled NH4+ or NO3- when present in the soil, and again released it to growing barley plants. We will report on the relationship between particle size, increased nutrient content, and plant accessibility of the nutrients associated with the co-composted biochar, and analyse the extent to which the strongly sorbed nutrients on the biochar may be 'invisible

  13. Protein ubiquitination via dehydroalanine: development and insights into the diastereoselective 1,4-addition step.

    PubMed

    Meledin, Roman; Mali, Sachitanand M; Singh, Sumeet K; Brik, Ashraf

    2016-06-01

    We report a strategy for site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the preparation of ubiquitin conjugates bearing a very close mimic of the native isopeptide bond. Our approach relies on the selective formation of Dha followed by conjugation with hexapeptide bearing a thiol handle derived from the C-terminal of ubiquitin. Subsequently, the resulting synthetic intermediate undergoes native chemical ligation with the complementary part of the ubiquitin polypeptide. It has been proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral protein environment may influence such an addition step. In the protein context these questions remain open and no experimental evidence was provided as to how such a protein environment affects the diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid chromatography analysis were performed. Moreover, the obtained peptide diastereomers were compared with identical synthetic peptides having defined stereogenic centers, which enabled the determination of the configuration of the isopeptide mimic in each diastereomer. Our study, which offers a new method for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the stereoselectivity of the addition step to Dha for chemical protein modifications. PMID:27143624

  14. Transcriptional Profiling of Dibenzo[def,p]chrysene-induced Spleen Atrophy Provides Mechanistic Insights into its Immunotoxicity in MutaMouse.

    PubMed

    Chepelev, Nikolai L; Long, Alexandra S; Williams, Andrew; Kuo, Byron; Gagné, Rémi; Kennedy, Dean A; Phillips, David H; Arlt, Volker M; White, Paul A; Yauk, Carole L

    2016-01-01

    Dibenzo[def,p]chrysene (DBC) is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) examined to date. We investigated the immunotoxicity of DBC, manifested as spleen atrophy, following acute exposure of adult MutaMouse males by oral gavage. Mice were exposed to 0, 2.0, 6.2, or 20.0 mg DBC /kg-bw per day, for 3 days. Genotoxic endpoints (DBC-DNA adducts and lacZ mutant frequency in spleen and bone marrow, and red blood cell micronucleus frequency) and global gene expression changes were measured. All of the genotoxicity measures increased in a dose-dependent manner in spleen and bone marrow. Gene expression analysis showed that DBC activates p53 signaling pathways related to cellular growth and proliferation, which was evident even at the low dose. Strikingly, the expression profiles of DBC exposed mouse spleens were highly inversely correlated with the expression profiles of the only published toxicogenomics dataset of enlarged mouse spleen. This analysis suggested a central role for Bnip3l, a pro-apoptotic protein involved in negative regulation of erythroid maturation. RT-PCR confirmed expression changes in several genes related to apoptosis, iron metabolism, and aryl hydrocarbon receptor signaling that are regulated in the opposite direction during spleen atrophy versus benzo[a]pyrene-mediated splenomegaly. In addition, benchmark dose modeling of toxicogenomics data yielded toxicity estimates that are very close to traditional toxicity endpoints. This work illustrates the power of toxicogenomics to reveal rich mechanistic information for immunotoxic compounds and its ability to provide information that is quantitatively similar to that derived from standard toxicity methods in health risk assessment. PMID:26496743

  15. Time-Resolved In Situ Measurements During Rapid Alloy Solidification: Experimental Insight for Additive Manufacturing

    NASA Astrophysics Data System (ADS)

    McKeown, Joseph T.; Zweiacker, Kai; Liu, Can; Coughlin, Daniel R.; Clarke, Amy J.; Baldwin, J. Kevin; Gibbs, John W.; Roehling, John D.; Imhoff, Seth D.; Gibbs, Paul J.; Tourret, Damien; Wiezorek, Jörg M. K.; Campbell, Geoffrey H.

    2016-03-01

    Additive manufacturing (AM) of metals and alloys is becoming a pervasive technology in both research and industrial environments, though significant challenges remain before widespread implementation of AM can be realized. In situ investigations of rapid alloy solidification with high spatial and temporal resolutions can provide unique experimental insight into microstructure evolution and kinetics that are relevant for AM processing. Hypoeutectic thin-film Al-Cu and Al-Si alloys were investigated using dynamic transmission electron microscopy to monitor pulsed-laser-induced rapid solidification across microsecond timescales. Solid-liquid interface velocities measured from time-resolved images revealed accelerating solidification fronts in both alloys. The observed microstructure evolution, solidification product, and presence of a morphological instability at the solid-liquid interface in the Al-4 at.%Cu alloy are related to the measured interface velocities and small differences in composition that affect the thermophysical properties of the alloys. These time-resolved in situ measurements can inform and validate predictive modeling efforts for AM.

  16. Time-Resolved In Situ Measurements During Rapid Alloy Solidification: Experimental Insight for Additive Manufacturing

    DOE PAGESBeta

    McKeown, Joseph T.; Zweiacker, Kai; Liu, Can; Coughlin, Daniel R.; Clarke, Amy J.; Baldwin, J. Kevin; Gibbs, John W.; Roehling, John D.; Imhoff, Seth D.; Gibbs, Paul J.; et al

    2016-01-27

    In research and industrial environments, additive manufacturing (AM) of metals and alloys is becoming a pervasive technology, though significant challenges remain before widespread implementation of AM can be realized. In situ investigations of rapid alloy solidification with high spatial and temporal resolutions can provide unique experimental insight into microstructure evolution and kinetics that are relevant for AM processing. Hypoeutectic thin-film Al–Cu and Al–Si alloys were investigated using dynamic transmission electron microscopy to monitor pulsed-laser-induced rapid solidification across microsecond timescales. Solid–liquid interface velocities measured from time-resolved images revealed accelerating solidification fronts in both alloys. We observed microstructure evolution, solidification product, andmore » presence of a morphological instability at the solid–liquid interface in the Al–4 at.%Cu alloy are related to the measured interface velocities and small differences in composition that affect the thermophysical properties of the alloys. These time-resolved in situ measurements can inform and validate predictive modeling efforts for AM.« less

  17. Redox chemistry of Mycobacterium tuberculosis alkylhydroperoxide reductase E (AhpE): Structural and mechanistic insight into a mycoredoxin-1 independent reductive pathway of AhpE via mycothiol.

    PubMed

    Kumar, Arvind; Balakrishna, Asha Manikkoth; Nartey, Wilson; Manimekalai, Malathy Sony Subramanian; Grüber, Gerhard

    2016-08-01

    Mycobacterium tuberculosis (Mtb) has the ability to persist within the human host for a long time in a dormant stage and re-merges when the immune system is compromised. The pathogenic bacterium employs an elaborate antioxidant defence machinery composed of the mycothiol- and thioredoxin system in addition to a superoxide dismutase, a catalase, and peroxiredoxins (Prxs). Among the family of Peroxiredoxins, Mtb expresses a 1-cysteine peroxiredoxin, known as alkylhydroperoxide reductase E (MtAhpE), and defined as a potential tuberculosis drug target. The reduced MtAhpE (MtAhpE-SH) scavenges peroxides to become converted to MtAhpE-SOH. To provide continuous availability of MtAhpE-SH, MtAhpE-SOH has to become reduced. Here, we used NMR spectroscopy to delineate the reduced (MtAhpE-SH), sulphenic (MtAhpE-SOH) and sulphinic (MtAhpE-SO2H) states of MtAhpE through cysteinyl-labelling, and provide for the first time evidence of a mycothiol-dependent mechanism of MtAhpE reduction. This is confirmed by crystallographic studies, wherein MtAhpE was crystallized in the presence of mycothiol and the structure was solved at 2.43Å resolution. Combined with NMR-studies, the crystallographic structures reveal conformational changes of important residues during the catalytic cycle of MtAhpE. In addition, alterations of the overall protein in solution due to redox modulation are observed by small angle X-ray scattering (SAXS) studies. Finally, by employing SAXS and dynamic light scattering, insight is provided into the most probable physiological oligomeric state of MtAhpE necessary for activity, being also discussed in the context of concerted substrate binding inside the dimeric MtAhpE. PMID:27417938

  18. Microscopic Investigation of Chemoselectivity in Ag-Pt-Fe3O4 Heterotrimer Formation: Mechanistic Insights and Implications for Controlling High-Order Hybrid Nanoparticle Morphology.

    PubMed

    Hodges, James M; Morse, James R; Williams, Mary Elizabeth; Schaak, Raymond E

    2015-12-16

    Three-component hybrid nanoparticle heterotrimers, which are important multifunctional constructs that underpin diverse applications, are commonly synthesized by growing a third domain off of a two-component heterodimer seed. However, because heterodimer seeds expose two distinct surfaces that often can both support nucleation and growth, selectively targeting one particular surface is critical for exclusively accessing a desired configuration. Understanding and controlling nucleation and growth therefore enables the rational formation of high-order hybrid nanoparticles. Here, we report an in-depth microscopic investigation that probes the chemoselective addition of Ag to Pt-Fe3O4 heterodimer seeds to form Ag-Pt-Fe3O4 heterotrimers. We find that the formation of the Ag-Pt-Fe3O4 heterotrimers initiates with indiscriminate Ag nucleation onto both the Pt and Fe3O4 surfaces of Pt-Fe3O4, followed by surface diffusion and coalescence of Ag onto the Pt surface to form the Ag-Pt-Fe3O4 product. Control experiments reveal that the size of the Ag domain of Ag-Pt-Fe3O4 correlates with the overall surface area of the Pt-Fe3O4 seeds, which is consistent with the coalescence of Ag through a surface-mediated process and can also be exploited to tune the size of the Ag domain. Additionally, we observe that small iron oxide islands on the Pt surface of the Pt-Fe3O4 seeds, deposited during the formation of Pt-Fe3O4, define the morphology of the Ag domain, which in turn influences its optical properties. These results provide unprecedented microscopic insights into the pathway by which Ag-Pt-Fe3O4 heterotrimer nanoparticles form and uncover new design guidelines for the synthesis of high-order hybrid nanoparticles with precisely targeted morphologies and properties. PMID:26599998

  19. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  20. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

  1. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

    PubMed Central

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

  2. Catalytic and mechanistic insights of the low-temperature selective oxidation of methane over Cu-promoted Fe-ZSM-5.

    PubMed

    Hammond, Ceri; Jenkins, Robert L; Dimitratos, Nikolaos; Lopez-Sanchez, Jose Antonio; ab Rahim, Mohd Hasbi; Forde, Michael M; Thetford, Adam; Murphy, Damien M; Hagen, Henk; Stangland, Eric E; Moulijn, Jacob M; Taylor, Stuart H; Willock, David J; Hutchings, Graham J

    2012-12-01

    The partial oxidation of methane to methanol presents one of the most challenging targets in catalysis. Although this is the focus of much research, until recently, approaches had proceeded at low catalytic rates (<10 h(-1)), not resulted in a closed catalytic cycle, or were unable to produce methanol with a reasonable selectivity. Recent research has demonstrated, however, that a system composed of an iron- and copper-containing zeolite is able to catalytically convert methane to methanol with turnover frequencies (TOFs) of over 14,000 h(-1) by using H(2)O(2) as terminal oxidant. However, the precise roles of the catalyst and the full mechanistic cycle remain unclear. We hereby report a systematic study of the kinetic parameters and mechanistic features of the process, and present a reaction network consisting of the activation of methane, the formation of an activated hydroperoxy species, and the by-production of hydroxyl radicals. The catalytic system in question results in a low-energy methane activation route, and allows selective C(1)-oxidation to proceed under intrinsically mild reaction conditions. PMID:23150452

  3. Synthetic applications of Pd(II)-catalyzed C-H carboxylation and mechanistic insights: expedient routes to anthranilic acids, oxazolinones, and quinazolinones.

    PubMed

    Giri, Ramesh; Lam, Jonathan K; Yu, Jin-Quan

    2010-01-20

    A Pd(II)-catalyzed reaction protocol for the carboxylation of ortho-C-H bonds in anilides to form N-acyl anthranilic acids has been developed. This reaction procedure provides a novel and efficient strategy for the rapid assembly of biologically and pharmaceutically significant molecules, such as benzoxazinones and quinazolinones, from simple anilides without installing and removing an external directing group. The reaction conditions are also amenable to the carboxylation of N-phenyl pyrrolidinones. A monomeric palladacycle containing p-toluenesulfonate as an anionic ligand has been characterized by X-ray crystallography, and the crucial role of p-toluenesulfonic acid in the activation of C-H bonds in the presence of carbon monoxide is discussed. Identification of two key intermediates, a mixed anhydride and benzoxazinone formed by reductive elimination from organometallic Ar(CO)Pd(II)-OTs species, provides mechanistic evidence for a dual-reaction pathway. PMID:20000840

  4. Cellular and molecular mechanistic insight into the DNA-damaging potential of few-layer graphene in human primary endothelial cells.

    PubMed

    Sasidharan, Abhilash; Swaroop, Siddharth; Chandran, Parwathy; Nair, Shantikumar; Koyakutty, Manzoor

    2016-07-01

    Despite graphene being proposed for a multitude of biomedical applications, there is a dearth in the fundamental cellular and molecular level understanding of how few-layer graphene (FLG) interacts with human primary cells. Herein, using human primary umbilical vein endothelial cells as model of vascular transport, we investigated the basic mechanism underlying the biological behavior of graphene. Mechanistic toxicity studies using a battery of cell based assays revealed an organized oxidative stress paradigm involving cytosolic reactive oxygen stress, mitochondrial superoxide generation, lipid peroxidation, glutathione oxidation, mitochondrial membrane depolarization, enhanced calcium efflux, all leading to cell death by apoptosis/necrosis. We further investigated the effect of graphene interactions using cDNA microarray analysis and identified potential adverse effects by down regulating key genes involved in DNA damage response and repair mechanisms. Single cell gel electrophoresis assay/Comet assay confirmed the DNA damaging potential of graphene towards human primary cells. PMID:26970024

  5. Mechanistic Insights into Cofactor-Dependent Coupling of RNA Folding and mRNA Transcription/Translation by a Cobalamin Riboswitch.

    PubMed

    Polaski, Jacob T; Holmstrom, Erik D; Nesbitt, David J; Batey, Robert T

    2016-05-01

    Riboswitches are mRNA elements regulating gene expression in response to direct binding of a metabolite. While these RNAs are increasingly well understood with respect to interactions between receptor domains and their cognate effector molecules, little is known about the specific mechanistic relationship between metabolite binding and gene regulation by the downstream regulatory domain. Using a combination of cell-based, biochemical, and biophysical techniques, we reveal the specific RNA architectural features enabling a cobalamin-dependent hairpin loop docking interaction between receptor and regulatory domains. Furthermore, these data demonstrate that docking kinetics dictate a regulatory response involving the coupling of translation initiation to general mechanisms that control mRNA abundance. These results yield a comprehensive picture of how RNA structure in the riboswitch regulatory domain enables kinetically constrained ligand-dependent regulation of gene expression. PMID:27117410

  6. Selective Alcohol Oxidation by a Copper TEMPO Catalyst: Mechanistic Insights by Simultaneously Coupled Operando EPR/UV-Vis/ATR-IR Spectroscopy.

    PubMed

    Rabeah, Jabor; Bentrup, Ursula; Stößer, Reinhard; Brückner, Angelika

    2015-09-28

    The first coupled operando EPR/UV-Vis/ATR-IR spectroscopy setup for mechanistic studies of gas-liquid phase reactions is presented and exemplarily applied to the well-known copper/TEMPO-catalyzed (TEMPO=(2,2,6,6-tetramethylpiperidin-1-yl)oxyl) oxidation of benzyl alcohol. In contrast to previous proposals, no direct redox reaction between TEMPO and Cu(I) /Cu(II) has been detected. Instead, the role of TEMPO is postulated to be the stabilization of a (bpy)(NMI)Cu(II) -O2 (⋅-) -TEMPO (bpy=2,2'-bipyridine, NMI=N-methylimidazole) intermediate formed by electron transfer from Cu(I) to molecular O2 . PMID:26174141

  7. Regioselectivity of radical additions to substituted alkenes: insight from conceptual density functional theory.

    PubMed

    De Vleeschouwer, Freija; Jaque, Pablo; Geerlings, Paul; Toro-Labbé, Alejandro; De Proft, Frank

    2010-08-01

    Radical additions to substituted alkenes are among the most important reactions in radical chemistry. Nonetheless, there is still some controversy in the literature about the factors that affect the rate and regioselectivity in these addition reactions. In this paper, the orientation of (nucleophilic) radical additions to electron-rich, -neutral, and -poor monosubstituted substrates (11 reactions in total) is investigated through the use of chemical concepts and reactivity descriptors. The regioselectivity of the addition of nucleophilic radicals on electron-rich and -neutral alkenes is thermodynamically controlled. An excellent correlation of 94% is found between the differences in activation barriers and in product stabilities (unsubstituted versus substituted site attack). Polar effects at the initial stage of the reaction play a significant role when electron-poor substrates are considered, lowering the extent of regioselectivity toward the unsubstituted sites, as predicted from the stability differences. This is nicely confirmed through an analysis for each of the 11 reactions using the spin-polarized dual descriptor, matching electrophilic and nucleophilic regions. PMID:20614876

  8. Suppression of acetylpolyamine oxidase by selected AP-1 members regulates DNp73 abundance: mechanistic insights for overcoming DNp73-mediated resistance to chemotherapeutic drugs.

    PubMed

    Bunjobpol, W; Dulloo, I; Igarashi, K; Concin, N; Matsuo, K; Sabapathy, K

    2014-08-01

    Enhanced resistance to chemotherapy has been correlated with high levels of Delta-Np73 (DNp73), an anti-apoptotic protein of the p53 tumor-suppressor family which inhibits the pro-apoptotic members such as p53 and TAp73. Although genotoxic drugs have been shown to induce DNp73 degradation, lack of mechanistic understanding of this process precludes strategies to enhance the targeting of DNp73 and improve treatment outcomes. Antizyme (Az) is a mediator of ubiquitin-independent protein degradation regulated by the polyamine biosynthesis pathway. We show here that acetylpolyamine oxidase (PAOX), a catabolic enzyme of this pathway, upregulates DNp73 levels by suppressing its degradation via the Az pathway. Conversely, downregulation of PAOX activity by siRNA-mediated knockdown or chemical inhibition leads to DNp73 degradation in an Az-dependent manner. PAOX expression is suppressed by several genotoxic drugs, via selected members of the activator protein-1 (AP-1) transcription factors, namely c-Jun, JunB and FosB, which are required for stress-mediated DNp73 degradation. Finally, chemical- and siRNA-mediated inhibition of PAOX significantly reversed the resistant phenotype of DNp73-overexpressing cancer cells to genotoxic drugs. Together, these data define a critical mechanism for the regulation of DNp73 abundance, and reveal that inhibition of PAOX could widen the therapeutic index of cytotoxic drugs and overcome DNp73-mediated chemoresistance in tumors. PMID:24722210

  9. Enantioselective rhodium-catalyzed [2 + 2 + 2] cycloadditions of terminal alkynes and alkenyl isocyanates: mechanistic insights lead to a unified model that rationalizes product selectivity.

    PubMed

    Dalton, Derek M; Oberg, Kevin M; Yu, Robert T; Lee, Ernest E; Perreault, Stéphane; Oinen, Mark Emil; Pease, Melissa L; Malik, Guillaume; Rovis, Tomislav

    2009-11-01

    This manuscript describes the development and scope of the asymmetric rhodium-catalyzed [2 + 2 + 2] cycloaddition of terminal alkynes and alkenyl isocyanates leading to the formation of indolizidine and quinolizidine scaffolds. The use of phosphoramidite ligands proved crucial for avoiding competitive terminal alkyne dimerization. Both aliphatic and aromatic terminal alkynes participate well, with product selectivity a function of both the steric and electronic character of the alkyne. Manipulation of the phosphoramidite ligand leads to tuning of enantio- and product selectivity, with a complete turnover in product selectivity seen with aliphatic alkynes when moving from Taddol-based to biphenol-based phosphoramidites. Terminal and 1,1-disubstituted olefins are tolerated with nearly equal efficacy. Examination of a series of competition experiments in combination with analysis of reaction outcome shed considerable light on the operative catalytic cycle. Through a detailed study of a series of X-ray structures of rhodium(cod)chloride/phosphoramidite complexes, we have formulated a mechanistic hypothesis that rationalizes the observed product selectivity. PMID:19817441

  10. Enantioselective Rhodium-Catalyzed [2+2+2] Cycloadditions of Terminal Alkynes and Alkenyl Isocyanates: Mechanistic Insights Lead to a Unified Model that Rationalizes Product Selectivity

    PubMed Central

    Dalton, Derek M.; Oberg, Kevin M.; Yu, Robert T.; Lee, Ernest E.; Perreault, Stéphane; Oinen, Mark Emil; Pease, Melissa L.; Malik, Guillaume; Rovis, Tomislav

    2009-01-01

    This manuscript describes the development and scope of the asymmetric rhodium-catalyzed [2+2+2] cycloaddition of terminal alkynes and alkenyl isocyanates leading to the formation of indolizidine and quinolizidine scaffolds. The use of phosphoramidite ligands proved crucial for avoiding competitive terminal alkyne dimerization. Both aliphatic and aromatic terminal alkynes participate well, with product selectivity a function of both the steric and electronic character of the alkyne. Manipulation of the phosphoramidite ligand leads to tuning of enantio- and product selectivity, with a complete turnover in product selectivity seen with aliphatic alkynes when moving from Taddol-based to biphenol-based phosphoramidites. Terminal and 1,1-disubstituted olefins are tolerated with nearly equal efficacy. Examination of a series of competition experiments in combination with analysis of reaction outcome shed considerable light on the operative catalytic cycle. Through a detailed study of a series of X-ray structures of rhodium(cod)chloride/phosphoramidite complexes, we have formulated a mechanistic hypothesis that rationalizes the observed product selectivity. PMID:19817441

  11. Identification of the dominant photochemical pathways and mechanistic insights to the ultrafast ligand exchange of Fe(CO)5 to Fe(CO)4EtOH

    PubMed Central

    Kunnus, K.; Josefsson, I.; Rajkovic, I.; Schreck, S.; Quevedo, W.; Beye, M.; Weniger, C.; Grübel, S.; Scholz, M.; Nordlund, D.; Zhang, W.; Hartsock, R. W.; Gaffney, K. J.; Schlotter, W. F.; Turner, J. J.; Kennedy, B.; Hennies, F.; de Groot, F. M. F.; Techert, S.; Odelius, M.; Wernet, Ph.; Föhlisch, A.

    2016-01-01

    We utilized femtosecond time-resolved resonant inelastic X-ray scattering and ab initio theory to study the transient electronic structure and the photoinduced molecular dynamics of a model metal carbonyl photocatalyst Fe(CO)5 in ethanol solution. We propose mechanistic explanation for the parallel ultrafast intra-molecular spin crossover and ligation of the Fe(CO)4 which are observed following a charge transfer photoexcitation of Fe(CO)5 as reported in our previous study [Wernet et al., Nature 520, 78 (2015)]. We find that branching of the reaction pathway likely happens in the 1A1 state of Fe(CO)4. A sub-picosecond time constant of the spin crossover from 1B2 to 3B2 is rationalized by the proposed 1B2 → 1A1 → 3B2 mechanism. Ultrafast ligation of the 1B2 Fe(CO)4 state is significantly faster than the spin-forbidden and diffusion limited ligation process occurring from the 3B2 Fe(CO)4 ground state that has been observed in the previous studies. We propose that the ultrafast ligation occurs via 1B2 → 1A1 → 1A′ Fe(CO)4EtOH pathway and the time scale of the 1A1 Fe(CO)4 state ligation is governed by the solute-solvent collision frequency. Our study emphasizes the importance of understanding the interaction of molecular excited states with the surrounding environment to explain the relaxation pathways of photoexcited metal carbonyls in solution. PMID:26958587

  12. The ternary complex of PrnB (the second enzyme in the pyrrolnitrin biosynthesis pathway), tryptophan, and cyanide yields new mechanistic insights into the indolamine dioxygenase superfamily.

    PubMed

    Zhu, Xiaofeng; van Pée, Karl-Heinz; Naismith, James H

    2010-07-01

    Pyrrolnitrin (3-chloro-4-(2'-nitro-3'-chlorophenyl)pyrrole) is a broad-spectrum antifungal compound isolated from Pseudomonas pyrrocinia. Four enzymes (PrnA, PrnB, PrnC, and PrnD) are required for pyrrolnitrin biosynthesis from tryptophan. PrnB rearranges the indole ring of 7-Cl-l-tryptophan and eliminates the carboxylate group. PrnB shows robust activity in vivo, but in vitro activity for PrnB under defined conditions remains undetected. The structure of PrnB establishes that the enzyme belongs to the heme b-dependent indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) family. We report the cyanide complex of PrnB and two ternary complexes with both l-tryptophan or 7-Cl-l-tryptophan and cyanide. The latter two complexes are essentially identical and mimic the likely catalytic ternary complex that occurs during turnover. In the cyanide ternary complexes, a loop previously disordered becomes ordered, contributing to the binding of substrates. The conformations of the bound tryptophan substrates are changed from that seen previously in the binary complexes. In l-tryptophan ternary complex, the indole ring now adopts the same orientation as seen in the PrnB binary complexes with other tryptophan substrates. The amide and carboxylate group of the substrate are orientated in a new conformation. Tyr(321) and Ser(332) play a key role in binding these groups. The structures suggest that catalysis requires an l-configured substrate. Isothermal titration calorimetry data suggest d-tryptophan does not bind after cyanide (or oxygen) coordinates with the distal (or sixth) site of heme. This is the first ternary complex with a tryptophan substrate of a member of the tryptophan dioxygenase superfamily and has mechanistic implications. PMID:20421301

  13. Identification of the dominant photochemical pathways and mechanistic insights to the ultrafast ligand exchange of Fe(CO)5 to Fe(CO)4EtOH.

    PubMed

    Kunnus, K; Josefsson, I; Rajkovic, I; Schreck, S; Quevedo, W; Beye, M; Weniger, C; Grübel, S; Scholz, M; Nordlund, D; Zhang, W; Hartsock, R W; Gaffney, K J; Schlotter, W F; Turner, J J; Kennedy, B; Hennies, F; de Groot, F M F; Techert, S; Odelius, M; Wernet, Ph; Föhlisch, A

    2016-07-01

    We utilized femtosecond time-resolved resonant inelastic X-ray scattering and ab initio theory to study the transient electronic structure and the photoinduced molecular dynamics of a model metal carbonyl photocatalyst Fe(CO)5 in ethanol solution. We propose mechanistic explanation for the parallel ultrafast intra-molecular spin crossover and ligation of the Fe(CO)4 which are observed following a charge transfer photoexcitation of Fe(CO)5 as reported in our previous study [Wernet et al., Nature 520, 78 (2015)]. We find that branching of the reaction pathway likely happens in the (1)A1 state of Fe(CO)4. A sub-picosecond time constant of the spin crossover from (1)B2 to (3)B2 is rationalized by the proposed (1)B2 → (1)A1 → (3)B2 mechanism. Ultrafast ligation of the (1)B2 Fe(CO)4 state is significantly faster than the spin-forbidden and diffusion limited ligation process occurring from the (3)B2 Fe(CO)4 ground state that has been observed in the previous studies. We propose that the ultrafast ligation occurs via (1)B2 → (1)A1 → (1)A' Fe(CO)4EtOH pathway and the time scale of the (1)A1 Fe(CO)4 state ligation is governed by the solute-solvent collision frequency. Our study emphasizes the importance of understanding the interaction of molecular excited states with the surrounding environment to explain the relaxation pathways of photoexcited metal carbonyls in solution. PMID:26958587

  14. Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles: A mechanistic insight.

    PubMed

    Ragelle, Héloïse; Colombo, Stefano; Pourcelle, Vincent; Vanvarenberg, Kevin; Vandermeulen, Gaëlle; Bouzin, Caroline; Marchand-Brynaert, Jacqueline; Feron, Olivier; Foged, Camilla; Préat, Véronique

    2015-08-10

    Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvβ3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvβ3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems. PMID:25989603

  15. Identification of the dominant photochemical pathways and mechanistic insights to the ultrafast ligand exchange of Fe(CO)5 to Fe(CO)4EtOH

    DOE PAGESBeta

    Kunnus, K.; Josefsson, I.; Rajkovic, I.; Schreck, S.; Quevedo, W.; Beye, M.; Weniger, C.; Grübel, S.; Scholz, M.; Nordlund, D.; et al

    2016-02-09

    We utilized femtosecond time-resolved resonant inelastic X-ray scattering and ab initio theory to study the transient electronic structure and the photoinduced molecular dynamics of a model metal carbonyl photocatalyst Fe(CO)5 in ethanol solution. We propose mechanistic explanation for the parallel ultrafast intra-molecular spin crossover and ligation of the Fe(CO)4 which are observed following a charge transfer photoexcitation of Fe(CO)5 as reported in our previous study [Wernet et al., Nature 520, 78 (2015)]. We find that branching of the reaction pathway likely happens in the 1A1 state of Fe(CO)4. A sub-picosecond time constant of the spin crossover from 1B2 tomore » 3B2 is rationalized by the proposed 1B2 → 1A1 → 3B2 mechanism. Ultrafast ligation of the 1B2 Fe(CO)4 state is significantly faster than the spin-forbidden and diffusion limited ligation process occurring from the 3B2 Fe(CO)4 ground state that has been observed in the previous studies. We propose that the ultrafast ligation occurs via 1B2 → 1A1 → 1A' Fe(CO)4EtOH pathway and the time scale of the 1A1 Fe(CO)4 state ligation is governed by the solute-solvent collision frequency. In conclusion, our study emphasizes the importance of understanding the interaction of molecular excited states with the surrounding environment to explain the relaxation pathways of photoexcited metal carbonyls in solution.« less

  16. Aircraft-Produced Ice Particles (APIPs): Additional Results and Further Insights.

    NASA Astrophysics Data System (ADS)

    Woodley, William L.; Gordon, Glenn; Henderson, Thomas J.; Vonnegut, Bernard; Rosenfeld, Daniel; Detwiler, Andrew

    2003-05-01

    This paper presents new results from studies of aircraft-produced ice particles (APIPs) in supercooled fog and clouds. Nine aircraft, including a Beech King Air 200T cloud physics aircraft, a Piper Aztec, a Cessna 421-C, two North American T-28s, an Aero Commander, a Piper Navajo, a Beech Turbo Baron, and a second four-bladed King Air were involved in the tests. The instrumented King Air served as the monitoring aircraft for trails of ice particles created, or not created, when the other aircraft were flown through clouds at various temperatures and served as both the test and monitoring aircraft when it itself was tested. In some cases sulfur hexafluoride (SF6) gas was released by the test aircraft during its test run and was detected by the King Air during its monitoring passes to confirm the location of the test aircraft wake. Ambient temperatures for the tests ranged between 5° and 12°C. The results confirm earlier published results and provide further insights into the APIPs phenomenon. The King Air at ambient temperatures less than 8°C can produce APIPs readily. The Piper Aztec and the Aero Commander also produced APIPs under the test conditions in which they were flown. The Cessna 421, Piper Navajo, and Beech Turbo Baron did not. The APIPs production potential of a T-28 is still indeterminate because a limited range of conditions was tested. Homogeneous nucleation in the adiabatically cooled regions where air is expanding around the rapidly rotating propeller tips is the cause of APIPs. An equation involving the propeller efficiency, engine thrust, and true airspeed of the aircraft is used along with the published thrust characteristics of the propellers to predict when the aircraft will produce APIPs. In most cases the predictions agree well with the field tests. Of all of the aircraft tested, the Piper Aztec, despite its small size and low horsepower, was predicted to be the most prolific producer of APIPs, and this was confirmed in field tests. The

  17. Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP Mechanistic Insights into a Minimalistic E1 Enzyme

    SciTech Connect

    Bacik, John-Paul; Walker, John R.; Ali, Mohsin; Schimmer, Aaron D.; Dhe-Paganon, Sirano

    2010-08-30

    E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys{sup 250}) is part of the adenylation domain in an {alpha}-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.

  18. New Insights on Tropical Ozone from SHADOZ (Southern Hemisphere Additional Ozonesondes) Profiles

    NASA Technical Reports Server (NTRS)

    Thompson, Anne M.

    2004-01-01

    The SHADOZ (Southern Hemisphere Additional Ozonesondes) ozone sounding network was initiated in 1998 to improve the coverage of tropical in-situ ozone measurements for satellite validation, algorithm development and related process studies. Over 2000 soundings have been archived at the website, , for 12 stations: Ascension Island; Nairobi and Malindi, Kenya; Irene, South Africa; Reunion Island, Watukosek, Java; Fiji; Tahiti; American Samoa; San Cristobal, Galapagos; Natal, Brazil; Paramaribo, Surinam. Key results from SHADOZ will be described from among the following: 1) By using ECC sondes with similar procedures, 5-10% accuracy and precision (1-sigma) of the sonde total ozone measurement was achieved; 2) Week-to-week variability in tropospheric ozone is so great that statistics are frequently not Gaussian; most stations vary up to a factor of 3 in tropospheric column over the course of a year; 3) Longitudinal variability in tropospheric ozone profiles is a consistent feature, with a 10-15 DU column-integrated difference between Atlantic and Pacific sites; this causes a "zonal wave-one" feature in total ozone. 4) The ozone record from Paramaribo, Surinam (6N, 55W) is a marked contrast to southern tropical ozone because Surinam is often north of the Intertropical Convergence Zone; 5) Indian Ocean region pollution may contribute up to half of the excess ozone observed in the south tropical Atlantic paradox in the December-January-February period of the year.

  19. Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing.

    PubMed

    Mei, R; Herschlag, D

    1996-05-01

    Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction. PMID:8639540

  20. Investigation of the biological properties of Cinnulin PF in the context of diabetes: mechanistic insights by genome-wide mRNA-Seq analysis

    PubMed Central

    Rafehi, Haloom; Ververis, Katherine; Balcerczyk, Aneta; Ziemann, Mark; Ooi, Jenny; Hu, Sean; Kwa, Faith A. A.; Loveridge, Shanon J.; Georgiadis, George T.; El-Osta, Assam; Karagiannis, Tom C.

    2012-01-01

    The accumulating evidence of the beneficial effects of cinnamon (Cinnamomum burmanni) in type-2 diabetes, a chronic age-associated disease, has prompted the commercialisation of various supplemental forms of the spice. One such supplement, Cinnulin PF®, represents the water soluble fraction containing relatively high levels of the double-linked procyanidin type-A polymers of flavanoids. The overall aim of this study was to utilize genome-wide mRNA-Seq analysis to characterise the changes in gene expression caused by Cinnulin PF in immortalised human keratinocytes and microvascular endothelial cells, which are relevant with respect to diabetic complications. In summary, our findings provide insights into the mechanisms of action of Cinnulin PF in diabetes and diabetic complications. More generally, we identify relevant candidate genes which could provide the basis for further investigation. PMID:22953038

  1. Investigation of the biological properties of Cinnulin PF in the context of diabetes: mechanistic insights by genome-wide mRNA-Seq analysis.

    PubMed

    Rafehi, Haloom; Ververis, Katherine; Balcerczyk, Aneta; Ziemann, Mark; Ooi, Jenny; Hu, Sean; Kwa, Faith A A; Loveridge, Shanon J; Georgiadis, George T; El-Osta, Assam; Karagiannis, Tom C

    2012-01-01

    The accumulating evidence of the beneficial effects of cinnamon (Cinnamomum burmanni) in type-2 diabetes, a chronic age-associated disease, has prompted the commercialisation of various supplemental forms of the spice. One such supplement, Cinnulin PF(®), represents the water soluble fraction containing relatively high levels of the double-linked procyanidin type-A polymers of flavanoids. The overall aim of this study was to utilize genome-wide mRNA-Seq analysis to characterise the changes in gene expression caused by Cinnulin PF in immortalised human keratinocytes and microvascular endothelial cells, which are relevant with respect to diabetic complications. In summary, our findings provide insights into the mechanisms of action of Cinnulin PF in diabetes and diabetic complications. More generally, we identify relevant candidate genes which could provide the basis for further investigation. PMID:22953038

  2. Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa

    SciTech Connect

    Matsuyama, Bruno Y.; Krasteva, Petya V.; Baraquet, Claudine; Harwood, Caroline S.; Sondermann, Holger; Navarro, Marcos V. A. S.

    2015-12-28

    Pseudomonas aeruginosa, an opportunistic pathogen that can cause fatal chronic infections, relies on the intracellular second-messenger c-di-GMP to form robust multicellular biofilms during host tissue colonization. c-di-GMP is sensed directly by the transcription regulator FleQ, which inversely regulates flagellar motility and exopolysaccharide secretion to secure a planktonic to sessile life-form transition. FleQ belongs to the diverse family of AAA+ ATPase enhancer-binding proteins, but how its noncanonical function on transcriptional regulation is controlled by c-di-GMP remains enigmatic. Here, we report structural and functional data that identify an unusual mode of c-di-GMP recognition accompanied by a major quaternary structure reorganization. Our analyses offer a consensus to previous studies and unique insights into the mechanism of action of FleQ and FleQ-like proteins.

  3. Insights into the Halogen Oxidative Addition Reaction to Dinuclear Gold(I) Di(NHC) Complexes.

    PubMed

    Baron, Marco; Tubaro, Cristina; Basato, Marino; Isse, Abdirisak Ahmed; Gennaro, Armando; Cavallo, Luigi; Graiff, Claudia; Dolmella, Alessandro; Falivene, Laura; Caporaso, Lucia

    2016-07-11

    Gold(I) dicarbene complexes [Au2 (MeIm-Y-ImMe)2 ](PF6 )2 (Y=CH2 (1), (CH2 )2 (2), (CH2 )4 (4), MeIm=1-methylimidazol-2-ylidene) react with iodine to give the mixed-valence complex [Au(MeIm-CH2 -ImMe)2 AuI2 ](PF6 )2 (1 a(I) ) and the gold(III) complexes [Au2 I4 (MeIm-Y-ImMe)2 ](PF6 )2 (2 c(I) and 4 c(I) ). Reaction of complexes 1 and 2 with an excess of ICl allows the isolation of the tetrachloro gold(III) complexes [Au2 Cl4 (MeIm-CH2 -ImMe)2 ](PF6 )2 (1 c(Cl) ) and [Au2 Cl4 (MeIm-(CH2 )2 -ImMe)2 ](Cl)2 (2 c(Cl) -Cl) (as main product); remarkably in the case of complex 2, the X-ray molecular structure of the crystals also shows the presence of I-Au-Cl mixed-sphere coordination. The same type of coordination has been observed in the main product of the reaction of complexes 3 or 4 with ICl. The study of the reactivity towards the oxidative addition of halogens to a large series of dinuclear bis(dicarbene) gold(I) complexes has been extended and reviewed. The complexes react with Cl2 , Br2 and I2 to give the successive formation of the mixed-valence gold(I)/gold(III) n a(X) and gold(III) n c(X) (excluding compound 1 c(I) ) complexes. However, complex 3 affords with Cl2 and Br2 the gold(II) complex 3 b(X) [Au2 X2 (MeIm-(CH2 )3 -ImMe)2 ](PF6 )2 (X=Cl, Br), which is the predominant species over compound 3 c(X) even in the presence of free halogen. The observed different relative stabilities of the oxidised complexes of compounds 1 and 3 have also been confirmed by DFT calculations. PMID:27297191

  4. Blocking the chaperone kinome pathway: Mechanistic insights into a novel dual inhibition approach for supra-additive suppression of malignant tumors

    SciTech Connect

    Grover, Abhinav; Shandilya, Ashutosh; Agrawal, Vibhuti; Pratik, Piyush; Bhasme, Divya; Bisaria, Virendra S.; Sundar, Durai

    2011-01-07

    Research highlights: {yields} Withaferin A and 17-DMAG synergistically inhibit the Hsp90-Cdc37 chaperone pair. {yields} Binding of WA to Cdc37 cleft suppresses its kinase binding activity. {yields} 17-DMAG binding to the association complex results in H-bonds with 60% clustering. {yields} The ligands' bound complex was found structurally and thermodynamically stable. -- Abstract: The chaperone Hsp90 is involved in regulating the stability and activation state of more than 200 'client' proteins and takes part in the cancer diseased states. The major clientele-protein kinases depend on Hsp90 for their proper folding and functioning. Cdc37, a kinase targeting co-chaperone of Hsp90, mediates the interactions between Hsp90 and protein kinases. Targeting of Cdc37 has the prospect of delivering predominantly kinase-selective molecular responses as compared to the current pharmacologic Hsp90 inhibitors. The present work reports a bio-computational study carried out with the aim of exploring the dual inhibition of Hsp90/Cdc37 chaperone/co-chaperone association complex by the naturally occurring drug candidates withaferin A and 17-DMAG along with their possible modes of action. Our molecular docking studies reveal that withaferin A in combination with 17-DMAG can act as potent chaperone system inhibitors. The structural and thermodynamic stability of the ligands' bound complex was also observed from molecular dynamics simulations in water. Our results suggest a novel tumor suppressive action mechanism of herbal ligands which can be looked forward for further clinical investigations for possible anticancer drug formulations.

  5. Silver vanadium diphosphate Ag{sub 2}VP{sub 2}O{sub 8}: Electrochemistry and characterization of reduced material providing mechanistic insights

    SciTech Connect

    Takeuchi, Esther S.; Lee, Chia-Ying; Cheng, Po-Jen; Menard, Melissa C.; Marschilok, Amy C.; Takeuchi, Kenneth J.

    2013-04-15

    Silver vanadium phosphorous oxides (Ag{sub w}V{sub x}P{sub y}O{sub z}) are notable battery cathode materials due to their high energy density and demonstrated ability to form in-situ Ag metal nanostructured electrically conductive networks within the cathode. While analogous silver vanadium diphosphate materials have been prepared, electrochemical evaluations of these diphosphate based materials have been limited. We report here the first electrochemical study of a silver vanadium diphosphate, Ag{sub 2}VP{sub 2}O{sub 8}, where the structural differences associated with phosphorous oxides versus diphosphates profoundly affect the associated electrochemistry. Reminiscent of Ag{sub 2}VO{sub 2}PO{sub 4} reduction, in-situ formation of silver metal nanoparticles was observed with reduction of Ag{sub 2}VP{sub 2}O{sub 8}. However, counter to Ag{sub 2}VO{sub 2}PO{sub 4} reduction, Ag{sub 2}VP{sub 2}O{sub 8} demonstrates a significant decrease in conductivity upon continued electrochemical reduction. Structural analysis contrasting the crystallography of the parent Ag{sub 2}VP{sub 2}O{sub 8} with that of the proposed Li{sub 2}VP{sub 2}O{sub 8} reduction product is employed to gain insight into the observed electrochemical reduction behavior, where the structural rigidity associated with the diphosphate anion may be associated with the observed particle fracturing upon deep electrochemical reduction. Further, the diphosphate anion structure may be associated with the high thermal stability of the partially reduced Ag{sub 2}VP{sub 2}O{sub 8} materials, which bodes well for enhanced safety of batteries incorporating this material. - Graphical abstract: Structure and galvanostatic intermittent titration-type test data for silver vanadium diphosphate, Ag{sub 2}VP{sub 2}O{sub 8}. Highlights: ► First electrochemical study of a silver vanadium diphosphate, Ag{sub 2}VP{sub 2}O{sub 8}. ► In-situ formation of Ag{sup 0} nanoparticles was observed upon electrochemical reduction.

  6. Mechanistic insight into conjugated N-N bond cleavage by Rh(III)-catalyzed redox-neutral C-H activation of pyrazolones.

    PubMed

    Wu, Weirong; Liu, Yuxia; Bi, Siwei

    2015-08-14

    Density functional theory (DFT) calculations have been performed to investigate the detailed mechanism of Rh(III)-catalyzed redox-neutral C-H activation of pyrazolones with PhC≡CPh. It is found that (1) the methylene C-H activation is prior to the phenyl C-H activation, (2) the N-N bond cleavage is realized via Rh(III) → Rh(I) → Rh(III) rather than via Rh(III) → Rh(V) → Rh(III). The zwitterionic Rh(I) complex is identified to be a key intermediate in promoting the N-N bond cleavage. (3) Different from the Rh(III)-catalyzed hydrazine-directed C-H activation for indole synthesis, the rate-determining step of the reaction studied in this work is the Rh(III) → Rh(I) → Rh(III) process resulting in the N-N bond cleavage rather than the alkyne insertion step. The present theoretical study provides new insight into the mechanism of the conjugated N-N bond cleavage. PMID:26138233

  7. Selective catalytic reduction of NO over Fe-ZSM-5: mechanistic insights by operando HERFD-XANES and valence-to-core X-ray emission spectroscopy.

    PubMed

    Boubnov, Alexey; Carvalho, Hudson W P; Doronkin, Dmitry E; Günter, Tobias; Gallo, Erik; Atkins, Andrew J; Jacob, Christoph R; Grunwaldt, Jan-Dierk

    2014-09-17

    An in-depth understanding of the active site requires advanced operando techniques and the preparation of defined catalysts. We elucidate here the mechanism of the selective catalytic reduction of NO by NH3 (NH3-SCR) over a Fe-ZSM-5 zeolite catalyst. 1.3 wt % Fe-ZSM-5 with low nuclearity Fe sites was synthesized, tested in the SCR reaction and characterized by UV-vis, X-ray absorption near edge structure (XANES), and extended X-ray absorption fine structure (EXAFS) spectroscopy. Next, this defined Fe-zeolite catalyst was studied by complementary high-energy-resolution fluorescence-detected XANES (HERFD-XANES) and valence-to-core X-ray emission spectroscopy (V2C XES) under different model in situ and realistic working (operando) conditions identical to the catalyst test bench including the presence of water vapor. HERFD-XANES uncovered that the coordination (between 4 and 5), geometry (tetrahedral, partly 5-fold), and oxidation state of the Fe centers (reduced in NH3, partly in SCR mixture, slight reduction in NO) strongly changed. V2C XES supported by DFT calculations provided important insight into the chemical nature of the species adsorbed on Fe sites. The unique combination of techniques applied under realistic reaction conditions and the corresponding catalytic data unraveled the adsorption of ammonia via oxygen on the iron site. The derived reaction model supports a mechanism where adsorbed NOx reacts with ammonia coordinated to the Fe(3+) site yielding Fe(2+) whose reoxidation is slow. PMID:25105343

  8. Simvastatin and Dipentyl Phthalate Lower Ex Vivo Testicular Testosterone Production and Exhibit Additive Effects on Testicular Testosterone and Gene Expression Via Distinct Mechanistic Pathways in the Fetal Rat

    PubMed Central

    Beverly, Brandiese E. J.; Lambright, Christy S.; Furr, Johnathan R.; Sampson, Hunter; Wilson, Vickie S.; McIntyre, Barry S.; Foster, Paul M. D.; Travlos, Gregory; Gray, L. Earl

    2014-01-01

    Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and consequently, lowering fetal androgen and Insl3 hormone levels. Simvastatin (SMV) is a cholesterol-lowering drug that directly inhibits HMG-CoA reductase. SMV may also disrupt steroid biosynthesis, but through a different mode of action (MOA) than the PEs. As cholesterol is a precursor of steroid hormone biosynthesis, we hypothesized that in utero exposure to SMV during the critical period of sex differentiation would lower fetal testicular testosterone (T) production without affecting genes involved in cholesterol and androgen synthesis and transport. Secondly, we hypothesized that a mixture of SMV and a PE, which may have different MOAs, would reduce testosterone levels in an additive manner. Pregnant Sprague Dawley rats were dosed orally with SMV, dipentyl phthalate (DPeP), or SMV plus DPeP from gestational days 14-18, and fetuses were evaluated on GD18. On GD18, SMV lowered fetal T production and serum triglycerides, low density lipoprotein, high density lipoprotein, and total cholesterol levels, and downregulated two genes in the fetal testis that were different from those altered by PEs. When SMV and DPeP were administered as a mixture, fetal T production was significantly reduced in an additive manner, thus demonstrating that a mixture of chemicals can induce additive effects on fetal T production even though they display different MOAs. PMID:25055962

  9. Mechanistic Insights into Radical-Mediated Oxidation of Tryptophan from ab Initio Quantum Chemistry Calculations and QM/MM Molecular Dynamics Simulations.

    PubMed

    Wood, Geoffrey P F; Sreedhara, Alavattam; Moore, Jamie M; Wang, John; Trout, Bernhardt L

    2016-05-12

    An assessment of the mechanisms of (•)OH and (•)OOH radical-mediated oxidation of tryptophan was performed using density functional theory calculations and ab initio plane-wave Quantum Mechanics/Molecular Mechanics (QM/MM) molecular dynamics simulations. For the (•)OH reactions, addition to the pyrrole ring at position 2 is the most favored site with a barrierless reaction in the gas phase. The subsequent degradation of this adduct through a H atom transfer to water was intermittently observed in aqueous-phase molecular dynamics simulations. For the (•)OOH reactions, addition to the pyrrole ring at position 2 is the most favored pathway, in contrast to the situation in the model system ethylene, where concerted addition to the double bond is preferred. From the (•)OOH position 2 adduct QM/MM simulations show that formation of oxy-3-indolanaline occurs readily in an aqueous environment. The observed transformation starts from an initial rupture of the O-O bond followed by a H atom transfer with the accompanying loss of an (•)OH radical to solution. Finally, classical molecular dynamics simulations were performed to equate observed differential oxidation rates of various tryptophan residues in monoclonal antibody fragments. It was found that simple parameters derived from simulation correlate well with the experimental data. PMID:27082439

  10. Experimental and computational mechanistic investigation of chlorocarbene additions to bridgehead carbene-anti-Bredt systems: noradamantylcarbene-adamantene and adamantylcarbene-homoadamantene.

    PubMed

    Hare, Stephanie R; Orman, Marina; Dewan, Faizunnahar; Dalchand, Elizabeth; Buzard, Camilla; Ahmed, Sadia; Tolentino, Julia C; Sethi, Ulweena; Terlizzi, Kelly; Houferak, Camille; Stein, Aliza M; Stedronsky, Alexandra; Thamattoor, Dasan M; Tantillo, Dean J; Merrer, Dina C

    2015-05-15

    Cophotolysis of noradamantyldiazirine with the phenanthride precursor of dichlorocarbene or phenylchlorodiazirine in pentane at room temperature produces noradamantylethylenes in 11% yield with slight diastereoselectivity. Cophotolysis of adamantyldiazirine with phenylchlorodiazirine in pentane at room temperature generates adamantylethylenes in 6% yield with no diastereoselectivity. (1)H NMR showed the reaction of noradamantyldiazirine + phenylchlorodiazirine to be independent of solvent, and the rate of noradamantyldiazirine consumption correlated with the rate of ethylene formation. Laser flash photolysis showed that reaction of phenylchlorocarbene + adamantene was independent of adamantene concentration. The reaction of phenylchlorocarbene + homoadamantene produces the ethylene products with k = 9.6 × 10(5) M(-1) s(-1). Calculations at the UB3LYP/6-31+G(d,p) and UM062X/6-31+G(d,p)//UB3LYP/6-31+G(d,p) levels show the formation of exocyclic ethylenes to proceed (a) on the singlet surface via stepwise addition of phenylchlorocarbene (PhCCl) to bridgehead alkenes adamantene and homoadamantene, respectively, producing an intermediate singlet diradical in each case, or (b) via addition of PhCCl to the diazo analogues of noradamantyl- and adamantyldiazirine. Preliminary direct dynamics calculations on adamantene + PhCCl show a high degree of recrossing (68%), indicative of a flat transition state surface. Overall, 9% of the total trajectories formed noradamantylethylene product, each proceeding via the computed singlet diradical. PMID:25902301

  11. Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family.

    PubMed

    Mills, Stephen J; Silvander, Camilla; Cozier, Gyles; Trésaugues, Lionel; Nordlund, Pär; Potter, Barry V L

    2016-03-01

    The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3',4,4',5,5'-hexakisphosphate [BiPh(3,3',4,4',5,5')P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3',4,4',5,5')P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3',4,4',5,5')P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B-BiPh(3,3',4,4',5,5')P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed "moving metal" mechanism. PMID:26854536

  12. Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family

    PubMed Central

    2016-01-01

    The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3′,4,4′,5,5′-hexakisphosphate [BiPh(3,3′,4,4′,5,5′)P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3′,4,4′,5,5′)P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3′,4,4′,5,5′)P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B–BiPh(3,3′,4,4′,5,5′)P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed “moving metal” mechanism

  13. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

    SciTech Connect

    Katika, Madhumohan R.; Hendriksen, Peter J.M.; Shao, Jia; Loveren, Henk van; Peijnenburg, Ad

    2012-10-01

    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  14. Imidazolium Sulfonates as Environmental-Friendly Catalytic Systems for the Synthesis of Biologically Active 2-Amino-4H-chromenes: Mechanistic Insights.

    PubMed

    Velasco, Jacinto; Pérez-Mayoral, Elena; Calvino-Casilda, Vanesa; López-Peinado, Antonio J; Bañares, Miguel A; Soriano, Elena

    2015-09-10

    Ionic Liquids (ILs) are valuable reaction media extremely useful in industrial sustainable organic synthesis. We describe here the study on the multicomponent reaction (MCR) between salicylaldehyde (2) and ethyl cyanoacetate (3), catalyzed by imidazolium sulfonates, to form chromenes 1, a class of heterocyclic scaffolds exhibiting relevant biological activity. We have clarified the reaction mechanism by combining the experimental results with computational studies. The results reported herein suggest that both the imidazolium core and the sulfonate anions in the selected ILs are involved in the reaction course acting as hydrogen bond donors and acceptors, respectively. Contrarily to the most widely accepted mechanism through initial Knoevenagel condensation, the most favorable reaction pathway consists of an aldolic reaction between reagents followed by heterocyclization, subsequent dehydration, and, finally, the Michael addition of the second molecule of ethyl cyanoacetate (3) to yield the chromenes 1. PMID:26301460

  15. Enhancement of antimicrobial activities of whole and sub-fractionated white tea by addition of copper (II) sulphate and vitamin C against Staphylococcus aureus; a mechanistic approach

    PubMed Central

    2011-01-01

    . Investigations to establish which WTF component/s and in what proportions additives are most effective against target organisms are warranted. PMID:22093997

  16. Heteroleptic tin(II) initiators for the ring-opening (co)polymerization of lactide and trimethylene carbonate: mechanistic insights from experiments and computations.

    PubMed

    Wang, Lingfang; Kefalidis, Christos E; Sinbandhit, Sourisak; Dorcet, Vincent; Carpentier, Jean-François; Maron, Laurent; Sarazin, Yann

    2013-09-27

    The tin(II) complexes {LO(x)}Sn(X) ({LO(x)}(-) =aminophenolate ancillary) containing amido (1-4), chloro (5), or lactyl (6) coligands (X) promote the ring-opening polymerization (ROP) of cyclic esters. Complex 6, which models the first insertion of L-lactide, initiates the living ROP of L-LA on its own, but the amido derivatives 1-4 require the addition of alcohol to do so. Upon addition of one to ten equivalents of iPrOH, precatalysts 1-4 promote the ROP of trimethylene carbonate (TMC); yet, hardly any activity is observed if tert-butyl (R)-lactate is used instead of iPrOH. Strong inhibition of the reactivity of TMC is also detected for the simultaneous copolymerization of L-LA and TMC, or for the block copolymerization of TMC after that of L-LA. Experimental and computational data for the {LO(x)}Sn(OR)complexes (OR=lactyl or lactidyl) replicating the active species during the tin(II)-mediated ROP of L-LA demonstrate that the formation of a five-membered chelate is largely favored over that of an eight-membered one, and that it constitutes the resting state of the catalyst during this (co)polymerization. Comprehensive DFT calculations show that, out of the four possible monomer insertion sequences during simultaneous copolymerization of L-LA and TMC: 1) TMC then TMC, 2) TMC then L-LA, 3) L-LA then L-LA, and 4) L-LA then TMC, the first three are possible. By contrast, insertion of L-LA followed by that of TMC (i.e., insertion sequence 4) is endothermic by +1.1 kcal mol(-1), which compares unfavorably with consecutive insertions of two L-LA units (i.e., insertion sequence 3) (-10.2 kcal mol(-1)). The copolymerization of L-LA and TMC thus proceeds under thermodynamic control. PMID:23955851

  17. Mechanistic insight of photo-induced aggregation of chicken egg white lysozyme: the interplay between hydrophobic interactions and formation of intermolecular disulfide bonds.

    PubMed

    Xie, Jinbing; Qin, Meng; Cao, Yi; Wang, Wei

    2011-08-01

    Recently, it was reported that ultraviolet (UV) illumination could trigger the unfolding of proteins by disrupting the buried disulfide bonds. However, the consequence of such unfolding has not been adequately evaluated. Here, we report that unfolded chicken egg white lysozyme (CEWL) triggered by UV illumination can form uniform globular aggregates as confirmed by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The assembling process of such aggregates was also monitored by several other methods, such as circular dichroism, fluorescence spectroscopy, mass spectrometry based on chymotrypsin digestion, ANS-binding assay, Ellman essay, and SDS-PAGE. Our finding is that due to the dissociation of the native disulfide bonds by UV illumination, CEWL undergoes drastic conformational changes resulting in the exposure of some hydrophobic residues and free thiols. Subsequently, these partially unfolded molecules self-assemble into small granules driven by intermolecular hydrophobic interaction. With longer UV illumination or longer incubation time, these granules can further self-assemble into larger globular aggregates. The combined effects from both the hydrophobic interaction and the formation of intermolecular disulfide bonds dominate this process. Additionally, similar aggregation behavior can also be found in other three typical disulfide-bonded proteins, that is, α-lactalbumin, RNase A, and bovine serum albumin. Thus, we propose that such aggregation behavior might be a general mechanism for some disulfide-bonded proteins under UV irradiation. PMID:21661057

  18. Mechanistic insight into peroxydisulfate reactivity: Oxidation of the cis,cis-[Ru(bpy)2(OH2)]2O4+ "Blue Dimmer"

    DOE PAGESBeta

    Hurst, James K.; Roemeling, Margo D.; Lymar, Sergei V.

    2015-04-10

    One-electron oxidation of the μ-oxo dimer (cis,cis-[RuIII(bpy)2(OH2)]2O4+, {3,3}) to {3,4} by S2O82- can be described by three concurrent reaction pathways corresponding to the three protic forms of {3,3}. Free energy correlations of the rate constants, transient species dynamics determined by pulse radiolysis, and medium and temperature dependencies of the alkaline pathway all suggest that the rate determining step in these reactions is a strongly non-adiabatic dissociative electron transfer within a precursor ion pair leading to the {3,4}|SO42-|SO4•- ion triple. As deduced from the SO4•- scavenging experiments with 2-propanol, the SO4•- radical then either oxidizes {3,4} to {4,4} within the ionmore » triple, effecting a net two-electron oxidation of {3,3}, or escapes in solution with ~25 % probability to react with additional {3,3} and {3,4}, that is, effecting sequential one-electron oxidations. The reaction model presented also invokes rapid {3,3} + {4,4} → 2{3,4} comproportionation, for which kcom ~5×107 M-1 s-1 was independently measured. The model provides an explanation for the observation that despite favorable energetics, no oxidation beyond the {3,4} state was detected. As a result, the indiscriminate nature of oxidation by SO4•- indicates that its fate must be quantitatively determined when using S2O82- as an oxidant« less

  19. Mechanistic Insight into Ketone α-Alkylation with Unactivated Olefins via C-H Activation Promoted by Metal-Organic Cooperative Catalysis (MOCC): Enriching the MOCC Chemistry.

    PubMed

    Dang, Yanfeng; Qu, Shuanglin; Tao, Yuan; Deng, Xi; Wang, Zhi-Xiang

    2015-05-20

    Metal-organic cooperative catalysis (MOCC) has been successfully applied for hydroacylation of olefins with aldehydes via directed C(sp(2))-H functionalization. Most recently, it was reported that an elaborated MOCC system, containing Rh(I) catalyst and 7-azaindoline (L1) cocatalyst, could even catalyze ketone α-alkylation with unactivated olefins via C(sp(3))-H activation. Herein we present a density functional theory study to understand the mechanism of the challenging ketone α-alkylation. The transformation uses IMesRh(I)Cl(L1)(CH2═CH2) as an active catalyst and proceeds via sequential seven steps, including ketone condensation with L1, giving enamine 1b; 1b coordination to Rh(I) active catalyst, generating Rh(I)-1b intermediate; C(sp(2))-H oxidative addition, leading to a Rh(III)-H hydride; olefin migratory insertion into Rh(III)-H bond; reductive elimination, generating Rh(I)-1c(alkylated 1b) intermediate; decoordination of 1c, liberating 1c and regenerating Rh(I) active catalyst; and hydrolysis of 1c, furnishing the final α-alkylation product 1d and regenerating L1. Among the seven steps, reductive elimination is the rate-determining step. The C-H bond preactivation via agostic interaction is crucial for the bond activation. The mechanism rationalizes the experimental puzzles: why only L1 among several candidates performed perfectly, whereas others failed, and why Wilkinson's catalyst commonly used in MOCC systems performed poorly. Based on the established mechanism and stimulated by other relevant experimental reactions, we attempted to enrich MOCC chemistry computationally, exemplifying how to develop new organic catalysts and proposing L7 to be an alternative for L1 and demonstrating the great potential of expanding the hitherto exclusive use of Rh(I)/Rh(III) manifold to Co(0)/Co(II) redox cycling in developing MOCC systems. PMID:25915086

  20. Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity.

    PubMed

    Nilsson, Lina; Larsson, Andreas; Begum, Afshan; Iakovleva, Irina; Carlsson, Marcus; Brännström, Kristoffer; Sauer-Eriksson, A Elisabeth; Olofsson, Anders

    2016-01-01

    Amyloid formation of the plasma protein transthyretin (TTR) has been linked to familial amyloid polyneuropathy and senile systemic amyloidosis. Binding of ligands within its natural hormone binding site can stabilize the tetrameric structure and impair amyloid formation. We have recently shown that the flavonoid luteolin stabilizes TTR in human plasma with a very high selectivity. Luteolin, however, is inactivated in vivo via glucuronidation for which the preferred site is the hydroxy group at position 7 on its aromatic A-ring. We have evaluated the properties of two luteolin variants in which the 7-hydroxy group has been exchanged for a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut). Using an in vitro model, based on human liver microsomes, we verified that these modifications increase the persistence of the drug. Crystal structure determinations show that 7-Cl-Lut binds similarly to luteolin. The larger MeO substituent cannot be accommodated within the same space as the chlorine or hydroxy group and as a result 7-MeO-Lut binds in the opposite direction with the methoxy group in position 7 facing the solvent. Both 7-Cl-Lut and 7-MeO-Lut qualify as high-affinity binders, but in contrast to luteolin, they display a highly non-specific binding to other plasma components. The binding of the two conformations and the key-interactions to TTR are discussed in detail. Taken together, these results show a proof-of-concept that the persistence of luteolin towards enzymatic modification can be increased. We reveal two alternative high-affinity binding modes of luteolin to TTR and that modification in position 7 is restricted only to small substituents if the original orientation of luteolin should be preserved. In addition, the present work provides a general and convenient method to evaluate the efficacy of TTR-stabilizing drugs under conditions similar to an in vivo environment. PMID:27050398

  1. Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity

    PubMed Central

    Nilsson, Lina; Larsson, Andreas; Begum, Afshan; Iakovleva, Irina; Carlsson, Marcus; Brännström, Kristoffer; Sauer-Eriksson, A. Elisabeth; Olofsson, Anders

    2016-01-01

    Amyloid formation of the plasma protein transthyretin (TTR) has been linked to familial amyloid polyneuropathy and senile systemic amyloidosis. Binding of ligands within its natural hormone binding site can stabilize the tetrameric structure and impair amyloid formation. We have recently shown that the flavonoid luteolin stabilizes TTR in human plasma with a very high selectivity. Luteolin, however, is inactivated in vivo via glucuronidation for which the preferred site is the hydroxy group at position 7 on its aromatic A-ring. We have evaluated the properties of two luteolin variants in which the 7-hydroxy group has been exchanged for a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut). Using an in vitro model, based on human liver microsomes, we verified that these modifications increase the persistence of the drug. Crystal structure determinations show that 7-Cl-Lut binds similarly to luteolin. The larger MeO substituent cannot be accommodated within the same space as the chlorine or hydroxy group and as a result 7-MeO-Lut binds in the opposite direction with the methoxy group in position 7 facing the solvent. Both 7-Cl-Lut and 7-MeO-Lut qualify as high-affinity binders, but in contrast to luteolin, they display a highly non-specific binding to other plasma components. The binding of the two conformations and the key-interactions to TTR are discussed in detail. Taken together, these results show a proof-of-concept that the persistence of luteolin towards enzymatic modification can be increased. We reveal two alternative high-affinity binding modes of luteolin to TTR and that modification in position 7 is restricted only to small substituents if the original orientation of luteolin should be preserved. In addition, the present work provides a general and convenient method to evaluate the efficacy of TTR-stabilizing drugs under conditions similar to an in vivo environment. PMID:27050398

  2. Mechanistic Insight into Peroxydisulfate Reactivity: Oxidation of the cis,cis-[Ru(bpy)2(OH2)]2O(4+) "Blue Dimer".

    PubMed

    Hurst, James K; Roemeling, Margo D; Lymar, Sergei V

    2015-06-18

    One-electron oxidation of the μ-oxo dimer (cis,cis-[Ru(III)(bpy)2(OH2)]2O(4+), {3,3}) to {3,4} by S2O8(2-) can be described by three concurrent reaction pathways corresponding to the three protic forms of {3,3}. Free energy correlations of the rate constants, transient species dynamics determined by pulse radiolysis, and medium and temperature dependencies of the alkaline pathway all suggest that the rate-determining step in these reactions is a strongly nonadiabatic dissociative electron transfer within a precursor ion pair leading to the {3,4}|SO4(2-)|SO4(•-) ion triple. As deduced from the SO4(•-) scavenging experiments with 2-propanol, the SO4(•-) radical then either oxidizes {3,4} to {4,4} within the ion triple, effecting a net two-electron oxidation of {3,3}, or escapes in solution with ∼25% probability to react with additional {3,3} and {3,4}, that is, effecting sequential one-electron oxidations. The reaction model presented also invokes rapid {3,3} + {4,4} → 2{3,4} comproportionation, for which kcom ∼5 × 10(7) M(-1) s(-1) was independently measured. The model provides an explanation for the observation that, despite favorable energetics, no oxidation beyond the {3,4} state was detected. The indiscriminate nature of oxidation by SO4(•-) indicates that its fate must be quantitatively determined when using S2O8(2-) as an oxidant. PMID:25819370

  3. Mechanistic Insights into Carbonyl-Directed Rhodium-Catalyzed Hydroboration: ab Initio Study of a Cyclic γ,δ-Unsaturated Amide.

    PubMed

    Yang, Zhao-Di; Pal, Rhitankar; Hoang, Gia L; Zeng, Xiao Cheng; Takacs, James M

    2014-03-01

    A two-point binding mechanism for the cationic rhodium(I)-catalyzed carbonyl-directed catalytic asymmetric hydroboration of a cyclic γ,δ-unsaturated amide is investigated using density functional theory. Geometry optimizations and harmonic frequency calculations for the model reaction are carried out using the basis set 6-31+G** for C, O, P, B, N, and H and LANL2DZ for Rh atoms. The Gibbs free energy of each species in THF solvent is obtained based on the single-point energy computed using the PCM model at the ECP28MWB/6-311+G(d,p) level plus the thermal correction to Gibbs free energy by deducting translational entropy contribution. The Rh-catalyzed reaction cycle involves the following sequence of events: (1) chelation of the cyclic γ,δ-unsaturated amide via alkene and carbonyl complexation in a model active catalytic species, [Rh(L2)2S2](+), (2) oxidative addition of pinacol borane (pinBH), (3) migratory insertion of the alkene double bond into Rh-H (preferred pathway) or Rh-B bond, (4) isomerization of the resulting intermediate, and finally, (5) reductive elimination to form the B-C or H-C bond with regeneration of the catalyst. Free energy profiles for potential pathways leading to the major γ-borylated product are computed and discussed in detail. The potential pathways considered include (1) pathways proceeding via migratory insertion into the Rh-H bond (pathways I, I-1, and I-2), (2) a potential pathway proceeding via migratory insertion into the Rh-B bond (pathway II), and two potential competing routes to a β-borylated byproduct (pathway III). The results find that the Rh-H migratory insertion pathway I-2, followed in sequence by an unanticipated isomerization via amide rotation and reductive elimination, is the most favorable reaction pathway. A secondary consequence of amide rotation is access to a competing β-hydride elimination pathway. The pathways computed in this study are supported by and help explain related experimental results. PMID

  4. Structural Insights into an Oxalate-producing Serine Hydrolase with an Unusual Oxyanion Hole and Additional Lyase Activity.

    PubMed

    Oh, Juntaek; Hwang, Ingyu; Rhee, Sangkee

    2016-07-15

    In Burkholderia species, the production of oxalate, an acidic molecule, is a key event for bacterial growth in the stationary phase. Oxalate plays a central role in maintaining environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In the phytopathogen Burkholderia glumae, two enzymes are responsible for oxalate production. First, the enzyme oxalate biosynthetic component A (ObcA) catalyzes the formation of a tetrahedral C6-CoA adduct from the substrates acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalate, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from Burkholderia thailandensis and Burkholderia pseudomallei Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain despite no sequence homology with ObcB. We report the crystal structure of Obc1 in its apo and glycerol-bound form at 2.5 Å and 2.8 Å resolution, respectively. The Obc1 N-terminal domain is essentially identical both in structure and function to that of ObcA. Its C-terminal domain has an α/β hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggested that His-934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis and an example of divergent evolution of the α/β hydrolase fold, which has both hydrolase and lyase activity. PMID:27226606

  5. Nanoparticles for heterogeneous catalysis: new mechanistic insights.

    PubMed

    Schauermann, Swetlana; Nilius, Niklas; Shaikhutdinov, Shamil; Freund, Hans-Joachim

    2013-08-20

    Metallic nanoparticles finely dispersed over oxide supports have found use as heterogeneous catalysts in many industries including chemical manufacturing, energy-related applications and environmental remediation. The compositional and structural complexity of such nanosized systems offers many degrees of freedom for tuning their catalytic properties. However, fully rational design of heterogeneous catalysts based on an atomic-level understanding of surface processes remains an unattained goal in catalysis research. Researchers have used surface science methods and metal single crystals to explore elementary processes in heterogeneous catalysis. In this Account, we use more realistic materials that capture part of the complexity inherent to industrial catalysts. We assess the impacts on the overall catalytic performance of characteristics such as finite particle size, particle structure, particle chemical composition, flexibility of atoms in clusters, and metal-support interactions. To prepare these materials, we grew thin oxide films on metal single crystals under ultrahigh vacuum conditions and used these films as supports for metallic nanoparticles. We present four case studies on specifically designed materials with properties that expand our atomic-level understanding of surface chemistry. Specifically, we address (1) the effect of dopants in the oxide support on the growth of metal nanoclusters; (2) the effects of size and structural flexibility of metal clusters on the binding energy of gas-phase adsorbates and their catalytic activity; (3) the role of surface modifiers, such as carbon, on catalytic activity and selectivity; and (4) the structural and compositional changes of the active surface as a result of strong metal-support interaction. Using these examples, we demonstrate how studies of complex nanostructured materials can help revealing atomic processes at the solid-gas interface of heterogeneous catalysts. Among our findings is that doping of oxide materials opens promising routes to alter the morphology and electronic properties of supported metal particles and to induce the direct dissociation and reaction of molecules bound to the oxide surface. Also, the small size and atomic flexibility of metal clusters can have an important influence on gas adsorption and catalytic performance. PMID:23252628

  6. Mechanistic investigations of CO-photoextrusion and oxidative addition reactions of early transition-metal carbonyls: (η(5)-C5H5)M(CO)4 (M = V, Nb, Ta).

    PubMed

    Su, Shih-Hao; Su, Ming-Der

    2016-06-28

    The mechanisms for the photochemical Si-H bond activation reaction are studied theoretically using a model system of the group 5 organometallic compounds, η(5)-CpM(CO)4 (M = V, Nb, and Ta), with the M06-2X method and the Def2-SVPD basis set. Three types of reaction pathways that lead to final insertion products are identified. The structures of the intersystem crossings, which play a central role in these photo-activation reactions, are determined. The intermediates and transitional structures in either the singlet or triplet states are also calculated to provide a mechanistic explanation of the reaction pathways. All of the potential energy surfaces for the group 5 η(5)-CpM(CO)4 complexes are quite similar. In particular, the theoretical evidence suggests that after irradiation using light, η(5)-CpM(CO)4 quickly loses one CO ligand to yield two tricarbonyls, in either the singlet or the triplet states. The triplet tricarbonyl 16-electron intermediates, ([η(5)-CpM(CO)3](3)), play a key role in the formation of the final oxidative addition product, η(5)-CpM(CO)3(H)(SiMe3). However, the singlet counterparts, ([η(5)-CpM(CO)3](1)), play no role in the formation of the final product molecule, but their singlet metal centers interact weakly with solvent molecules ((Me3)SiH) to produce alkyl-solvated organometallic complexes, which are observable experimentally. This theoretical evidence is in accordance with the available experimental observations. PMID:27265245

  7. Performance modeling of the Ballard Mark IV solid polymer electrolyte fuel cell. 1: Mechanistic model development

    SciTech Connect

    Amphlett, J.C.; Baumert, R.M.; Mann, R.F.; Peppley, B.A.; Roberge, P.R. ); Harris, T.J. )

    1995-01-01

    A parametric model predicting the performance of a solid polymer electrolyte, proton exchange membrane (PEM) fuel cell has been developed using a combination of mechanistic and empirical modeling techniques. This paper details the mechanistic model development. Mass transport properties are considered in the mechanistic development via Stefan-Maxwell equations. Thermodynamic equilibrium potentials are defined using the Nernst equation. Activation overvoltages are defined via a Tafel equation, and internal resistance are defined via the Nernst-Planck equation, leading to a definition of ohmic overvoltage via an Ohm's law equation. The mechanistic model cannot adequately model fuel cell performance, since several simplifying approximations have been used in order to facilitate model development. Additionally, certain properties likely to be observed in operational fuel cells, such as thermal gradients, have not been considered. Nonetheless, the insights gained from the mechanistic assessment of fuel cell processes were found to give the resulting empirical model a firmer theoretical basis than many of the models presently available in the literature. Correlation of the empirical model to actual experimental data was very good.

  8. Oxidative addition of carbon-carbon bonds to gold.

    PubMed

    Joost, Maximilian; Estévez, Laura; Miqueu, Karinne; Amgoune, Abderrahmane; Bourissou, Didier

    2015-04-20

    The oxidative addition of strained CC bonds (biphenylene, benzocyclobutenone) to DPCb (diphosphino-carborane) gold(I) complexes is reported. The resulting cationic organogold(III) complexes have been isolated and fully characterized. Experimental conditions can be adjusted to obtain selectively acyl gold(III) complexes resulting from oxidative addition of either the C(aryl)C(O) or C(alkyl)C(O) bond of benzocyclobutenone. DFT calculations provide mechanistic insight into this unprecedented transformation. PMID:25727203

  9. Additional insights on the bastadins: isolation of analogues from the sponge Ianthella cf. reticulata and exploration of the oxime configurations.

    PubMed

    Calcul, Laurent; Inman, Wayne D; Morris, Alexi A; Tenney, Karen; Ratnam, Joseline; McKerrow, James H; Valeriote, Frederick A; Crews, Phillip

    2010-03-26

    The focus of this study is on the bastadin class of bromotyrosine derivatives, commonly isolated from Ianthella marine sponges, and is the first report on the secondary metabolites from Ianthella cf. reticulata. Two new bastadins were isolated, (E,Z)-bastadin 19 (1a), a diastereoisomer of the known (E,E)-bastadin 19 (1b), and dioxepine bastadin 3 (2), an unusual dibenzo-1,3-dioxepine. A bastadin NMR database was created and assisted in the structure determination of 1b and 2 and the rapid dereplication of 10 other known compounds including bastadins 2-9 (3-10), 13 (11), and 19 (1a). The geometry of the 2-(hydroxyimino)-N-alkylamide chains, a chemical feature present in all bastadins, was further probed, and new insights regarding the natural oxime configuration are discussed. Bastadins possessing (E,Z)-, (Z,E)-, or (E,E)-dioxime configurations could be artifacts of isolation or storage in solution. Therefore, this point was explored by photochemical and thermal isomerization studies, as well as molecular mechanics calculations. Bastadins 13 (11) and 19 (1a) exhibited moderate inhibition against Trypanosoma brucei, and bastadin 4 (5) was cytotoxic to HCT-116 colon cancer cells. PMID:20102170

  10. Dehydrofluorination of Hydrofluorocarbons by Titanium Alkylidynes via Sequential C-H/C-F Bond Activation Reactions. A Synthetic, Structural, and Mechanistic Study of 1,2-CH Bond Addition and [beta]-Fluoride Elimination

    SciTech Connect

    Fout, A.R.; Scott, J.; Miller, D.L.; Bailey, B.C.; Pink, M.; Mindiola, D.J.

    2009-01-07

    The neopentylidene-neopentyl complex (PNP)Ti=CH{sup t}Bu(CH{sub 2}{sup t}Bu) (1); (PNP{sup -} = N[2-P(CHMe{sub 2}){sub 2}-4-methylphenyl]{sub 2}) extrudes neopentane in neat fluorobenzene under mild conditions (25 C) to generate the transient titanium alkylidyne (PNP)Ti-C{sup t}Bu (A), which subsequently undergoes regioselective 1,2-CH bond addition of a fluorobenzene across the Ti-C linkage to generate (PNP)Ti=CH{sup t}Bu(o-FC{sub 6}H{sub 4}) (2). Kinetic and mechanistic studies suggest that the C-H activation process is pseudo-first-order in titanium, with the {alpha}-hydrogen abstraction being the rate-determining step and the post-rate-determining step being the C-H bond activation of fluorobenzene. At 100 C complex 2 does not equilibrate back to A and the preference for C-H activation in benzene versus fluorobenzene is 2:3, respectively. Compound 1 also reacts readily, and in most cases cleanly, with a series of hydrofluoroarenes (HAr{sub F}), to form a family of alkylidene-arylfluoride derivatives of the type (PNP)Ti=CH{sup t}Bu(Ar{sub F}). Thermolysis of the latter compounds generates the titanium alkylidene-fluoride (PNP)Ti=CH{sup t}Bu(F) (14) by a {beta}-fluoride elimination, concurrent with formation of o-benzyne. {beta}-Fluoride elimination to yield 14 occurs from 2 under elevated temperatures with k{sub average} = 4.96(16) x 10{sup -5} s{sup -1} and with activation parameters {Delta}H{sub {-+}} = 29(1) kcal/mol and {Delta}S{sub {-+}} = -3(4) cal/mol {center_dot}K. It was found that {beta}-fluoride elimination is accelerated when electron-rich groups are adjacent to the fluoride group, thus implying that a positive charge buildup at the arylfluoride ring occurs in the activated complex of 2. The alkylidene derivative (PNP)Ti=CHSiMe{sub 3}(CH{sub 2}SiMe{sub 3}) (15) also undergoes {alpha}-hydrogen abstraction to form the putative (PNP)Ti'-CSiMe{sub 3} (B) at higher temperatures (>70 C) and dehydrofluorinates the same series of HArF when the reaction

  11. Insights into the Electronic Structure of Ozone and Sulfur Dioxide from Generalized Valence Bond Theory: Addition of Hydrogen Atoms.

    PubMed

    Lindquist, Beth A; Takeshita, Tyler Y; Dunning, Thom H

    2016-05-01

    Ozone (O3) and sulfur dioxide (SO2) are valence isoelectronic species, yet their properties and reactivities differ dramatically. In particular, O3 is highly reactive, whereas SO2 is chemically relatively stable. In this paper, we investigate serial addition of hydrogen atoms to both the terminal atoms of O3 and SO2 and to the central atom of these species. It is well-known that the terminal atoms of O3 are much more amenable to bond formation than those of SO2. We show that the differences in the electronic structure of the π systems in the parent triatomic species account for the differences in the addition of hydrogen atoms to the terminal atoms of O3 and SO2. Further, we find that the π system in SO2, which is a recoupled pair bond dyad, facilitates the addition of hydrogen atoms to the sulfur atom, resulting in stable HSO2 and H2SO2 species. PMID:27070292

  12. Insight into the reaction mechanisms for oxidative addition of strong σ bonds to an Al(i) center.

    PubMed

    Zhang, Xiangfei; Cao, Zexing

    2016-06-21

    The oxidation addition of a series of σ H-X bonds (X = H, B, C, Si, N, P, and O) to a single Al(i) supported by a (NacNac)(-) bidentate ligand ((NacNac)(-) = [ArNC(Me)CHC(Me)NAr](-) and Ar = 2,6-(i)Pr2C6H3) has been explored through extensive DFT calculations. The presented results show that activation and addition of these σ bonds follow various reaction mechanisms, in which hydride transfer, proton transfer, and Al-X bond coupling steps are involved. The predicted free energy barriers for these oxidative additions range from 8 to 32 kcal mol(-1), and all the reactions are remarkably favorable thermodynamically. However, sterically hindered ligands, for most reactants, make the formation of the initial reactant complex difficult and may reduce the efficiency of the reaction. Calculations reveal a strong dependence of the reaction mechanism and low-energy channel on the bonding features of X-H and the local structural environments. PMID:27249667

  13. Conjugate Addition of 3-Buytn-2-one to Anilines in Ethanol: Alkene Geometric Insights through In Situ FTIR Monitoring.

    PubMed

    Chisholm, David R; Valentine, Roy; Pohl, Ehmke; Whiting, Andrew

    2016-09-01

    A convenient, mild and effective conjugate addition of 3-butyn-2-one to a variety of anilines in ethanol is reported. The reaction was monitored and characterized through in situ FTIR, and the dynamics of the facile E/Z alkene geometry interconversion of the resultant aniline-derived enaminones was explored through NMR, FTIR and X-ray crystallography. A straightforward purification protocol that employs direct Kugelrohr distillation was identified, and the method was further extended to other amines and ynones, allowing rapid access to these interesting compounds. PMID:27467234

  14. Food for Thought ... Mechanistic Validation

    PubMed Central

    Hartung, Thomas; Hoffmann, Sebastian; Stephens, Martin

    2013-01-01

    Summary Validation of new approaches in regulatory toxicology is commonly defined as the independent assessment of the reproducibility and relevance (the scientific basis and predictive capacity) of a test for a particular purpose. In large ring trials, the emphasis to date has been mainly on reproducibility and predictive capacity (comparison to the traditional test) with less attention given to the scientific or mechanistic basis. Assessing predictive capacity is difficult for novel approaches (which are based on mechanism), such as pathways of toxicity or the complex networks within the organism (systems toxicology). This is highly relevant for implementing Toxicology for the 21st Century, either by high-throughput testing in the ToxCast/ Tox21 project or omics-based testing in the Human Toxome Project. This article explores the mostly neglected assessment of a test's scientific basis, which moves mechanism and causality to the foreground when validating/qualifying tests. Such mechanistic validation faces the problem of establishing causality in complex systems. However, pragmatic adaptations of the Bradford Hill criteria, as well as bioinformatic tools, are emerging. As critical infrastructures of the organism are perturbed by a toxic mechanism we argue that by focusing on the target of toxicity and its vulnerability, in addition to the way it is perturbed, we can anchor the identification of the mechanism and its verification. PMID:23665802

  15. Insights into Tropical Tropospheric Ozone from the 1998-2000 SHADOZ (Southern Hemisphere Additional Ozonesondes) Data Record

    NASA Technical Reports Server (NTRS)

    Thompson, Anne M.; Witte, Jacquelyn C.; Oltmans, Samuel J.; Schmidlin, Francis J.; Volker, W.; Kirchhoff, J. H.; Posny, Franaoise; Gert, J.; Coetzee, R.; Hoegger, Bruno; Bhartia, P. K. (Technical Monitor)

    2002-01-01

    We describe the first overview of total, stratospheric and tropospheric ozone in the southern hemisphere tropics based on a three year, ten site record of ozone soundings from the Southern Hemisphere Additional Ozonesondes (SHADOZ) network. Observations covering 1998-2000 were made over Ascension Island; Nairobi, Kenya; Irene, South Africa; Reunion Island; Watukosek, Java; Fiji; Tahiti; American Samoa; San Cristobal, Galapagos; Natal, Brazil. The ozone data, with simultaneous temperature profiles to approximately 7 hPa and relative humidity to approximately 200 hPa, are at an archive: http://code9l6. gsfc.nasa.gov/Data_services/shadoz. Prominent features are highly variable tropospheric ozone, a zonal wave-one pattern in total (and tropospheric) column ozone, and signatures of the Quasi-Biennial Oscillation (QBO) in stratospheric ozone. Total, stratospheric and tropospheric column ozone amounts usually peak between August and November and are lowest in the first half of the year. Tropospheric ozone variability over the Indian and Pacific Ocean displays influences of the waning 1997-1998 Indian Ocean Dipole and ENSO (El Nino / Southern Oscillation), seasonal convection and pollution transport from Africa. Tropospheric ozone over the Atlantic Basin reflects regional subsidence and recirculation as well as pollution ozone from biomass burning.

  16. Genomic-scale comparison of sequence- and structure-based methods of function prediction: Does structure provide additional insight?

    PubMed Central

    Fetrow, Jacquelyn S.; Siew, Naomi; Di Gennaro, Jeannine A.; Martinez-Yamout, Maria; Dyson, H. Jane; Skolnick, Jeffrey

    2001-01-01

    A function annotation method using the sequence-to-structure-to-function paradigm is applied to the identification of all disulfide oxidoreductases in the Saccharomyces cerevisiae genome. The method identifies 27 sequences as potential disulfide oxidoreductases. All previously known thioredoxins, glutaredoxins, and disulfide isomerases are correctly identified. Three of the 27 predictions are probable false-positives. Three novel predictions, which subsequently have been experimentally validated, are presented. Two additional novel predictions suggest a disulfide oxidoreductase regulatory mechanism for two subunits (OST3 and OST6) of the yeast oligosaccharyltransferase complex. Based on homology, this prediction can be extended to a potential tumor suppressor gene, N33, in humans, whose biochemical function was not previously known. Attempts to obtain a folded, active N33 construct to test the prediction were unsuccessful. The results show that structure prediction coupled with biochemically relevant structural motifs is a powerful method for the function annotation of genome sequences and can provide more detailed, robust predictions than function prediction methods that rely on sequence comparison alone. PMID:11316881

  17. New Insights into the Detection of Sulfur Trioxide Anion Radical by Spin Trapping: Radical Trapping versus Nucleophilic Addition

    PubMed Central

    Ranguelova, Kalina; Mason, Ronald P.

    2009-01-01

    It has recently been proposed that (bi)sulfite (hydrated sulfur dioxide) reacts with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in biological systems via a nonradical, nucleophilic reaction, implying that the radical adduct (DMPO/•SO3−) formation in these systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical (•SO3−). Here, the one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase/H2O2 has been re-investigated by ESR spin trapping with DMPO and oxygen uptake studies in order to obtain further evidence for the radical reaction mechanism. In the case of ESR experiments, the signal of DMPO/•SO3− radical adduct was detected, and the initial rate of its formation was calculated. Support for the radical pathway via •SO3− was obtained from the stoichiometry between the amount of consumed molecular oxygen and the amount of (bi)sulfite oxidized to sulfate (SO42−). When DMPO was incubated with (bi)sulfite, oxygen consumption was completely inhibited due to the efficiency of DMPO trapping. In the absence of DMPO, the initial rate of oxygen and H2O2 consumption was determined to be half of the initial rate of DMPO/•SO3− radical adduct formation as determined by ESR, demonstrating that DMPO forms the radical adduct by trapping the •SO3− exclusively. We conclude that DMPO is not susceptible to artifacts arising from nonradical chemistry (nucleophilic addition) except when both (bi)sulfite and DMPO concentrations are at nonphysiological levels of at least 0.1 M and the incubations are for longer time periods. PMID:19362142

  18. Palladium-Catalyzed C8-Selective C–H Arylation of Quinoline N-Oxides: Insights into the Electronic, Steric, and Solvation Effects on the Site Selectivity by Mechanistic and DFT Computational Studies

    PubMed Central

    2015-01-01

    We report herein a palladium-catalyzed C–H arylation of quinoline N-oxides that proceeds with high selectivity in favor of the C8 isomer. This site selectivity is unusual for palladium, since all of the hitherto described methods of palladium-catalyzed C–H functionalization of quinoline N-oxides are highly C2 selective. The reaction exhibits a broad synthetic scope with respect to quinoline N-oxides and iodoarenes and can be significantly accelerated to subhour reaction times under microwave irradiation. The C8-arylation method can be carried out on a gram scale and has excellent functional group tolerance. Mechanistic and density functional theory (DFT) computational studies provide evidence for the cyclopalladation pathway and describe key parameters influencing the site selectivity. PMID:25580364

  19. Mechanistic analysis of challenge-response experiments.

    PubMed

    Shotwell, M S; Drake, K J; Sidorov, V Y; Wikswo, J P

    2013-09-01

    We present an application of mechanistic modeling and nonlinear longitudinal regression in the context of biomedical response-to-challenge experiments, a field where these methods are underutilized. In this type of experiment, a system is studied by imposing an experimental challenge, and then observing its response. The combination of mechanistic modeling and nonlinear longitudinal regression has brought new insight, and revealed an unexpected opportunity for optimal design. Specifically, the mechanistic aspect of our approach enables the optimal design of experimental challenge characteristics (e.g., intensity, duration). This article lays some groundwork for this approach. We consider a series of experiments wherein an isolated rabbit heart is challenged with intermittent anoxia. The heart responds to the challenge onset, and recovers when the challenge ends. The mean response is modeled by a system of differential equations that describe a candidate mechanism for cardiac response to anoxia challenge. The cardiac system behaves more variably when challenged than when at rest. Hence, observations arising from this experiment exhibit complex heteroscedasticity and sharp changes in central tendency. We present evidence that an asymptotic statistical inference strategy may fail to adequately account for statistical uncertainty. Two alternative methods are critiqued qualitatively (i.e., for utility in the current context), and quantitatively using an innovative Monte-Carlo method. We conclude with a discussion of the exciting opportunities in optimal design of response-to-challenge experiments. PMID:23859366

  20. Near-infrared and ultraviolet induced isomerization of crotonic acid in N{sub 2} and Xe cryomatrices: First observation of two high-energy trans C–O conformers and mechanistic insights

    SciTech Connect

    Kuş, Nihal; Fausto, Rui

    2014-12-21

    E-crotonic acid was isolated in cryogenic solid N{sub 2} and xenon matrices, and subjected to Laser ultraviolet (UV) and near-infrared (NIR) irradiations. In the deposited matrices, the two low-energy cis C–O E-cc and E-ct conformers, which are the only forms significantly populated in the gas phase, were observed. UV irradiation (λ= 250 nm) of the compound in N{sub 2} matrix allows for experimental detection, not just of the two low-energy cis C–O isomers of Z-crotonic acid previously observed in the experiments carried out in argon matrix [Z-cc and Z-ct; R. Fausto, A. Kulbida, and O. Schrems, J. Chem. Soc., Faraday Trans. 91, 3755–3770 (1995)] but also of the never observed before high-energy forms of both E- and Z-crotonic acids bearing the carboxylic acid group in the trans arrangement (E-tc and Z-tc conformers). In turn, NIR irradiation experiments in the N{sub 2} matrix allow to produce the high-energy E-tc trans C–O conformer in a selective way, from the initially deposited E-cc form. The vibrational signatures of all the 6 rotameric structures of the crotonic acids experimentally observed, including those of the new trans C–O forms, were determined and the individual spectra fully assigned, also with support of theoretically obtained data. On the other hand, as found before for the compound isolated in argon matrix, the experiments performed in xenon matrix failed to experimental detection of the trans C–O forms. This demonstrates that in noble gas matrices these forms are not stable long enough to allow for their observation by steady state spectroscopy techniques. In these matrices, the trans C–O forms convert spontaneously into their cis C–O counterparts, by tunnelling. Some mechanistic details of the studied processes were extracted and discussed.

  1. Quantitative assessment of biological impact using transcriptomic data and mechanistic network models

    SciTech Connect

    Thomson, Ty M.; Sewer, Alain; Martin, Florian; Belcastro, Vincenzo; Frushour, Brian P.; Gebel, Stephan; Park, Jennifer; Schlage, Walter K.; Talikka, Marja; Vasilyev, Dmitry M.; Westra, Jurjen W.; Hoeng, Julia; Peitsch, Manuel C.

    2013-11-01

    Exposure to biologically active substances such as therapeutic drugs or environmental toxicants can impact biological systems at various levels, affecting individual molecules, signaling pathways, and overall cellular processes. The ability to derive mechanistic insights from the resulting system responses requires the integration of experimental measures with a priori knowledge about the system and the interacting molecules therein. We developed a novel systems biology-based methodology that leverages mechanistic network models and transcriptomic data to quantitatively assess the biological impact of exposures to active substances. Hierarchically organized network models were first constructed to provide a coherent framework for investigating the impact of exposures at the molecular, pathway and process levels. We then validated our methodology using novel and previously published experiments. For both in vitro systems with simple exposure and in vivo systems with complex exposures, our methodology was able to recapitulate known biological responses matching expected or measured phenotypes. In addition, the quantitative results were in agreement with experimental endpoint data for many of the mechanistic effects that were assessed, providing further objective confirmation of the approach. We conclude that our methodology evaluates the biological impact of exposures in an objective, systematic, and quantifiable manner, enabling the computation of a systems-wide and pan-mechanistic biological impact measure for a given active substance or mixture. Our results suggest that various fields of human disease research, from drug development to consumer product testing and environmental impact analysis, could benefit from using this methodology. - Highlights: • The impact of biologically active substances is quantified at multiple levels. • The systems-level impact integrates the perturbations of individual networks. • The networks capture the relationships between

  2. Reaction Coordinates and Mechanistic Hypothesis Tests.

    PubMed

    Peters, Baron

    2016-05-27

    Reaction coordinates are integral to several classic rate theories that can (a) predict kinetic trends across conditions and homologous reactions, (b) extract activation parameters with a clear physical interpretation from experimental rates, and (c) enable efficient calculations of free energy barriers and rates. New trajectory-based rare events methods can provide rates directly from dynamical trajectories without a reaction coordinate. Trajectory-based frameworks can also generate ideal (but abstract) reaction coordinates such as committors and eigenfunctions of the master equation. However, rates and mechanistic insights obtained from trajectory-based methods and abstract coordinates are not readily generalized across simulation conditions or reaction families. We discuss methods for identifying physically meaningful reaction coordinates, including committor analysis, variational transition state theory, Kramers-Langer-Berezhkovskii-Szabo theory, and statistical inference methods that can use path sampling data to screen, mix, and optimize thousands of trial coordinates. Special focus is given to likelihood maximization and inertial likelihood maximization approaches. PMID:27090846

  3. Reaction Coordinates and Mechanistic Hypothesis Tests

    NASA Astrophysics Data System (ADS)

    Peters, Baron

    2016-05-01

    Reaction coordinates are integral to several classic rate theories that can (a) predict kinetic trends across conditions and homologous reactions, (b) extract activation parameters with a clear physical interpretation from experimental rates, and (c) enable efficient calculations of free energy barriers and rates. New trajectory-based rare events methods can provide rates directly from dynamical trajectories without a reaction coordinate. Trajectory-based frameworks can also generate ideal (but abstract) reaction coordinates such as committors and eigenfunctions of the master equation. However, rates and mechanistic insights obtained from trajectory-based methods and abstract coordinates are not readily generalized across simulation conditions or reaction families. We discuss methods for identifying physically meaningful reaction coordinates, including committor analysis, variational transition state theory, Kramers-Langer-Berezhkovskii-Szabo theory, and statistical inference methods that can use path sampling data to screen, mix, and optimize thousands of trial coordinates. Special focus is given to likelihood maximization and inertial likelihood maximization approaches.

  4. Water-soluble NHC-Cu catalysts: applications in click chemistry, bioconjugation and mechanistic analysis.

    PubMed

    Díaz Velázquez, Heriberto; Ruiz García, Yara; Vandichel, Matthias; Madder, Annemieke; Verpoort, Francis

    2014-12-14

    Copper(I)-catalyzed 1,3-dipolar cycloaddition of azides and terminal alkynes (CuAAC), better known as "click" reaction, has triggered the use of 1,2,3-triazoles in bioconjugation, drug discovery, materials science and combinatorial chemistry. Here we report a new series of water-soluble catalysts based on N-heterocyclic carbene (NHC)-Cu complexes which are additionally functionalized with a sulfonate group. The complexes show superior activity towards CuAAC reactions and display a high versatility, enabling the production of triazoles with different substitution patterns. Additionally, successful application of these complexes in bioconjugation using unprotected peptides acting as DNA binding domains was achieved for the first time. Mechanistic insight into the reaction mechanism is obtained by means of state-of-the-art first principles calculations. PMID:25251642

  5. Mechanistic Studies in Friction and Wear of Bulk Materials

    NASA Astrophysics Data System (ADS)

    Sawyer, W. Gregory; Argibay, Nicolas; Burris, David L.; Krick, Brandon A.

    2014-07-01

    From the context of a contemporary understanding of the phenomenological origins of friction and wear of materials, we review insightful contributions from recent experimental investigations of three classes of materials that exhibit uniquely contrasting tribological behaviors: metals, polymers, and ionic solids. We focus on the past decade of research by the community to better understand the correlations between environment parameters, materials properties, and tribological behavior in systems of increasingly greater complexity utilizing novel synthesis and in situ experimental techniques. In addition to such review, and a half-century after seminal publications on the subject, we present recently acquired evidence linking anisotropy in friction response with anisotropy in wear behavior of crystalline ionic solids as a function of crystallographic orientation. Although the tribological behaviors of metals, polymers, and ionic solids differ widely, it is increasingly more evident that the mechanistic origins (such as fatigue, corrosion, abrasion, and adhesion) are essentially the same. However, we hope to present a clear and compelling argument favoring the prominent and irreplaceable role of in situ experimental techniques as a bridge between fundamental atomistic and molecular processes and emergent behaviors governing tribological contacts.

  6. A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells.

    PubMed Central

    Shumway, Stuart D; Miyamoto, Shigeki

    2004-01-01

    Inducible activation of the transcription factor NF-kappaB (nuclear factor kappaB) is classically mediated by proteasomal degradation of its associated inhibitors, IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. However, certain B-lymphocytes maintain constitutively nuclear NF-kappaB activity (a p50-c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IkappaBalpha, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IkappaBalpha degradation and constitutive p50-c-Rel activity. Domain analysis of IkappaBalpha by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IkappaBalpha represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IkappaBbeta, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IkappaBbeta to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IkappaBalpha that was required for complete targeting. Nevertheless, the NF-kappaB pool associated with IkappaBbeta now became constitutively active by virtue of IkappaBbeta instability in these cells. These findings further support the notion that IkappaB instability governs the maintenance of constitutive p50-c-Rel activity in certain B-cells via a unique degradation pathway. PMID:14763901

  7. Mechanistic insight into peroxydisulfate reactivity: Oxidation of the cis,cis-[Ru(bpy)2(OH2)]2O4+ "Blue Dimmer"

    SciTech Connect

    Hurst, James K.; Roemeling, Margo D.; Lymar, Sergei V.

    2015-04-10

    One-electron oxidation of the μ-oxo dimer (cis,cis-[RuIII(bpy)2(OH2)]2O4+, {3,3}) to {3,4} by S2O82- can be described by three concurrent reaction pathways corresponding to the three protic forms of {3,3}. Free energy correlations of the rate constants, transient species dynamics determined by pulse radiolysis, and medium and temperature dependencies of the alkaline pathway all suggest that the rate determining step in these reactions is a strongly non-adiabatic dissociative electron transfer within a precursor ion pair leading to the {3,4}|SO42-|SO4•- ion triple. As deduced from the SO4•- scavenging experiments with 2-propanol, the SO4•- radical then either oxidizes {3,4} to {4,4} within the ion triple, effecting a net two-electron oxidation of {3,3}, or escapes in solution with ~25 % probability to react with additional {3,3} and {3,4}, that is, effecting sequential one-electron oxidations. The reaction model presented also invokes rapid {3,3} + {4,4} → 2{3,4} comproportionation, for which kcom ~5×107 M-1 s-1 was independently measured. The model provides an explanation for the observation that despite favorable energetics, no oxidation beyond the {3,4} state was detected. As a result, the indiscriminate nature of oxidation by SO4•- indicates that its fate must be quantitatively determined when using S2O82- as an oxidant

  8. Capillary Forces between Floating Objects: Superhydrophobic Surfaces Provide Mechanistic Insight

    NASA Astrophysics Data System (ADS)

    Zhang, Minchao; McCarthy, Thomas J.; Crosby, Alfred J.

    When two floating objects are close, they will either move towards or away from one another to minimize the energy caused by the overlap of the liquid/air interfacial deformations. Capillary forces cause these behaviors, but directly relating the interfacial deformations and the capillary interactions hasn't been explored experimentally. We choose a liquid marble, which has a superhydrophobic surface, as a free floating object and a fixed ``wall'' with carefully controlled contact angle as another object to generate two deformations at the interface. When the liquid marble is close to the wall, the two deformations interact, causing changes in the Laplace pressure at the interface and a reconfiguration of the interface. In response, the liquid marble moves either towards or away from the wall. Using image analysis of videos recording the liquid marble position as a function of time, we measured the liquid marble to wall distance and determine the spatio-temporal relationships. Furthermore, capillary forces were calculated from the velocities and accelerations. Based on this data, we present a new explanation for the capillary interactions from the perspective of Laplace pressure changing induced the reconfiguration of the interfacial deformations.

  9. Structural and mechanistic insights into Mps1 kinase activation

    SciTech Connect

    Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

    2010-11-05

    Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

  10. Daphnia magna ecotoxicogenomics provides mechanistic insights into metal toxicity.

    PubMed

    Poynton, Helen C; Varshavsky, Julia R; Chang, Bonnie; Cavigiolio, Giorgio; Chan, Sarah; Holman, Patricia S; Loguinov, Alexandre V; Bauer, Darren J; Komachi, Kelly; Theil, Elizabeth C; Perkins, Edward J; Hughes, Owen; Vulpe, Chris D

    2007-02-01

    Toxicogenomics has provided innovative approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to understand the modes of toxicity in environmentally relevant organisms. Daphnia magna, a small aquatic crustacean, is considered a "keystone" species in ecological food webs and is an indicator species for toxicant exposure. Our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and uncovering potential modes of action in D. magna. Using a custom D. magna cDNA microarray, we identified distinct expression profiles in response to sublethal copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. By integrating gene expression profiling into an environmentally important organism, this study provides experimental support for the utility of ecotoxicogenomics. PMID:17328222

  11. Mechanistic insights into interaction of humic acid with silver nanoparticles.

    PubMed

    Manoharan, Vijayan; Ravindran, Aswathy; Anjali, C H

    2014-01-01

    Humic acid (HA) is one of the major components of the natural organic matter present in the environment that alters the fate and behavior of silver nanoparticles (Ag NPs). Transformation of Ag NPs happens upon interaction with HA, thereby, changing both physical and chemical properties. Fluorescence spectroscopy and scanning electron microscopy (SEM) were used to analyze the interaction of Ag NPs with HA. In pH and time-dependent studies, the near field electro dynamical environment of Ag NPs influenced the fluorescence of HA, indicated by fluorescence enhancement. SEM revealed not only morphological changes, but also significant reduction in size of Ag NPs after interaction with HA. Based on these studies, a probable mechanism was proposed for the interaction of HA with Ag NPs, suggesting the possible transformation that these nanoparticles can undergo in the environment. PMID:23801156

  12. Mechanistic insight into ligand binding to G-quadruplex DNA

    PubMed Central

    Di Leva, Francesco Saverio; Novellino, Ettore; Cavalli, Andrea; Parrinello, Michele; Limongelli, Vittorio

    2014-01-01

    Specific guanine-rich regions in human genome can form higher-order DNA structures called G-quadruplexes, which regulate many relevant biological processes. For instance, the formation of G-quadruplex at telomeres can alter cellular functions, inducing apoptosis. Thus, developing small molecules that are able to bind and stabilize the telomeric G-quadruplexes represents an attractive strategy for antitumor therapy. An example is 3-(benzo[d]thiazol-2-yl)-7-hydroxy-8-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2H-chromen-2-one (compound 1), recently identified as potent ligand of the G-quadruplex [d(TGGGGT)]4 with promising in vitro antitumor activity. The experimental observations are suggestive of a complex binding mechanism that, despite efforts, has defied full characterization. Here, we provide through metadynamics simulations a comprehensive understanding of the binding mechanism of 1 to the G-quadruplex [d(TGGGGT)]4. In our calculations, the ligand explores all the available binding sites on the DNA structure and the free-energy landscape of the whole binding process is computed. We have thus disclosed a peculiar hopping binding mechanism whereas 1 is able to bind both to the groove and to the 3’ end of the G-quadruplex. Our results fully explain the available experimental data, rendering our approach of great value for further ligand/DNA studies. PMID:24753420

  13. Inhibition of insulin fibrillation by osmolytes: Mechanistic Insights

    NASA Astrophysics Data System (ADS)

    Choudhary, Sinjan; Kishore, Nand; Hosur, Ramakrishna V.

    2015-11-01

    We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50-250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies.

  14. Enhanced Identification of Transcriptional Enhancers Provides Mechanistic Insights into Diseases.

    PubMed

    Murakawa, Yasuhiro; Yoshihara, Masahito; Kawaji, Hideya; Nishikawa, Miki; Zayed, Hatem; Suzuki, Harukazu; Fantom Consortium; Hayashizaki, Yoshihide

    2016-02-01

    Enhancers are distal cis-regulatory DNA elements that increase the expression of target genes. Various experimental and computational approaches including chromatin signature profiling have been developed to predict enhancers on a genome-wide scale, although each method has its advantages and disadvantages. Here we overview an emerging method to identify transcribed enhancers at exceedingly high nucleotide resolution based on enhancer RNA transcripts captured by Cap Analysis of Gene Expression (CAGE) technology. We further argue that disease-causative regulatory mutations at enhancers are increasingly recognized, emphasizing the importance of enhancer identification in functional and clinical genomics including, but not limited to, genome-wide association studies (GWASs) and cancer genomics studies. PMID:26780995

  15. Inhibition of insulin fibrillation by osmolytes: Mechanistic Insights

    PubMed Central

    Choudhary, Sinjan; Kishore, Nand; Hosur, Ramakrishna V.

    2015-01-01

    We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50–250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies. PMID:26616401

  16. Mechanistic insights into the regulation of metabolic enzymes by acetylation

    PubMed Central

    2012-01-01

    The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

  17. Insights into the mechanistic photodissociation of methyl formate

    SciTech Connect

    Cui Ganglong; Zhang Feng; Fang Weihai

    2010-01-21

    In this work, we studied the photodissociation dynamics of methyl formate (CH{sub 3}OC(O)H) using state-of-the-art multireference configuration interaction with single and double excitation and the complete active space self-consistent field methods. It was found that the direct {alpha}-C-O bond cleavage in the first excited singlet state (S{sub 1}) is the dominant dissociation channel, consistent with the recent experiment [S. H. Lee, J. Chem. Phys. 129, 194304 (2008)]. This cleavage mechanism is different from that for aldehydes/ketones where it occurs in the lowest triplet state (T{sub 1}) as a result of the S{sub 1}{yields}T{sub 1} intersystem crossing. On the basis of comparison to the {alpha}-bond fission in the asymmetrically substituted aliphatic carbonyl compounds studied previously, we suggest the photolytic reaction of CH{sub 3}OC(O)H as a special type of Norrish type I reaction.

  18. Mechanistic insights into the recycling machine of the SNARE complex.

    PubMed

    Zhao, Minglei; Wu, Shenping; Zhou, Qiangjun; Vivona, Sandro; Cipriano, Daniel J; Cheng, Yifan; Brunger, Axel T

    2015-02-01

    Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes. PMID:25581794

  19. Mechanistic insights into the recycling machine of the SNARE complex

    PubMed Central

    Zhao, Minglei; Wu, Shenping; Zhou, Qiangjun; Vivona, Sandro; Cipriano, Daniel J.; Cheng, Yifan; Brunger, Axel T.

    2014-01-01

    Summary Evolutionarily conserved SNARE (Soluble N-ethylmaleimide sensitive factor Attachment protein REceptors) proteins form a complex that drives fusion between membranes in eukaryotes. SNARE complexes are disassembled by the ATPase NSF (N-ethylmaleimide Sensitive Factor), together with SNAP (Soluble NSF Attachment Protein) proteins, making individual SNAREs available for a subsequent round of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometer resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains, to pseudo four-fold symmetry of the SNARE complex. SNAPs are interacting with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes. PMID:25581794

  20. Mechanistic insights into filamentous phage integration in Vibrio cholerae

    PubMed Central

    Das, Bhabatosh

    2014-01-01

    Vibrio cholerae, the etiological agent of acute diarrhoeal disease cholera, harbors large numbers of lysogenic filamentous phages, contribute significantly to the host pathogenesis and provide fitness factors to the pathogen that help the bacterium to survive in natural environment. Most of the vibriophage genomes are not equipped with integrase and thus exploit two host-encoded tyrosine recombinases, XerC and XerD, for lysogenic conversion. Integration is site-specific and it occurs at dimer resolution site (dif) of either one or both chromosomes of V. cholerae. Each dif sequence contains two recombinase-binding sequences flanking a central region. The integration follows a sequential strand exchanges between dif and attP sites within a DNA-protein complex consisting of one pair of each recombinase and two DNA fragments. During entire process of recombination, both the DNA components and recombinases of the synaptic complex keep transiently interconnected. Within the context of synaptic complex, both of the actuated enzymes mediate cleavage of phosphodiester bonds. First cleavage generates a phosphotyrosyl-linked recombinase-DNA complex at the recombinase binding sequence and free 5′-hydroxyl end at the first base of the central region. Following the cleavage, the exposed bases with 5′-hydroxyl ends of the central region of dif and attP sites melt from their complementary strands and react with the recombinase-DNA phosphotyrosyl linkage of their recombining partner. Subsequent ligation between dif and attP strands requires complementary base pair interactions at the site of phosphodiester bond formation. Integration mechanism is mostly influenced by the compatibility of dif and attP sequences. dif sites are highly conserved across bacterial phyla. Different phage genomes have different attP sequences; therefore they rely on different mechanisms for integration. Here, I review our current understanding of integration mechanisms used by the vibriophages. PMID:25506341

  1. Inhibition of insulin fibrillation by osmolytes: Mechanistic insights.

    PubMed

    Choudhary, Sinjan; Kishore, Nand; Hosur, Ramakrishna V

    2015-01-01

    We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50-250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies. PMID:26616401

  2. PTN signaling: Components and mechanistic insights in human ovarian cancer.

    PubMed

    Sethi, Geetika; Kwon, Youngjoo; Burkhalter, Rebecca J; Pathak, Harsh B; Madan, Rashna; McHugh, Sarah; Atay, Safinur; Murthy, Smruthi; Tawfik, Ossama W; Godwin, Andrew K

    2015-12-01

    Molecular vulnerabilities represent promising candidates for the development of targeted therapies that hold the promise to overcome the challenges encountered with non-targeted chemotherapy for the treatment of ovarian cancer. Through a synthetic lethality screen, we previously identified pleiotrophin (PTN) as a molecular vulnerability in ovarian cancer and showed that siRNA-mediated PTN knockdown induced apoptotic cell death in epithelial ovarian cancer (EOC) cells. Although, it is well known that PTN elicits its pro-tumorigenic effects through its receptor, protein tyrosine phosphatase receptor Z1 (PTPRZ1), little is known about the potential importance of this pathway in the pathogenesis of ovarian cancer. In this study, we show that PTN is expressed, produced, and secreted in a panel of EOC cell lines. PTN levels in serous ovarian tumor tissues are on average 3.5-fold higher relative to normal tissue and PTN is detectable in serum samples of patients with EOC. PTPRZ1 is also expressed and produced by EOC cells and is found to be up-regulated in serous ovarian tumor tissue relative to normal ovarian surface epithelial tissue (P < 0.05). Gene silencing of PTPRZ1 in EOC cell lines using siRNA-mediated knockdown shows that PTPRZ1 is essential for viability and results in significant apoptosis with no effect on the cell cycle phase distribution. In order to determine how PTN mediates survival, we silenced the gene using siRNA mediated knockdown and performed expression profiling of 36 survival-related genes. Through computational mapping of the differentially expressed genes, members of the MAPK (mitogen-activated protein kinase) family were found to be likely effectors of PTN signaling in EOC cells. Our results provide the first experimental evidence that PTN and its signaling components may be of significance in the pathogenesis of epithelial ovarian cancer and provide a rationale for clinical evaluation of MAPK inhibitors in PTN and/or PTPRZ1 expressing ovarian tumors. PMID:25418856

  3. Thermodynamic Analysis of Thermal Hysteresis: Mechanistic Insights into Biological Antifreezes

    PubMed Central

    Wang, Sen; Amornwittawat, Natapol; Wen, Xin

    2012-01-01

    Antifreeze proteins (AFPs) bind to ice crystal surfaces and thus inhibit the ice growth. The mechanism for how AFPs suppress freezing is commonly modeled as an adsorption-inhibition process by the Gibbs-Thomson effect. Here we develop an improved adsorption-inhibition model for AFP action based on the thermodynamics of impurity adsorption on the crystal surfaces. We demonstrate the derivation of a realistic relationship between surface protein coverage and the protein concentration. We show that the improved model provides a quantitatively better fit to the experimental antifreeze activities of AFPs from distinct structural classes, including fish and insect AFPs, in a wide range of concentrations. Our theoretical results yielded the adsorption coefficients of the AFPs on ice, suggesting that, despite the distinct difference in their antifreeze activities and structures, the affinities of the AFPs to ice are very close and the mechanism of AFP action is a kinetically controlled, reversible process. The applications of the model to more complex systems along with its potential limitations are also discussed. PMID:22822266

  4. Describing dengue epidemics: Insights from simple mechanistic models

    NASA Astrophysics Data System (ADS)

    Aguiar, Maíra; Stollenwerk, Nico; Kooi, Bob W.

    2012-09-01

    We present a set of nested models to be applied to dengue fever epidemiology. We perform a qualitative study in order to show how much complexity we really need to add into epidemiological models to be able to describe the fluctuations observed in empirical dengue hemorrhagic fever incidence data offering a promising perspective on inference of parameter values from dengue case notifications.

  5. Answering evolutionary questions: A guide for mechanistic biologists.

    PubMed

    Masel, Joanna; Promislow, Daniel E L

    2016-07-01

    The questions and methods of molecular biology and evolutionary biology are clearly distinct, yet a unified approach can lead to deep insights. Unfortunately, attempts to unify these approaches are fraught with pitfalls. In this informal series of questions and answers, we offer the mechanistically oriented biologist a set of steps to come up with evolutionarily reasonable and meaningful hypotheses. We emphasize the critical power and importance of carefully constructed null hypotheses, and we illustrate our ideas with examples representing a range of topics, from the biology of aging, to protein structure, to speciation, and more. We also stress the importance of mathematics as the lingua franca for biologists of all stripes, and encourage mechanistic biologists to seek out quantitative collaborators to build explicit mathematical models, making their assumptions explicit, and their logic clear and testable. Biologists in all realms of inquiry stand to gain from strong bridges between our disciplines. PMID:27151396

  6. Mechanistic investigations of shuttle glow

    NASA Technical Reports Server (NTRS)

    Caledonia, G. E.; Holtzclaw, K. W.; Krech, R. H.; Sonnenfroh, D. M.; Leone, A.; Blumberg, W. A. M.

    1993-01-01

    A series of laboratory measurements have been performed in order to provide a mechanistic interpretation for the visible shuttle glow. These studies involved interactions of an 8 km/s oxygen atom beam with both contaminant dosed surfaces and gaseous targets. We conclude that visible shuttle glow arises from surface mediated O + NO recombination via a Langmuir-Hinshelwood mechanism and that the gas-phase exchange reaction O + N2 - NO + N provides a viable source of precursor NO above surfaces oriented in the ram direction.

  7. Mechanistic investigations of shuttle glow

    SciTech Connect

    Caledonia, G.E.; Holtzclaw, K.W.; Krech, R.H.; Sonnenfroh, D.M. ); Leone, A. ); Blumber, W.A.M. )

    1993-03-01

    A series of laboratory measurements have been performed in order to provide a mechanistic interpretation for the visible shuttle glow. These studies involved interactions in an 8 km/s oxygen atom beam with both contaminant dosed surfaces and gaseous targets. The authors conclude that visible shuttle glow arises from surface mediated O + NO recombination via a Langmuir-Hinshelwood mechanism and that the gas-phase exchange reaction O + N[sub 2] [r arrow] NO + N provides a viable source of precursor NO above surfaces oriented in the ram direction. 35 refs., 4 figs.

  8. Additional Insights on the Bastadins – Isolation of Analogs from the Sponge Ianthella cf. reticulata and Exploration of the Oxime Configurations†

    PubMed Central

    Calcul, Laurent; Inman, Wayne D.; Morris, Alexi A.; Tenney, Karen; Ratnam, Joseline; McKerrow, James H.; Valeriote, Frederick A.; Crews, Phillip

    2015-01-01

    The focus of this study was on the bastadin class of bromotyrosine derivatives, commonly isolated from Ianthella marine sponges, and is the first report on the secondary metabolites from Ianthella cf. reticulata. Two new bastadins were isolated, (E,Z)-bastadin 19 (1b), a diastereoisomer of the known (E,E)-bastadin 19 (1a), and dioxepine bastadin 3 (2), an unusual dibenzo-1,3-dioxepine. A bastadin NMR database was created and assisted in the structure determination of 1b, 2 and the rapid dereplication of ten other known compounds including bastadins 2–9 (3-10), 13 (11) and 19 (1a). The geometry of the 2-(hydroxyimino)-N-alkylamide chains, a chemical feature present in all bastadins, was further probed and new insights regarding the natural oxime configuration are discussed. Bastadins possessing (E,Z), (Z,E) or (E,E)-dioxime configurations could be artifacts of isolation or storage in solution. Therefore, this point was explored by photochemical and thermal isomerization studies, as well as molecular mechanics calculations. Bastadins 13 (11) and 19 (1a) exhibited moderate inhibition against Trypanosoma brucei and bastadin 4 (5) was cytotoxic to HCT-116 colon cancer cells. PMID:20102170

  9. Insight into self-discharge of layered lithium-rich oxide cathode in carbonate-based electrolytes with and without additive

    NASA Astrophysics Data System (ADS)

    Li, Jianhui; Xing, Lidan; Zhang, Liping; Yu, Le; Fan, Weizhen; Xu, Mengqing; Li, Weishan

    2016-08-01

    Self-discharge behavior of layered lithium-rich oxide as cathode of lithium ion battery in a carbonated-based electrolyte is understood, and a simple boron-containing compound, trimethyl borate (TMB), is used as an electrolyte additive to suppress this self-discharge. It is found that layered lithium-rich oxide charged under 4.8 V in additive-free electrolyte suffers severe self-discharge and TMB is an effective electrolyte additive for self-discharge suppression. Physical characterizations from XRD, SEM, TEM, XPS and ICP-MS demonstrate that the crystal structure of the layered lithium-rich oxide collapses due to the chemical interaction between the charged oxide and electrolyte. When TMB is applied, the structural integrity of the oxide is maintained due to the protective cathode film generated from the preferential oxidation of TMB.

  10. Mechanistic investigations reveal that dibromobimane extrudes sulfur from biological sulfhydryl sources other than hydrogen sulfide† †Electronic supplementary information (ESI) available: Experimental details, pH stability data for BTE, NMR spectra. See DOI: 10.1039/c4sc01875c Click here for additional data file.

    PubMed Central

    Montoya, Leticia A.; Shen, Xinggui; McDermott, James J.

    2015-01-01

    Hydrogen sulfide (H2S) has emerged as an important biological signaling molecule in the last decade. During the growth of this field, significant controversy has arisen centered on the physiological concentrations of H2S. Recently, a monobromobimane (mBB) method has been developed for the quantification of different biologically-relevant sulfide pools. Based on the prevalence of the mBB method for sulfide quantification, we expand on this method to report the use of dibromobimane (dBB) for sulfide quantification. Reaction of H2S with dBB results in formation of highly-fluorescent bimane thioether (BTE), which is readily quantifiable by HPLC. Additionally, the reaction of sulfide with dBB to form BTE is significantly faster than the reaction of sulfide with mBB to form sulfide dibimane. Using the dBB method, BTE levels as low as 0.6 pM can be detected. Upon use of the dBB method in wild-type and CSE–/– mice, however, dBB reports significantly higher sulfide levels than those measured using mBB. Further investigation revealed that dBB is able to extract sulfur from other sulfhydryl sources including thiols. Based on mechanistic studies, we demonstrate that dBB extracts sulfur from thiols with α- or β-hydrogens, thus leading to higher BTE formation than from sulfide alone. Taken together, the dBB method is a highly sensitive method for H2S but is not compatible for use in studies in which other thiols are present. PMID:25632344

  11. HTGR Mechanistic Source Terms White Paper

    SciTech Connect

    Wayne Moe

    2010-07-01

    The primary purposes of this white paper are: (1) to describe the proposed approach for developing event specific mechanistic source terms for HTGR design and licensing, (2) to describe the technology development programs required to validate the design methods used to predict these mechanistic source terms and (3) to obtain agreement from the NRC that, subject to appropriate validation through the technology development program, the approach for developing event specific mechanistic source terms is acceptable

  12. Genome-wide analysis of BMI in adolescents and young adults reveals additional insight into the effects of genetic loci over the life course

    PubMed Central

    Graff, Mariaelisa; Ngwa, Julius S.; Workalemahu, Tsegaselassie; Homuth, Georg; Schipf, Sabine; Teumer, Alexander; Völzke, Henry; Wallaschofski, Henri; Abecasis, Goncalo R.; Edward, Lakatta; Francesco, Cucca; Sanna, Serena; Scheet, Paul; Schlessinger, David; Sidore, Carlo; Xiao, Xiangjun; Wang, Zhaoming; Chanock, Stephen J.; Jacobs, Kevin B.; Hayes, Richard B.; Hu, Frank; Van Dam, Rob M.; Crout, Richard J.; Marazita, Mary L.; Shaffer, John R; Atwood, Larry D.; Fox, Caroline S.; Heard-Costa, Nancy L.; White, Charles; Choh, Audrey C.; Czerwinski, Stefan A.; Demerath, Ellen W.; Dyer, Thomas D.; Towne, Bradford; Amin, Najaf; Oostra, Ben A.; Van Duijn, Cornelia M.; Zillikens, M. Carola; Esko, Tõnu; Nelis, Mari; Nikopensius, Tit; Metspalu, Andres; Strachan, David P.; Monda, Keri; Qi, Lu; North, Kari E.; Cupples, L. Adrienne; Gordon-Larsen, Penny; Berndt, Sonja I.

    2013-01-01

    Genetic loci for body mass index (BMI) in adolescence and young adulthood, a period of high risk for weight gain, are understudied, yet may yield important insight into the etiology of obesity and early intervention. To identify novel genetic loci and examine the influence of known loci on BMI during this critical time period in late adolescence and early adulthood, we performed a two-stage meta-analysis using 14 genome-wide association studies in populations of European ancestry with data on BMI between ages 16 and 25 in up to 29 880 individuals. We identified seven independent loci (P < 5.0 × 10−8) near FTO (P = 3.72 × 10−23), TMEM18 (P = 3.24 × 10−17), MC4R (P = 4.41 × 10−17), TNNI3K (P = 4.32 × 10−11), SEC16B (P = 6.24 × 10−9), GNPDA2 (P = 1.11 × 10−8) and POMC (P = 4.94 × 10−8) as well as a potential secondary signal at the POMC locus (rs2118404, P = 2.4 × 10−5 after conditioning on the established single-nucleotide polymorphism at this locus) in adolescents and young adults. To evaluate the impact of the established genetic loci on BMI at these young ages, we examined differences between the effect sizes of 32 published BMI loci in European adult populations (aged 18–90) and those observed in our adolescent and young adult meta-analysis. Four loci (near PRKD1, TNNI3K, SEC16B and CADM2) had larger effects and one locus (near SH2B1) had a smaller effect on BMI during adolescence and young adulthood compared with older adults (P < 0.05). These results suggest that genetic loci for BMI can vary in their effects across the life course, underlying the importance of evaluating BMI at different ages. PMID:23669352

  13. Natural selection and mechanistic regularity.

    PubMed

    DesAutels, Lane

    2016-06-01

    In this article, I address the question of whether natural selection operates regularly enough to qualify as a mechanism of the sort characterized by Machamer, Darden, and Craver (2000). Contrary to an influential critique by Skipper and Millstein (2005), I argue that natural selection can be seen to be regular enough to qualify as an MDC mechanism just fine-as long as we pay careful attention to some important distinctions regarding mechanistic regularity and abstraction. Specifically, I suggest that when we distinguish between process vs. product regularity, mechanism-internal vs. mechanism-external sources of irregularity, and abstract vs. concrete regularity, we can see that natural selection is only irregular in senses that are unthreatening to its status as an MDC mechanism. PMID:26921876

  14. Palladium(0)/NHC-Catalyzed Reductive Heck Reaction of Enones: A Detailed Mechanistic Study.

    PubMed

    Raoufmoghaddam, Saeed; Mannathan, Subramaniyan; Minnaard, Adriaan J; de Vries, Johannes G; Reek, Joost N H

    2015-12-14

    We have studied the mechanism of the palladium-catalyzed reductive Heck reaction of para-substituted enones with 4-iodoanisole by using N,N-diisopropylethylamine (DIPEA) as the reductant. Kinetic studies and in situ spectroscopic analysis have provided a detailed insight into the reaction. Progress kinetic analysis demonstrated that neither catalyst decomposition nor product inhibition occurred during the catalysis. The reaction is first order in the palladium and aryl iodide, and zero order in the activated alkene, N-heterocyclic carbene (NHC) ligand, and DIPEA. The experiments with deuterated solvent ([D7]DMF) and deuterated base ([D15]Et3N) supported the role of the amine as a reductant in the reaction. The palladium complex [Pd(0)(NHC)(1)] has been identified as the resting state. The kinetic experiments by stopped-flow UV/Vis also revealed that the presence of the second substrate, benzylideneacetone 1, slows down the oxidative addition of 4-iodoanisole through its competing coordination to the palladium center. The kinetic and mechanistic studies indicated that the oxidative addition of the aryl iodide is the rate-determining step. Various scenarios for the oxidative addition step have been analyzed by using DFT calculations (bp86/def2-TZVP) that supported the inhibiting effect of substrate 1 by formation of resting state [Pd(0)(NHC)(1)] species at the cost of further increase in the energy barrier of the oxidative addition step. PMID:26561034

  15. Rational and Mechanistic Perspectives on Reinforcement Learning

    ERIC Educational Resources Information Center

    Chater, Nick

    2009-01-01

    This special issue describes important recent developments in applying reinforcement learning models to capture neural and cognitive function. But reinforcement learning, as a theoretical framework, can apply at two very different levels of description: "mechanistic" and "rational." Reinforcement learning is often viewed in mechanistic terms--as…

  16. Bridging Mechanistic and Phenomenological Models of Complex Biological Systems

    PubMed Central

    Transtrum, Mark K.; Qiu, Peng

    2016-01-01

    The inherent complexity of biological systems gives rise to complicated mechanistic models with a large number of parameters. On the other hand, the collective behavior of these systems can often be characterized by a relatively small number of phenomenological parameters. We use the Manifold Boundary Approximation Method (MBAM) as a tool for deriving simple phenomenological models from complicated mechanistic models. The resulting models are not black boxes, but remain expressed in terms of the microscopic parameters. In this way, we explicitly connect the macroscopic and microscopic descriptions, characterize the equivalence class of distinct systems exhibiting the same range of collective behavior, and identify the combinations of components that function as tunable control knobs for the behavior. We demonstrate the procedure for adaptation behavior exhibited by the EGFR pathway. From a 48 parameter mechanistic model, the system can be effectively described by a single adaptation parameter τ characterizing the ratio of time scales for the initial response and recovery time of the system which can in turn be expressed as a combination of microscopic reaction rates, Michaelis-Menten constants, and biochemical concentrations. The situation is not unlike modeling in physics in which microscopically complex processes can often be renormalized into simple phenomenological models with only a few effective parameters. The proposed method additionally provides a mechanistic explanation for non-universal features of the behavior. PMID:27187545

  17. Bridging Mechanistic and Phenomenological Models of Complex Biological Systems.

    PubMed

    Transtrum, Mark K; Qiu, Peng

    2016-05-01

    The inherent complexity of biological systems gives rise to complicated mechanistic models with a large number of parameters. On the other hand, the collective behavior of these systems can often be characterized by a relatively small number of phenomenological parameters. We use the Manifold Boundary Approximation Method (MBAM) as a tool for deriving simple phenomenological models from complicated mechanistic models. The resulting models are not black boxes, but remain expressed in terms of the microscopic parameters. In this way, we explicitly connect the macroscopic and microscopic descriptions, characterize the equivalence class of distinct systems exhibiting the same range of collective behavior, and identify the combinations of components that function as tunable control knobs for the behavior. We demonstrate the procedure for adaptation behavior exhibited by the EGFR pathway. From a 48 parameter mechanistic model, the system can be effectively described by a single adaptation parameter τ characterizing the ratio of time scales for the initial response and recovery time of the system which can in turn be expressed as a combination of microscopic reaction rates, Michaelis-Menten constants, and biochemical concentrations. The situation is not unlike modeling in physics in which microscopically complex processes can often be renormalized into simple phenomenological models with only a few effective parameters. The proposed method additionally provides a mechanistic explanation for non-universal features of the behavior. PMID:27187545

  18. An enhanced plant lipidomics method based on multiplexed liquid chromatography-mass spectrometry reveals additional insights into cold- and drought-induced membrane remodeling.

    PubMed

    Tarazona, Pablo; Feussner, Kirstin; Feussner, Ivo

    2015-11-01

    Within the lipidome of plants a few bulk molecular species hamper the detection of the rest, which are present at relatively low levels. In addition, low-abundance species are often masked by numerous isobaric interferences, such as those caused by isoelemental species and isotopologues. This scenario not only means that minor species are underrepresented, but also leads to potential misidentifications and limits the structural information gathered by lipidomics approaches. In order to overcome these limitations we have developed a multiplexed liquid chromatography-mass spectrometry lipidomics platform able to achieve an enhanced coverage of plant lipidomes. The platform is based on a single extraction step followed by a series of ultra-performance liquid chromatography separations. Post-column flow is then directed to both a triple quadrupole analyzer for targeted profiling and a time-of-flight analyzer for accurate mass analysis. As a proof of concept, plants were subjected to cold or drought, which are known to trigger widespread remodeling events in plant cell membranes. Analysis of the leaf lipidome yielded 393 molecular species within 23 different lipid classes. This enhanced coverage allowed us to identify lipid molecular species and even classes that are altered upon stress, allowing hypotheses on role of glycosylinositolphosphoceramides (GIPC), steryl glycosides (SG) and acylated steryl glycosides (ASG) in drought stress to be addressed and confirming the findings from numerous previous studies with a single, wide-ranging lipidomics approach. This extended our knowledge on membrane remodeling during the drought response, integrating sphingolipids and sterol lipids into the current glycerolipid-based model. PMID:26340975

  19. Advancements in the mechanistic understanding of the copper-catalyzed azide–alkyne cycloaddition

    PubMed Central

    2013-01-01

    Summary The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is one of the most broadly applicable and easy-to-handle reactions in the arsenal of organic chemistry. However, the mechanistic understanding of this reaction has lagged behind the plethora of its applications for a long time. As reagent mixtures of copper salts and additives are commonly used in CuAAC reactions, the structure of the catalytically active species itself has remained subject to speculation, which can be attributed to the multifaceted aggregation chemistry of copper(I) alkyne and acetylide complexes. Following an introductory section on common catalyst systems in CuAAC reactions, this review will highlight experimental and computational studies from early proposals to very recent and more sophisticated investigations, which deliver more detailed insights into the CuAAC’s catalytic cycle and the species involved. As diverging mechanistic views are presented in articles, books and online resources, we intend to present the research efforts in this field during the past decade and finally give an up-to-date picture of the currently accepted dinuclear mechanism of CuAAC. Additionally, we hope to inspire research efforts on the development of molecularly defined copper(I) catalysts with defined structural characteristics, whose main advantage in contrast to the regularly used precatalyst reagent mixtures is twofold: on the one hand, the characteristics of molecularly defined, well soluble catalysts can be tuned according to the particular requirements of the experiment; on the other hand, the understanding of the CuAAC reaction mechanism can be further advanced by kinetic studies and the isolation and characterization of key intermediates. PMID:24367437

  20. Advancements in the mechanistic understanding of the copper-catalyzed azide-alkyne cycloaddition.

    PubMed

    Berg, Regina; Straub, Bernd F

    2013-01-01

    The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is one of the most broadly applicable and easy-to-handle reactions in the arsenal of organic chemistry. However, the mechanistic understanding of this reaction has lagged behind the plethora of its applications for a long time. As reagent mixtures of copper salts and additives are commonly used in CuAAC reactions, the structure of the catalytically active species itself has remained subject to speculation, which can be attributed to the multifaceted aggregation chemistry of copper(I) alkyne and acetylide complexes. Following an introductory section on common catalyst systems in CuAAC reactions, this review will highlight experimental and computational studies from early proposals to very recent and more sophisticated investigations, which deliver more detailed insights into the CuAAC's catalytic cycle and the species involved. As diverging mechanistic views are presented in articles, books and online resources, we intend to present the research efforts in this field during the past decade and finally give an up-to-date picture of the currently accepted dinuclear mechanism of CuAAC. Additionally, we hope to inspire research efforts on the development of molecularly defined copper(I) catalysts with defined structural characteristics, whose main advantage in contrast to the regularly used precatalyst reagent mixtures is twofold: on the one hand, the characteristics of molecularly defined, well soluble catalysts can be tuned according to the particular requirements of the experiment; on the other hand, the understanding of the CuAAC reaction mechanism can be further advanced by kinetic studies and the isolation and characterization of key intermediates. PMID:24367437

  1. Data graphs and mechanistic explanation.

    PubMed

    Burnston, Daniel C

    2016-06-01

    It is a widespread assumption in philosophy of science that representations of data are not explanatory-that they are mere stepping stones towards an explanation, such as a representation of a mechanism. I draw on instances of representational and explanatory practice from mammalian chronobiology to suggest that this assumption is unsustainable. In many instances, biologists employ representations of data in explanatory ways that are not reducible to constraints on or evidence for representations of mechanisms. Data graphs are used to represent relationships between quantities across conditions, and often these representations are necessary for explaining particular aspects of the phenomena under study. The benefit of the analysis is two-fold. First, it provides a more accurate account of explanatory practice in broadly mechanistic investigation in biology. Second, it suggests that there is not an explanatorily "fundamental" type of representation in biology. Rather, the practice of explanation consists in the construction of different types of representations and their employment for distinct explanatory purposes. PMID:26871740

  2. Negative mechanistic reasoning in medical intervention assessment.

    PubMed

    Jerkert, Jesper

    2015-12-01

    Traditionally, mechanistic reasoning has been assigned a negligible role in standard EBM (evidence-based medicine) literature, although some recent authors have argued for an upgrade. Even so, the mechanistic reasoning that has received attention has almost exclusively been positive--both in an epistemic sense of claiming that there is a mechanistic chain and in a health-related sense of there being claimed benefits for the patient. Negative mechanistic reasoning has been neglected, both in the epistemic and in the health-related sense. I distinguish three main types of negative mechanistic reasoning and subsume them under a new definition of mechanistic reasoning in the context of assessing medical interventions. This definition is wider than a previous suggestion in the literature. Each negative type corresponds to a range of evidential strengths, and it is argued that there are differences with respect to typical evidential strengths. The variety of negative mechanistic reasoning should be acknowledged in EBM, and it presents a serious challenge to proponents of so-called medical hierarchies of evidence. PMID:26597869

  3. Mechanistic Considerations in the Synthesis of 2-Aryl-Indole Analogues under Bischler-Mohlau Conditions

    PubMed Central

    MacDonough, Matthew T.; Shi, Zhe; Pinney, Kevin G.

    2015-01-01

    Mechanistic insight into the pathway of the Bischler-Mohlau indole formation reaction is provided by isotopic labeling utilizing judicious incorporation of a 13C atom within the α-bromoacetophenone analogue reactant. The resulting rearranged 2-aryl indole, isolated as the major product, located the 13C isotope label at the methine carbon of the fused five-membered heterocyclic ring, which suggested that the mechanistic pathway of cyclization, in this specific example, required two equivalents of the aniline analogue reactant partner and proceeded through an imine intermediate rather than by direct formation of the corresponding 3-aryl indole accompanied by a concomitant 1,2-aryl shift rearrangement. PMID:26973358

  4. Mechanistic chemical perspective of hydrogen sulfide signaling.

    PubMed

    Nagy, Péter

    2015-01-01

    Hydrogen sulfide is now a well-appreciated master regulator in a diverse array of physiological processes. However, as a consequence of the rapid growth of the area, sulfide biology suffers from an increasing number of controversial observations and interpretations. A better understanding of the underlying molecular pathways of sulfide's actions is key to reconcile controversial issues, which calls for rigorous chemical/biochemical investigations. Protein sulfhydration and coordination/redox chemical interactions of sulfide with heme proteins are the two most extensively studied pathways in sulfide biochemistry. These pathways are important mediators of protein functions, generate bioactive sulfide metabolites, contribute to sulfide storage/trafficking and carry antioxidant functions. In addition, inorganic polysulfides, which are oxidative sulfide metabolites, are increasingly recognized as important players in sulfide biology. This chapter provides an overview of our mechanistic perspective on the reactions that govern (i) sulfide's bioavailability (including the delicate enzyme machineries that orchestrate sulfide production and consumption and the roles of the large sulfide-storing pools as biological buffers), (ii) biological significance and mechanisms of persulfide formation (including the reduction of disulfides, condensation with sulfenic acids, oxidation of thiols with polysulfides and radical-mediated pathways), (iii) coordination and redox chemical interactions of sulfide with heme proteins (including cytochrome c oxidase, hemoglobins, myoglobins and peroxidases), and (iv) the chemistry of polysulfides. PMID:25725513

  5. MECHANISTIC INDICATORS OF CHILDHOOD ASTHMA (MICA)

    EPA Science Inventory

    The US Environmental Protection Agency (EPA) is interested in the interplay of environmental and genetic factors on the development and exacerbation of asthma. The Mechanistic Indicators of Childhood Asthma (MICA) study will use exposure measurements and markers of environmental ...

  6. A framework for recognizing mechanistic reasoning in student scientific inquiry

    NASA Astrophysics Data System (ADS)

    Russ, Rosemary S.

    A central ambition of science education reform is to help students develop abilities for scientific inquiry. Education research is thus rightly focused on defining what constitutes "inquiry" and developing tools for assessing it. There has been progress with respect to particular aspects of inquiry, namely student abilities for controlled experimentation and scientific argumentation. However, we suggest that in addition to these frameworks for assessing the structure of inquiry we need frameworks for analyzing the substance of that inquiry. In this work we draw attention to and evaluate the substance of student mechanistic reasoning. Both within the history and philosophy of science and within science education research, scientific inquiry is characterized in part as understanding the causal mechanisms that underlie natural phenomena. The challenge for science education, however, is that there has not been the same progress with respect to making explicit what constitutes mechanistic reasoning as there has been in making explicit other aspects of inquiry. This dissertation attempts to address this challenge. We adapt an account of mechanism in professional research science to develop a framework for reliably recognizing mechanistic reasoning in student discourse. The coding scheme articulates seven specific aspects of mechanistic reasoning and can be used to systematically analyze narrative data for patterns in student thinking. It provides a tool for detecting quality reasoning that may be overlooked by more traditional assessments. We apply the mechanism coding scheme to video and written data from a range of student inquiries, from large group discussions among first grade students to the individual problem solving of graduate students. While the primary result of this work is the coding scheme itself and the finding that it provides a reliable means of analyzing transcript data for evidence of mechanistic thinking, the rich descriptions we develop in each case

  7. Myricetin arrests human telomeric G-quadruplex structure: a new mechanistic approach as an anticancer agent.

    PubMed

    Mondal, Soma; Jana, Jagannath; Sengupta, Pallabi; Jana, Samarjit; Chatterjee, Subhrangsu

    2016-07-19

    The use of small molecules to arrest G-quadruplex structure has become a potential strategy for the development and design of a new class of anticancer therapeutics. We have studied the interaction of myricetin, a plant flavonoid and a putative anticancer agent, with human telomeric G-quadruplex TTAGGG(TTAGGG)3 DNA. Reverse transcription PCR data revealed significant repression in hTERT expression in MCF-7 breast cancer cells upon increasing the concentration of myricetin. Further, we conducted a telomeric repeat amplification protocol assay to confirm the inhibition of telomerase by myricetin. Optical spectroscopic techniques like circular dichroism, UV spectroscopy and fluorescence spectroscopy revealed the formation of a stable myricetin-G-quadruplex complex. The thermodynamic parameters of myricetin-G-quadruplex complex formation, presented through isothermal titration calorimetry studies, indicate the binding process to be thermodynamically favorable. In addition, high resolution NMR spectroscopy in conjunction with molecular dynamics simulation is employed to provide detailed mechanistic insights into the binding in the myricetin-G-quadruplex complex at the atomic level. Our results thus propose a new mode of action of myricetin as an anticancer agent via arresting telomeric G-quadruplex structure. PMID:27249025

  8. Centrosome amplification, chromosomal instability and cancer: mechanistic, clinical and therapeutic issues.

    PubMed

    Cosenza, Marco Raffaele; Krämer, Alwin

    2016-01-01

    Centrosomes, the main microtubule-organizing centers in most animal cells, are of crucial importance for the assembly of a bipolar mitotic spindle and subsequent faithful segregation of chromosomes into two daughter cells. Centrosome abnormalities can be found in virtually all cancer types and have been linked to chromosomal instability (CIN) and tumorigenesis. Although our knowledge on centrosome structure, replication, and amplification has greatly increased within recent years, still only very little is known on nature, causes, and consequences of centrosome aberrations in primary tumor tissues. In this review, we summarize our current insights into the mechanistic link between centrosome aberrations, aneuploidy, CIN and tumorigenesis. Mechanisms of induction and cellular consequences of aneuploidy, tetraploidization and CIN, as well as origin and effects of supernumerary centrosomes will be discussed. In addition, animal models for both CIN and centrosome amplification will be outlined. Finally, we describe approaches to exploit centrosome amplification, aneuploidy and CIN for novel and specific anticancer treatment strategies based on the modulation of chromosome missegregation rates. PMID:26645976

  9. Mechanistic models in computational social science

    NASA Astrophysics Data System (ADS)

    Holme, Petter; Liljeros, Fredrik

    2015-09-01

    Quantitative social science is not only about regression analysis or, in general, data inference. Computer simulations of social mechanisms have an over 60 years long history. They have been used for many different purposes—to test scenarios, to test the consistency of descriptive theories (proof-of-concept models), to explore emergent phenomena, for forecasting, etc. In this essay, we sketch these historical developments, the role of mechanistic models in the social sciences and the influences from the natural and formal sciences. We argue that mechanistic computational models form a natural common ground for social and natural sciences, and look forward to possible future information flow across the social-natural divide.

  10. (Mechanistic examination of organometallic electron transfer reactions: Annual report, 1989)

    SciTech Connect

    Not Available

    1989-01-01

    Our mechanistic examination of electron transfer reactions between organometallic complexes has required data from our stopped-flow infrared spectrophotometer that was constructed in the first year. Our research on organometallic electron transfer reaction mechanisms was recognized by an invitation to the Symposium on Organometallic Reaction Mechanisms at the National ACS meeting in Miami. We have obtained a reasonable understanding of the electron transfer reactions between metal cations and anions and between metal carbonyl anions and metal carbonyl dimers. In addition we have begun to obtain data on the outer sphere electron transfer between metal carbonyl anions and coordination complexes and on reactions involving cluster anions.

  11. [Mechanistic examination of organometallic electron transfer reactions: Annual report, 1989

    SciTech Connect

    Not Available

    1989-12-31

    Our mechanistic examination of electron transfer reactions between organometallic complexes has required data from our stopped-flow infrared spectrophotometer that was constructed in the first year. Our research on organometallic electron transfer reaction mechanisms was recognized by an invitation to the Symposium on Organometallic Reaction Mechanisms at the National ACS meeting in Miami. We have obtained a reasonable understanding of the electron transfer reactions between metal cations and anions and between metal carbonyl anions and metal carbonyl dimers. In addition we have begun to obtain data on the outer sphere electron transfer between metal carbonyl anions and coordination complexes and on reactions involving cluster anions.

  12. Testing mechanistic models of growth in insects.

    PubMed

    Maino, James L; Kearney, Michael R

    2015-11-22

    Insects are typified by their small size, large numbers, impressive reproductive output and rapid growth. However, insect growth is not simply rapid; rather, insects follow a qualitatively distinct trajectory to many other animals. Here we present a mechanistic growth model for insects and show that increasing specific assimilation during the growth phase can explain the near-exponential growth trajectory of insects. The presented model is tested against growth data on 50 insects, and compared against other mechanistic growth models. Unlike the other mechanistic models, our growth model predicts energy reserves per biomass to increase with age, which implies a higher production efficiency and energy density of biomass in later instars. These predictions are tested against data compiled from the literature whereby it is confirmed that insects increase their production efficiency (by 24 percentage points) and energy density (by 4 J mg(-1)) between hatching and the attainment of full size. The model suggests that insects achieve greater production efficiencies and enhanced growth rates by increasing specific assimilation and increasing energy reserves per biomass, which are less costly to maintain than structural biomass. Our findings illustrate how the explanatory and predictive power of mechanistic growth models comes from their grounding in underlying biological processes. PMID:26609084

  13. Exploring Organic Mechanistic Puzzles with Molecular Modeling

    ERIC Educational Resources Information Center

    Horowitz, Gail; Schwartz, Gary

    2004-01-01

    The molecular modeling was used to reinforce more general skills such as deducing and drawing reaction mechanisms, analyzing reaction kinetics and thermodynamics and drawing reaction coordinate energy diagrams. This modeling was done through the design of mechanistic puzzles, involving reactions not familiar to the students.

  14. Mechanistic Indicators of Childhood Asthma (MICA) Study

    EPA Science Inventory

    The Mechanistic Indicators of Childhood Asthma (MICA) Study has been designed to incorporate state-of-the-art technologies to examine the physiological and environmental factors that interact to increase the risk of asthmatic responses. MICA is primarily a clinically-bases obser...

  15. Integrating mechanistic organism--environment interactions into the basic theory of community and evolutionary ecology.

    PubMed

    Baskett, Marissa L

    2012-03-15

    This paper presents an overview of how mechanistic knowledge of organism-environment interactions, including biomechanical interactions of heat, mass and momentum transfer, can be integrated into basic theoretical population biology through mechanistic functional responses that quantitatively describe how organisms respond to their physical environment. Integrating such functional responses into simple community and microevolutionary models allows scaling up of the organism-level understanding from biomechanics both ecologically and temporally. For community models, Holling-type functional responses for predator-prey interactions provide a classic example of the functional response affecting qualitative model dynamics, and recent efforts are expanding analogous models to incorporate environmental influences such as temperature. For evolutionary models, mechanistic functional responses dependent on the environment can serve as fitness functions in both quantitative genetic and game theoretic frameworks, especially those concerning function-valued traits. I present a novel comparison of a mechanistic fitness function based on thermal performance curves to a commonly used generic fitness function, which quantitatively differ in their predictions for response to environmental change. A variety of examples illustrate how mechanistic functional responses enhance model connections to biologically relevant traits and processes as well as environmental conditions and therefore have the potential to link theoretical and empirical studies. Sensitivity analysis of such models can provide biologically relevant insight into which parameters and processes are important to community and evolutionary responses to environmental change such as climate change, which can inform conservation management aimed at protecting response capacity. Overall, the distillation of detailed knowledge or organism-environment interactions into mechanistic functional responses in simple population

  16. Mechanistic fracture criteria for the failure of human cortical bone

    SciTech Connect

    Nalla, Ravi K.; Kinney, John H.; Ritchie, Robert O.

    2002-12-13

    A mechanistic understanding of fracture in human bone is critical to predicting fracture risk associated with age and disease. Despite extensive work, a mechanistic framework for describing how the underlying microstructure affects the failure mode in bone is lacking.

  17. Mechanistic information from analysis of molecular weight distributions of starch.

    PubMed

    Castro, Jeffrey V; Dumas, Céline; Chiou, Herbert; Fitzgerald, Melissa A; Gilbert, Robert G

    2005-01-01

    A methodology is developed for interpreting the molecular weight distributions of debranched amylopectin, based on techniques developed for quantitatively and qualitatively finding mechanistic information from the molecular weight distributions of synthetic polymers. If the only events occurring are random chain growth and stoppage (i.e., the rates are independent of degree of polymerization over the range in question), then the number of chains of degree of polymerization N, P(N), is linear in ln P(N) with a negative slope, where the slope gives the ratio of the stoppage and growth rates. This starting point suggests that mechanistic inferences can be made from a plot of lnP against N. Application to capillary electrophoresis data for the P(N) of debranched starch from across the major taxa, from bacteria (Escherichia coli), green algae (Chlamydomonas reinhardtii), mammals (Bos), and flowering plants (Oryza sativa, rice; Zea mays, maize; Triticum aestivum, wheat; Hordeum vulgare, barley; and Solanum tuberosum, potato), gives insights into the biosynthetic pathways, showing the differences and similarities of the alpha-1,4-glucans produced by the various species. Four characteristic regions for storage starch from the higher plants are revealed: (1) an initial increasing region corresponding to the formation of new branches, (2) a linear ln P region with negative slope, indicating random growth and stoppage, (3) a region corresponding to the formation of the crystalline lamellae and subsequent elongation of chains, and (4) a second linear ln P with negative slope region. Each region can be assigned to specific enzymatic processes in starch synthesis, including determining the ranges of degrees of polymerization which are subject to random and nonrandom processes. PMID:16004469

  18. Mechanistic study of a diazo dye degradation by Soybean Peroxidase

    PubMed Central

    2013-01-01

    Background Enzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published. Results The present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H2O2 concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway. Conclusion We report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H2O2 mediated degradation of the phenolic Trypan Blue. PMID:23711110

  19. A mechanistic detachment rate model to predict soil erodibility due to fluvial and seepage forces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The erosion rate of cohesive soils is typically computed using an excess shear stress model based on the applied fluvial shear stress. However, no mechanistic approaches are available for incorporating additional forces such as localized groundwater seepage forces into the excess shear stress model...

  20. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    NASA Astrophysics Data System (ADS)

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-01

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  1. A data-intensive approach to mechanistic elucidation applied to chiral anion catalysis

    PubMed Central

    Milo, Anat; Neel, Andrew J.; Toste, F. Dean; Sigman, Matthew S.

    2015-01-01

    Knowledge of chemical reaction mechanisms can facilitate catalyst optimization, but extracting that knowledge from a complex system is often challenging. Here we present a data-intensive method for deriving and then predictively applying a mechanistic model of an enantioselective organic reaction. As a validating case study, we selected an intramolecular dehydrogenative C-N coupling reaction, catalyzed by chiral phosphoric acid derivatives, in which catalyst-substrate association involves weak, non-covalent interactions. Little was previously understood regarding the structural origin of enantioselectivity in this system. Catalyst and substrate substituent effects were probed by systematic physical organic trend analysis. Plausible interactions between the substrate and catalyst that govern enantioselectivity were identified and supported experimentally, indicating that such an approach can afford an efficient means of leveraging mechanistic insight to optimize catalyst design. PMID:25678656

  2. Science insights.

    PubMed

    Tanabe, Kazuyuki

    2015-06-01

    "Below is an essay by Prof. Tanabe originally written in Japanese. It gives an insight to Prof. Tanabe's inquiring mind and his approach to science. He also seek, as always, to inspire and nudge the young to scientific discovery". PMID:25463310

  3. MECHANISTIC STUDIES OF IMPROVED FOAM EOR PROCESSES

    SciTech Connect

    William R. Rossen

    2003-03-31

    . We find that such behavior is consistent with earlier models of foam viscosity in tubes, and a modified model for the low-quality regime can account for this behavior. It is not yet clear why this new regime appears in some cases and not in others. Simple modeling suggests that the answer may have to do with the sensitivity of gas trapping to pressure gradient. Research on Task 3 continued to focus on foam generation at limited pressure gradient in sandpacks. We investigated the effects of permeability, surfactant concentration and liquid injection rates on foam generation. In addition, a careful review of published studies showed that repeated snap-off is not a plausible mechanism of foam generation in homogeneous porous media beyond the stage of initial drainage from a fully liquid-saturated state. Snap-off has been the focus of much research on foam generation and is incorporated into most mechanistic foam simulators. This finding should force a reconsideration of its role in foam generation and properties in porous media.

  4. Mechanistic facility safety and source term analysis

    SciTech Connect

    PLYS, M.G.

    1999-06-09

    A PC-based computer program was created for facility safety and source term analysis at Hanford The program has been successfully applied to mechanistic prediction of source terms from chemical reactions in underground storage tanks, hydrogen combustion in double contained receiver tanks, and proccss evaluation including the potential for runaway reactions in spent nuclear fuel processing. Model features include user-defined facility room, flow path geometry, and heat conductors, user-defined non-ideal vapor and aerosol species, pressure- and density-driven gas flows, aerosol transport and deposition, and structure to accommodate facility-specific source terms. Example applications are presented here.

  5. Mechanistic studies of carbon monoxide reduction

    SciTech Connect

    Geoffroy, G.L.

    1990-06-12

    The progress made during the current grant period (1 January 1988--1 April 1990) in three different areas of research is summarized. The research areas are: (1) oxidatively-induced double carbonylation reactions to form {alpha}-ketoacyl complexes and studies of the reactivity of the resulting compounds, (2) mechanistic studies of the carbonylation of nitroaromatics to form isocyanates, carbamates, and ureas, and (3) studies of the formation and reactivity of unusual metallacycles and alkylidene ligands supported on binuclear iron carbonyl fragments. 18 refs., 5 figs., 1 tab.

  6. Composite Nanomechanics: A Mechanistic Properties Prediction

    NASA Technical Reports Server (NTRS)

    Chamis, Christos C.; Handler, Louis M.; Manderscheid, Jane M.

    2007-01-01

    A unique mechanistic theory is described to predict the properties of nanocomposites. The theory is based on composite micromechanics with progressive substructuring down to a nanoscale slice of a nanofiber where all the governing equations are formulated. These equations hav e been programmed in a computer code. That computer code is used to predict 25 properties of a mononanofiber laminate. The results are pr esented graphically and discussed with respect to their practical sig nificance. Most of the results show smooth distributions. Results for matrix-dependent properties show bimodal through-the-thickness distr ibution with discontinuous changes from mode to mode.

  7. Composite Nanomechanics: A Mechanistic Properties Prediction

    NASA Technical Reports Server (NTRS)

    Chamis, Christos C.; Handler, Louis M.; Manderscheid, Jane M.

    2007-01-01

    A unique mechanistic theory is described to predict the properties of nanocomposites. The theory is based on composite micromechanics with progressive substructuring down to a nanoscale slice of a nanofiber where all the governing equations are formulated. These equations have been programmed in a computer code. That computer code is used to predict 25 properties of a mononanofiber laminate. The results are presented graphically and discussed with respect to their practical significance. Most of the results show smooth distributions. Results for matrix-dependent properties show bimodal through-the-thickness distribution with discontinuous changes from mode to mode.

  8. Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B from Amphitrite ornata†

    PubMed Central

    D’Antonio, Jennifer; D’Antonio, Edward L.; Thompson, Matthew K.; Bowden, Edmond F.; Franzen, Stefan; Smirnova, Tatyana; Ghiladi, Reza A.

    2010-01-01

    Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system. PMID:20545299

  9. A mechanistic stochastic framework for regulating bacterial cell division

    PubMed Central

    Ghusinga, Khem Raj; Vargas-Garcia, Cesar A.; Singh, Abhyudai

    2016-01-01

    How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size. PMID:27456660

  10. Equation-free mechanistic ecosystem forecasting using empirical dynamic modeling

    PubMed Central

    Ye, Hao; Beamish, Richard J.; Glaser, Sarah M.; Grant, Sue C. H.; Hsieh, Chih-hao; Richards, Laura J.; Schnute, Jon T.; Sugihara, George

    2015-01-01

    It is well known that current equilibrium-based models fall short as predictive descriptions of natural ecosystems, and particularly of fisheries systems that exhibit nonlinear dynamics. For example, model parameters assumed to be fixed constants may actually vary in time, models may fit well to existing data but lack out-of-sample predictive skill, and key driving variables may be misidentified due to transient (mirage) correlations that are common in nonlinear systems. With these frailties, it is somewhat surprising that static equilibrium models continue to be widely used. Here, we examine empirical dynamic modeling (EDM) as an alternative to imposed model equations and that accommodates both nonequilibrium dynamics and nonlinearity. Using time series from nine stocks of sockeye salmon (Oncorhynchus nerka) from the Fraser River system in British Columbia, Canada, we perform, for the the first time to our knowledge, real-data comparison of contemporary fisheries models with equivalent EDM formulations that explicitly use spawning stock and environmental variables to forecast recruitment. We find that EDM models produce more accurate and precise forecasts, and unlike extensions of the classic Ricker spawner–recruit equation, they show significant improvements when environmental factors are included. Our analysis demonstrates the strategic utility of EDM for incorporating environmental influences into fisheries forecasts and, more generally, for providing insight into how environmental factors can operate in forecast models, thus paving the way for equation-free mechanistic forecasting to be applied in management contexts. PMID:25733874

  11. Equation-free mechanistic ecosystem forecasting using empirical dynamic modeling.

    PubMed

    Ye, Hao; Beamish, Richard J; Glaser, Sarah M; Grant, Sue C H; Hsieh, Chih-Hao; Richards, Laura J; Schnute, Jon T; Sugihara, George

    2015-03-31

    It is well known that current equilibrium-based models fall short as predictive descriptions of natural ecosystems, and particularly of fisheries systems that exhibit nonlinear dynamics. For example, model parameters assumed to be fixed constants may actually vary in time, models may fit well to existing data but lack out-of-sample predictive skill, and key driving variables may be misidentified due to transient (mirage) correlations that are common in nonlinear systems. With these frailties, it is somewhat surprising that static equilibrium models continue to be widely used. Here, we examine empirical dynamic modeling (EDM) as an alternative to imposed model equations and that accommodates both nonequilibrium dynamics and nonlinearity. Using time series from nine stocks of sockeye salmon (Oncorhynchus nerka) from the Fraser River system in British Columbia, Canada, we perform, for the the first time to our knowledge, real-data comparison of contemporary fisheries models with equivalent EDM formulations that explicitly use spawning stock and environmental variables to forecast recruitment. We find that EDM models produce more accurate and precise forecasts, and unlike extensions of the classic Ricker spawner-recruit equation, they show significant improvements when environmental factors are included. Our analysis demonstrates the strategic utility of EDM for incorporating environmental influences into fisheries forecasts and, more generally, for providing insight into how environmental factors can operate in forecast models, thus paving the way for equation-free mechanistic forecasting to be applied in management contexts. PMID:25733874

  12. Mechanistic Perspectives of Maslinic Acid in Targeting Inflammation

    PubMed Central

    Yap, Wei Hsum; Lim, Yang Mooi

    2015-01-01

    Chronic inflammation drives the development of various pathological diseases such as rheumatoid arthritis, atherosclerosis, multiple sclerosis, and cancer. The arachidonic acid pathway represents one of the major mechanisms for inflammation. Prostaglandins (PGs) are lipid products generated from arachidonic acid by the action of cyclooxygenase (COX) enzymes and their activity is blocked by nonsteroidal anti-inflammatory drugs (NSAIDS). The use of natural compounds in regulation of COX activity/prostaglandins production is receiving increasing attention. In Mediterranean diet, olive oil and table olives contain significant dietary sources of maslinic acid. Maslinic acid is arising as a safe and novel natural pentacyclic triterpene which has protective effects against chronic inflammatory diseases in various in vivo and in vitro experimental models. Understanding the anti-inflammatory mechanism of maslinic acid is crucial for its development as a potential dietary nutraceutical. This review focuses on the mechanistic action of maslinic acid in regulating the inflammation pathways through modulation of the arachidonic acid metabolism including the nuclear factor-kappa B (NF-κB)/COX-2 expression, upstream protein kinase signaling, and phospholipase A2 enzyme activity. Further investigations may provide insight into the mechanism of maslinic acid in regulating the molecular targets and their associated pathways in response to specific inflammatory stimuli. PMID:26491566

  13. A mechanistic stochastic framework for regulating bacterial cell division.

    PubMed

    Ghusinga, Khem Raj; Vargas-Garcia, Cesar A; Singh, Abhyudai

    2016-01-01

    How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size. PMID:27456660

  14. Phylogenetic structure illuminates the mechanistic role of environmental heterogeneity in community organization.

    PubMed

    Stevens, Richard D; Gavilanez, Maria M; Tello, Juan S; Ray, David A

    2012-03-01

    1. Diversity begets diversity. Numerous published positive correlations between environmental heterogeneity and species diversity indicate ubiquity of this phenomenon. Nonetheless, most assessments of this relationship are phenomenological and provide little insight into the mechanism whereby such positive association results. 2. Two unresolved issues could better illuminate the mechanistic basis to diversity begets diversity. First, as environmental heterogeneity increases, both productivity and the species richness that contributes to that productivity often increase in a correlated fashion thus obscuring the primary driver. Second, it is unclear how species are added to communities as diversity increases and whether additions are trait based. 3. We examined these issues based on 31 rodent communities in the central Mojave Desert. At each site, we estimated rodent species richness and characterized environmental heterogeneity from the perspectives of standing primary productivity and number of seed resources. We further examined the phylogenetic structure of communities by estimating the mean phylogenetic distance (MPD) among species and by comparing empirical phylogenetic distances to those based on random assembly from a regional species pool. 4. The relationship between rodent species diversity and environmental heterogeneity was positive and significant. Moreover, diversity of resources accounted for more unique variation than did total productivity, suggesting that variety and not total amount of resource was the driver of increased rodent diversity. Relationships between environmental heterogeneity and phylogenetic distance were negative and significant; species were significantly phylogenetically over-dispersed in communities of low environmental heterogeneity and became more clumped as environmental heterogeneity increased. 5. Results suggest that species diversity increases with environmental heterogeneity because a wider variety of resources allow

  15. Food additives

    MedlinePlus

    Food additives are substances that become part of a food product when they are added during the processing or making of that food. "Direct" food additives are often added during processing to: Add nutrients ...

  16. Appropriateness of mechanistic and non-mechanistic models for the application of ultrafiltration to mixed waste

    SciTech Connect

    Foust, Henry; Ghosehajra, Malay

    2007-07-01

    This study asks two questions: (1) How appropriate is the use of a basic filtration equation to the application of ultrafiltration of mixed waste, and (2) How appropriate are non-parametric models for permeate rates (volumes)? To answer these questions, mechanistic and non-mechanistic approaches are developed for permeate rates and volumes associated with an ultrafiltration/mixed waste system in dia-filtration mode. The mechanistic approach is based on a filtration equation which states that t/V vs. V is a linear relationship. The coefficients associated with this linear regression are composed of physical/chemical parameters of the system and based the mass balance equation associated with the membrane and associated developing cake layer. For several sets of data, a high correlation is shown that supports the assertion that t/V vs. V is a linear relationship. It is also shown that non-mechanistic approaches, i.e., the use of regression models to are not appropriate. One models considered is Q(p) = a*ln(Cb)+b. Regression models are inappropriate because the scale-up from a bench scale (pilot scale) study to full-scale for permeate rates (volumes) is not simply the ratio of the two membrane surface areas. (authors)

  17. Mechanistic models of biofilm growth in porous media

    NASA Astrophysics Data System (ADS)

    Jaiswal, Priyank; Al-Hadrami, Fathiya; Atekwana, Estella A.; Atekwana, Eliot A.

    2014-07-01

    Nondestructive acoustics methods can be used to monitor in situ biofilm growth in porous media. In practice, however, acoustic methods remain underutilized due to the lack of models that can translate acoustic data into rock properties in the context of biofilm. In this paper we present mechanistic models of biofilm growth in porous media. The models are used to quantitatively interpret arrival times and amplitudes recorded in the 29 day long Davis et al. (2010) physical scale biostimulation experiment in terms of biofilm morphologies and saturation. The model pivots on addressing the sediment elastic behavior using the lower Hashin-Shtrikman bounds for grain mixing and Gassmann substitution for fluid saturation. The time-lapse P wave velocity (VP; a function of arrival times) is explained by a combination of two rock models (morphologies); "load bearing" which assumes the biofilm as an additional mineral in the rock matrix and "pore filling" which assumes the biofilm as an additional fluid phase in the pores. The time-lapse attenuation (QP-1; a function of amplitudes), on the other hand, can be explained adequately in two ways; first, through squirt flow where energy is lost from relative motion between rock matrix and pore fluid, and second, through an empirical function of porosity (φ), permeability (κ), and grain size. The squirt flow model-fitting results in higher internal φ (7% versus 5%) and more oblate pores (0.33 versus 0.67 aspect ratio) for the load-bearing morphology versus the pore-filling morphology. The empirical model-fitting results in up to 10% increase in κ at the initial stages of the load-bearing morphology. The two morphologies which exhibit distinct mechanical and hydraulic behavior could be a function of pore throat size. The biofilm mechanistic models developed in this study can be used for the interpretation of seismic data critical for the evaluation of biobarriers in bioremediation, microbial enhanced oil recovery, and CO2

  18. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  19. Structural and Mechanistic Roles of Novel Chemical Ligands on the SdiA Quorum-Sensing Transcription Regulator

    DOE PAGESBeta

    Nguyen, Y.; Nguyen, Nam X.; Rogers, Jamie L.; Liao, Jun; MacMillan, John B.; Jiang, Youxing; Sperandio, Vanessa

    2015-05-19

    Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not inmore » an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment. IMPORTANCE Quorum sensing refers to bacterial chemical signaling. The QS acyl homoserine lactone (AHL) signals are recognized by LuxR-type receptors that regulate gene transcription. However, some bacteria have orphan Lux

  20. Structural and Mechanistic Roles of Novel Chemical Ligands on the SdiA Quorum-Sensing Transcription Regulator

    SciTech Connect

    Nguyen, Y.; Nguyen, Nam X.; Rogers, Jamie L.; Liao, Jun; MacMillan, John B.; Jiang, Youxing; Sperandio, Vanessa

    2015-05-19

    Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not in an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment. IMPORTANCE Quorum sensing refers to bacterial chemical signaling. The QS acyl homoserine lactone (AHL) signals are recognized by LuxR-type receptors that regulate gene transcription. However, some bacteria have orphan LuxR-type receptors and

  1. GLUT, SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters.

    PubMed

    Deng, Dong; Yan, Nieng

    2016-03-01

    Glucose is the primary fuel to life on earth. Cellular uptake of glucose is a fundamental process for metabolism, growth, and homeostasis. Three families of secondary glucose transporters have been identified in human, including the major facilitator superfamily glucose facilitators GLUTs, the sodium-driven glucose symporters SGLTs, and the recently identified SWEETs. Structures of representative members or their prokaryotic homologs of all three families were obtained. This review focuses on the recent advances in the structural elucidation of the glucose transporters and the mechanistic insights derived from these structures, including the molecular basis for substrate recognition, alternating access, and stoichiometric coupling of co-transport. PMID:26650681

  2. Electron beam deposition for nanofabrication: Insights from surface science

    NASA Astrophysics Data System (ADS)

    Wnuk, J. D.; Rosenberg, S. G.; Gorham, J. M.; van Dorp, W. F.; Hagen, C. W.; Fairbrother, D. H.

    2011-02-01

    Electron beam induced deposition (EBID) is a direct-write lithographic technique that utilizes the dissociation of volatile precursors by a focused electron beam in a low vacuum environment to create nanostructures. Notable advantages of EBID over competing lithographic techniques are that it is a single step process that allows three-dimensional free-standing structures to be created, including features with single-nanometer scale dimensions. However, despite the inherent advantages of EBID, scientific and technological issues are impeding its development as an industrial nanofabrication tool. Perhaps the greatest single limitation of EBID is that metal-containing nanostructures deposited from organometallic precursors typically possess unacceptable levels of organic contamination which adversely affects the material's properties. In addition to the issue of purity, there is also a lack of understanding and quantitative information on the fundamental surface reactions and reaction cross-sections that are responsible for EBID. In this prospective, we describe how surface analytical techniques have begun to provide mechanistic and kinetic insights into the molecular level processes associated with EBID. This has been achieved by observing the effect of electron irradiation on nanometer thick films of organometallic precursors adsorbed onto solid substrates at low temperatures (< 200 K) under ultra-high vacuum conditions. Experimental observations include probing changes in surface composition, metal oxidation state, and the evolution of volatile species. Insights into surface reactions associated with purification strategies are also detailed. We also discuss unresolved scientific challenges and opportunities for future EBID research.

  3. Structural and functional analyses of the archaeal tRNA m2G/m22G10 methyltransferase aTrm11 provide mechanistic insights into site specificity of a tRNA methyltransferase that contains common RNA-binding modules.

    PubMed

    Hirata, Akira; Nishiyama, Seiji; Tamura, Toshihiro; Yamauchi, Ayano; Hori, Hiroyuki

    2016-07-27

    N(2)-methylguanosine is one of the most universal modified nucleosides required for proper function in transfer RNA (tRNA) molecules. In archaeal tRNA species, a specific S-adenosyl-L-methionine (SAM)-dependent tRNA methyltransferase (MTase), aTrm11, catalyzes formation of N(2)-methylguanosine and N(2),N(2)-dimethylguanosine at position 10. Here, we report the first X-ray crystal structures of aTrm11 from Thermococcus kodakarensis (Tko), of the apo-form, and of its complex with SAM. The structures show that TkoTrm11 consists of three domains: an N-terminal ferredoxinlike domain (NFLD), THUMP domain and Rossmann-fold MTase (RFM) domain. A linker region connects the THUMP-NFLD and RFM domains. One SAM molecule is bound in the pocket of the RFM domain, suggesting that TkoTrm11 uses a catalytic mechanism similar to that of other tRNA MTases containing an RFM domain. Furthermore, the conformation of NFLD and THUMP domains in TkoTrm11 resembles that of other tRNA-modifying enzymes specifically recognizing the tRNA acceptor stem. Our docking model of TkoTrm11-SAM in complex with tRNA, combined with biochemical analyses and pre-existing evidence, provides insights into the substrate tRNA recognition mechanism: The THUMP domain recognizes a 3'-ACCA end, and the linker region and RFM domain recognize the T-stem, acceptor stem and V-loop of tRNA, thereby causing TkoTrm11 to specifically identify its methylation site. PMID:27325738

  4. Progress in the Experimental Observation of Thiamin Diphosphate-bound Intermediates on Enzymes and Mechanistic Information Derived from these Observations

    PubMed Central

    Jordan, Frank; Nemeria, Natalia S.

    2014-01-01

    Thiamin diphosphate (ThDP), the vitamin B1 coenzyme is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/carbene/C2-carbanion of the thiazolium ring and the C2α-carbanion/enamine, once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of a several intermediates which are stable under acidic conditions, solid-state NMR and circular dichroism detection of the states of ionization and tautomerization of the 4′-aminopyrimidine moiety of ThDP in some of the intermediates. These methods also enabled in some cases determination of the rate-limiting step in the complex series of steps. This review is an update of a review with the same title published by the authors in 2005 in this Journal. Much progress has been made in the intervening decade in the identification of the intermediates and their application to gain additional mechanistic insight. PMID:15888311

  5. MECHANISTIC STUDIES OF IMPROVED FOAM EOR PROCESSES

    SciTech Connect

    William R. Rossen

    2004-06-14

    The objective of this research is to widen the application of foam to enhanced oil recovery (EOR) by investigating fundamental mechanisms of foams in porous media. This research will lay the groundwork for more applied research on foams for improved sweep efficiency in miscible gas, steam and surfactant-based EOR. Task 1 investigates the pore-scale interactions between foam bubbles and polymer molecules. Task 2 examines the mechanisms of gas trapping, and interaction between gas trapping and foam effectiveness. Task 3 investigates mechanisms of foam generation in porous media. Significant progress was made during this period on all three Tasks. Regarding Task 1, we continued comparisons of foam behavior in sandpacks with and without polymer and oil. As in our previous results, decane was moderately destabilizing to foam. Xanthan polymer did not stabilize foam in the presence of decane in this case. Rather, it appears to have destabilized foam, so that pressure gradient decreased in spite of the increase in aqueous-phase viscosity. Research on Task 2 included the first shake-down experiments with our new apparatus for gas-phase tracer tests for direct measurement of trapped-gas saturation with foam. In addition, we began to analyze CT images of gas-phase tracer in foam displacements, which offers an independent measure of trapped-gas fraction and insights into the roles of convection of tracer in flowing gas and diffusion into trapped gas. Research on Task 3 included foam generation experiments in heterogeneous sandpacks and beadpacks and modeling of discontinuous changes in state such as foam generation. The experiments found the same three regimes (coarse foam, strong foam, and intermediate regime) in heterogeneous sandpacks previously identified in homogeneous porous media. One implication is that there may be a minimum flow rate required for foam generation in even heterogeneous porous media. The dynamics in SAG foam processes in heterogeneous media are complex

  6. Comparative evaluation of statistical and mechanistic models of Escherichia coli at beaches in southern Lake Michigan

    USGS Publications Warehouse

    Safaie, Ammar; Wendzel, Aaron; Ge, Zhongfu; Nevers, Meredith; Whitman, Richard L.; Corsi, Steven R.; Phanikumar, Mantha S.

    2016-01-01

    Statistical and mechanistic models are popular tools for predicting the levels of indicator bacteria at recreational beaches. Researchers tend to use one class of model or the other, and it is difficult to generalize statements about their relative performance due to differences in how the models are developed, tested, and used. We describe a cooperative modeling approach for freshwater beaches impacted by point sources in which insights derived from mechanistic modeling were used to further improve the statistical models and vice versa. The statistical models provided a basis for assessing the mechanistic models which were further improved using probability distributions to generate high-resolution time series data at the source, long-term “tracer” transport modeling based on observed electrical conductivity, better assimilation of meteorological data, and the use of unstructured-grids to better resolve nearshore features. This approach resulted in improved models of comparable performance for both classes including a parsimonious statistical model suitable for real-time predictions based on an easily measurable environmental variable (turbidity). The modeling approach outlined here can be used at other sites impacted by point sources and has the potential to improve water quality predictions resulting in more accurate estimates of beach closures.

  7. Comparative Evaluation of Statistical and Mechanistic Models of Escherichia coli at Beaches in Southern Lake Michigan.

    PubMed

    Safaie, Ammar; Wendzel, Aaron; Ge, Zhongfu; Nevers, Meredith B; Whitman, Richard L; Corsi, Steven R; Phanikumar, Mantha S

    2016-03-01

    Statistical and mechanistic models are popular tools for predicting the levels of indicator bacteria at recreational beaches. Researchers tend to use one class of model or the other, and it is difficult to generalize statements about their relative performance due to differences in how the models are developed, tested, and used. We describe a cooperative modeling approach for freshwater beaches impacted by point sources in which insights derived from mechanistic modeling were used to further improve the statistical models and vice versa. The statistical models provided a basis for assessing the mechanistic models which were further improved using probability distributions to generate high-resolution time series data at the source, long-term "tracer" transport modeling based on observed electrical conductivity, better assimilation of meteorological data, and the use of unstructured-grids to better resolve nearshore features. This approach resulted in improved models of comparable performance for both classes including a parsimonious statistical model suitable for real-time predictions based on an easily measurable environmental variable (turbidity). The modeling approach outlined here can be used at other sites impacted by point sources and has the potential to improve water quality predictions resulting in more accurate estimates of beach closures. PMID:26825142

  8. Food additives.

    PubMed

    Berglund, F

    1978-01-01

    The use of additives to food fulfils many purposes, as shown by the index issued by the Codex Committee on Food Additives: Acids, bases and salts; Preservatives, Antioxidants and antioxidant synergists; Anticaking agents; Colours; Emulfifiers; Thickening agents; Flour-treatment agents; Extraction solvents; Carrier solvents; Flavours (synthetic); Flavour enhancers; Non-nutritive sweeteners; Processing aids; Enzyme preparations. Many additives occur naturally in foods, but this does not exclude toxicity at higher levels. Some food additives are nutrients, or even essential nutritents, e.g. NaCl. Examples are known of food additives causing toxicity in man even when used according to regulations, e.g. cobalt in beer. In other instances, poisoning has been due to carry-over, e.g. by nitrate in cheese whey - when used for artificial feed for infants. Poisonings also occur as the result of the permitted substance being added at too high levels, by accident or carelessness, e.g. nitrite in fish. Finally, there are examples of hypersensitivity to food additives, e.g. to tartrazine and other food colours. The toxicological evaluation, based on animal feeding studies, may be complicated by impurities, e.g. orthotoluene-sulfonamide in saccharin; by transformation or disappearance of the additive in food processing in storage, e.g. bisulfite in raisins; by reaction products with food constituents, e.g. formation of ethylurethane from diethyl pyrocarbonate; by metabolic transformation products, e.g. formation in the gut of cyclohexylamine from cyclamate. Metabolic end products may differ in experimental animals and in man: guanylic acid and inosinic acid are metabolized to allantoin in the rat but to uric acid in man. The magnitude of the safety margin in man of the Acceptable Daily Intake (ADI) is not identical to the "safety factor" used when calculating the ADI. The symptoms of Chinese Restaurant Syndrome, although not hazardous, furthermore illustrate that the whole ADI

  9. A unique dinuclear mixed V(V) oxo-peroxo complex in the structural speciation of the ternary V(V)-peroxo-citrate system. potential mechanistic and structural insight into the aqueous synthetic chemistry of dinuclear V(V)-citrate species with H2O2.

    PubMed

    Kaliva, M; Gabriel, C; Raptopoulou, C P; Terzis, A; Voyiatzis, G; Zervou, M; Mateescu, C; Salifoglou, A

    2011-11-21

    Diverse vanadium biological activities entail complex interactions with physiological target ligands in aqueous media and constitute the crux of the undertaken investigation at the synthetic level. Facile aqueous redox reactions, as well as nonredox reactions, of V(III) and V(V) with physiological citric acid and hydrogen peroxide, under pH-specific conditions, led to the synthesis and isolation of a well-formed crystalline material upon the addition of ethanol as the precipitating solvent. Elemental analysis pointed to the molecular formulation (NH4)4[(VO2){VO(O2)}(C6H5O7)2]·1.5H2O (1). Complex 1 was further characterized by Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR), Raman spectroscopy, cyclic voltammetry, and X-ray crystallography. The crystallographic structure of 1 reveals the presence of the first dinuclear V(V)-citrate complex with non-peroxo- and peroxo-containing V(V) ions, concurrently present within the basic VV2O2 core. The nonperoxo unit VO2+ and the peroxo unit VO(O2)+ are each coordinated to a triply deprotonated citrate ligand in a distinct coordination mode and coordination geometry around the V(V) ions. These units are similar to those in homodinuclear complexes bearing oxo or peroxo groups. The unique assembly of both units in the anion of 1 renders the latter as a potential intermediate in the peroxidation process, from [V2O4(C6H5O7)2]4– to [V2O2(O2)2(C6H6O7)2]2–. The transformation reactions of 1 establish its connection with several V(V) and V(IV) dinuclear species present in the aqueous distribution of the V(IV,V)-citrate systems. The shown position of 1 as an intermediate in the mechanism of H2O2 addition to dinuclear V(V)-citrate species portends its role in the complex aqueous distribution of species in the ternary V(V)-peroxo-citrate system and its potential reactivity in (bio)chemically relevant media. PMID:22029259

  10. Structural and Mechanistic Aspects of Copper Catalyzed Atom Transfer Radical Polymerization

    NASA Astrophysics Data System (ADS)

    Pintauer, Tomislav; Matyjaszewski, Krzysztof

    During the past decade, atom transfer radical polymerization (ATRP) has had a tremendous impact on the synthesis of macromolecules with well-defined compositions, architectures, and functionalities. Structural features of copper(I) and copper(II) complexes with bidentate, tridentate, tetradentate, and multidentate nitrogen-based ligands commonly utilized in ATRP are reviewed and discussed. Additionally, recent advances in mechanistic understanding of copper-mediated ATRP are outlined.

  11. Mechanistically based mapping of human cardiac fibrillation

    PubMed Central

    Zaman, Junaid A. B.

    2016-01-01

    Abstract The mechanisms underpinning human cardiac fibrillation remain elusive. In his 1913 paper ‘On dynamic equilibrium in the heart’, Mines proposed that an activation wave front could propagate repeatedly in a circle, initiated by a stimulus in the vulnerable period. While the dynamics of activation and recovery are central to cardiac fibrillation, these physiological data are rarely used in clinical mapping. Fibrillation is a rapid irregular rhythm with spatiotemporal disorder resulting from two fundamental mechanisms – sources in preferred cardiac regions or spatially diffuse self‐sustaining activity, i.e. with no preferred source. On close inspection, however, this debate may also reflect mapping technique. Fibrillation is initiated from triggers by regional dispersion in repolarization, slow conduction and wavebreak, then sustained by non‐uniform interactions of these mechanisms. Notably, optical mapping of action potentials in atrial fibrillation (AF) show spiral wave sources (rotors) in nearly all studies including humans, while most traditional electrogram analyses of AF do not. Techniques may diverge in fibrillation because electrograms summate non‐coherent waves within an undefined field whereas optical maps define waves with a visually defined field. Also fibrillation operates at the limits of activation and recovery, which are well represented by action potentials while fibrillatory electrograms poorly represent repolarization. We conclude by suggesting areas for study that may be used, until such time as optical mapping is clinically feasible, to improve mechanistic understanding and therapy of human cardiac fibrillation. PMID:26607671

  12. Mechanistically based mapping of human cardiac fibrillation.

    PubMed

    Narayan, Sanjiv M; Zaman, Junaid A B

    2016-05-01

    The mechanisms underpinning human cardiac fibrillation remain elusive. In his 1913 paper 'On dynamic equilibrium in the heart', Mines proposed that an activation wave front could propagate repeatedly in a circle, initiated by a stimulus in the vulnerable period. While the dynamics of activation and recovery are central to cardiac fibrillation, these physiological data are rarely used in clinical mapping. Fibrillation is a rapid irregular rhythm with spatiotemporal disorder resulting from two fundamental mechanisms - sources in preferred cardiac regions or spatially diffuse self-sustaining activity, i.e. with no preferred source. On close inspection, however, this debate may also reflect mapping technique. Fibrillation is initiated from triggers by regional dispersion in repolarization, slow conduction and wavebreak, then sustained by non-uniform interactions of these mechanisms. Notably, optical mapping of action potentials in atrial fibrillation (AF) show spiral wave sources (rotors) in nearly all studies including humans, while most traditional electrogram analyses of AF do not. Techniques may diverge in fibrillation because electrograms summate non-coherent waves within an undefined field whereas optical maps define waves with a visually defined field. Also fibrillation operates at the limits of activation and recovery, which are well represented by action potentials while fibrillatory electrograms poorly represent repolarization. We conclude by suggesting areas for study that may be used, until such time as optical mapping is clinically feasible, to improve mechanistic understanding and therapy of human cardiac fibrillation. PMID:26607671

  13. Potlining Additives

    SciTech Connect

    Rudolf Keller

    2004-08-10

    In this project, a concept to improve the performance of aluminum production cells by introducing potlining additives was examined and tested. Boron oxide was added to cathode blocks, and titanium was dissolved in the metal pool; this resulted in the formation of titanium diboride and caused the molten aluminum to wet the carbonaceous cathode surface. Such wetting reportedly leads to operational improvements and extended cell life. In addition, boron oxide suppresses cyanide formation. This final report presents and discusses the results of this project. Substantial economic benefits for the practical implementation of the technology are projected, especially for modern cells with graphitized blocks. For example, with an energy savings of about 5% and an increase in pot life from 1500 to 2500 days, a cost savings of $ 0.023 per pound of aluminum produced is projected for a 200 kA pot.

  14. Phosphazene additives

    SciTech Connect

    Harrup, Mason K; Rollins, Harry W

    2013-11-26

    An additive comprising a phosphazene compound that has at least two reactive functional groups and at least one capping functional group bonded to phosphorus atoms of the phosphazene compound. One of the at least two reactive functional groups is configured to react with cellulose and the other of the at least two reactive functional groups is configured to react with a resin, such as an amine resin of a polycarboxylic acid resin. The at least one capping functional group is selected from the group consisting of a short chain ether group, an alkoxy group, or an aryloxy group. Also disclosed are an additive-resin admixture, a method of treating a wood product, and a wood product.

  15. Mechanistic study of secondary organic aerosol components formed from nucleophilic addition reactions of methacrylic acid epoxide

    NASA Astrophysics Data System (ADS)

    Birdsall, A. W.; Miner, C. R.; Mael, L. E.; Elrod, M. J.

    2014-08-01

    Recently, methacrylic acid epoxide (MAE) has been proposed as a precursor to an important class of isoprene-derived compounds found in secondary organic aerosol (SOA): 2-methylglyceric acid (2-MG) and a set of oligomers, nitric acid esters and sulfuric acid esters related to 2-MG. However, the specific chemical mechanisms by which MAE could form these compounds have not been previously studied. In order to determine the relevance of these processes to atmospheric aerosol, MAE and 2-MG have been synthesized and a series of bulk solution-phase experiments aimed at studying the reactivity of MAE using nuclear magnetic resonance (NMR) spectroscopy have been performed. The present results indicate that the acid-catalyzed MAE reaction is more than 600 times slower than a similar reaction of an important isoprene-derived epoxide, but is still expected to be kinetically feasible in the atmosphere on more acidic SOA. The specific mechanism by which MAE leads to oligomers was identified, and the reactions of MAE with a number of atmospherically relevant nucleophiles were also investigated. Because the nucleophilic strengths of water, sulfate, alcohols (including 2-MG), and acids (including MAE and 2-MG) in their reactions with MAE were found to be of a similar magnitude, it is expected that a diverse variety of MAE + nucleophile product species may be formed on ambient SOA. Thus, the results indicate that epoxide chain reaction oligomerization will be limited by the presence of high concentrations of non-epoxide nucleophiles (such as water); this finding is consistent with previous environmental chamber investigations of the relative humidity-dependence of 2-MG-derived oligomerization processes and suggests that extensive oligomerization may not be likely on ambient SOA because of other competitive MAE reaction mechanisms.

  16. Mechanistic study of secondary organic aerosol components formed from nucleophilic addition reactions of methacrylic acid epoxide

    NASA Astrophysics Data System (ADS)

    Birdsall, A. W.; Miner, C. R.; Mael, L. E.; Elrod, M. J.

    2014-12-01

    Recently, methacrylic acid epoxide (MAE) has been proposed as a precursor to an important class of isoprene-derived compounds found in secondary organic aerosol (SOA): 2-methylglyceric acid (2-MG) and a set of oligomers, nitric acid esters, and sulfuric acid esters related to 2-MG. However, the specific chemical mechanisms by which MAE could form these compounds have not been previously studied with experimental methods. In order to determine the relevance of these processes to atmospheric aerosol, MAE and 2-MG have been synthesized and a series of bulk solution-phase experiments aimed at studying the reactivity of MAE using nuclear magnetic resonance (NMR) spectroscopy have been performed. The present results indicate that the acid-catalyzed MAE reaction is more than 600 times slower than a similar reaction of an important isoprene-derived epoxide, but is still expected to be kinetically feasible in the atmosphere on more acidic SOA. The specific mechanism by which MAE leads to oligomers was identified, and the reactions of MAE with a number of atmospherically relevant nucleophiles were also investigated. Because the nucleophilic strengths of water, sulfate, alcohols (including 2-MG), and acids (including MAE and 2-MG) in their reactions with MAE were found to be of similar magnitudes, it is expected that a diverse variety of MAE + nucleophile product species may be formed on ambient SOA. Thus, the results indicate that epoxide chain reaction oligomerization will be limited by the presence of high concentrations of non-epoxide nucleophiles (such as water); this finding is consistent with previous environmental chamber investigations of the relative humidity dependence of 2-MG-derived oligomerization processes and suggests that extensive oligomerization may not be likely on ambient SOA because of other competitive MAE reaction mechanisms.

  17. Black tea polyphenols: a mechanistic treatise.

    PubMed

    Butt, M S; Imran, A; Sharif, M K; Ahmad, Rabia Shabir; Xiao, Hang; Imran, M; Rsool, H A

    2014-01-01

    Dietary interventions are among the emerging trends to curtail physiological malfunctioning like cancer, diabetes, cardiac complications, etc. The essence of phytonutrients has developed the concept of nutraceuticals at the junction of diet health linkages. In this context, theaflavin & thearubigins are the oxidized derivatives of black tea catechins during fermentation having nutraceutical potential owing to esterification of hydroxyl ring with digallate esters. Theaflavin may influence activation of transcription factors such as NFnB or AP-1 that ultimately hinder the formation of nitric oxide expression gene. Likewise, black tea contains a unique amino acid theanine acts as neurotransmitter owing to its ability to cross the blood-brain barrier. Moreover, it boasts immunity by enhancing the disease-fighting ability of gamma delta T cells. Theaflavin & thearubigins act as safeguard against oxidative stress thereby effective in the cardiac functioning. The mechanistic approach of these antioxidants is likely to be associated with inhibition of redox sensitive transcription factors & pro-oxidant enzymes such as xanthine oxidase or nitric oxide synthase. However, their involvement in antioxidative enzyme induction as in glutathione-S-transferases is also well documented. They act as curative agent against numerous pathological disorders by disrupting the electron chain thus inhibiting the progression of certain ailments. Black tea polyphenols established themselves as strong antioxidants due to their standard one-electron potential, and their vitality is dependent on the concentration of polyphenols and pH for their inclusive execution. Present review is an attempt to enrich the readers regarding the health promoting aspects of black tea polyphenols. Concomitantly, it needs core attention of researchers for the exploitations of black tea flavanols as an important dietary constituent for the vulnerable segment. PMID:24499118

  18. Predictive toxicodynamics: Empirical/mechanistic approaches.

    PubMed

    Frazier, J M

    1997-10-01

    A major objective of the toxicological sciences is to predict the in vivo toxicological consequences of human exposure to pure chemicals, complex mixtures and commercial formulations. Historically, the experimental approach to this goal has been to investigate toxicological processes in whole animal models and extrapolate the results obtained to predict human risk using various extrapolation procedures (high-dose/low-dose extrapolation, interspecies extrapolation and route-to-route extrapolation). Can in vitro methods be more widely employed in quantitative risk assessment? One major limitation to the broader application of in vitro toxicity testing methods is the lack of validated techniques for the extrapolation of in vitro-derived toxicodynamic data to the in vivo situation. The objective of this paper is to describe some approaches to the development of techniques to extrapolate in vitro toxicity testing data to predict in vivo toxicological responses. An empirical approach within the context of a mechanistic framework is explored. The basic hypothesis is that the in vivo response can be constructed from a cellular toxicity factor that accounts for the cellular response and a toxicodynamic factor that relates toxicological events at the cellular level to the observable in vivo responses. A predictive paradigm to describe the in vivo acute target organ toxicity (hepatotoxicity) of a model chemical (cadmium) is discussed. The cellular toxicity factor is derived from in vitro toxicity testing studies using isolated rat hepatocytes. The toxicodynamic factor is derived through Biologically-Based Response (BBR) modelling techniques to predict target organ toxicity markers (i.e. plasma hepatic enzyme levels as markers for acute hepatotoxicity). The ultimate goal is to develop validated extrapolation procedures that can be applied to predicting target organ toxicity quantitatively in human populations based on in vitro toxicity studies using human cellular models. PMID

  19. The effect of the physical state of binders on high-shear wet granulation and granule properties: a mechanistic approach to understand the high-shear wet granulation process. part IV. the impact of rheological state and tip-speeds.

    PubMed

    Li, Jinjiang; Tao, Li; Buckley, David; Tao, Jing; Gao, Julia; Hubert, Mario

    2013-12-01

    The purpose of this study is to provide a mechanistic understanding concerning the effect of tip-speed on a granulation at various binder rheological states; the in situ rheological state of a binder was controlled by exposing a granulation blend to 96% relative humidity. This approach allowed us to investigate the impact of tip-speed on granule consolidation coupled with the in situ binder state, which was not possible using a conventional granulation approach. Experimentally, the rheological state of binders was characterized using a rheometer. Granule size and granule porosity were measured by Qicpic instrument and Mercury Intrusion Porosimetry, respectively. For the granulations containing binders at viscous state (PVP K17 and PVP K29/32), the granule size increased significantly with mixing time and the growth rate increased with tip-speed until 5.8 m/s; when binders were at viscoelastic state, tip-speed had no impact on granulation. Furthermore, the granule porosity was higher for granulation with binders at viscoelastic state (HPC and PVP K90), whereas it was lower for granulation with binders at viscous state. In addition, the impeller tip-speed had minimal impact on the porosity of the final granules. Finally, Ennis' model was used for interpreting results, providing mechanistic insights on granulation. PMID:24135976

  20. Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

    PubMed

    Brady, Pamlea N; Macnaughtan, Megan A

    2015-12-15

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. PMID:26342307

  1. Novel colloidal carriers for psoriasis: current issues, mechanistic insight and novel delivery approaches.

    PubMed

    Pradhan, Madhulika; Singh, Deependra; Singh, Manju Rawat

    2013-09-28

    Psoriasis is an autoimmune disorder of the skin with relapsing episodes of inflammation and hyperkeratosis. Numerous approaches have been explored to treat this dreadful disease using different antipsoriatic drugs with different modes of action and routes of administration. But, till date there is no cure for psoriasis due to lack of an ideal carrier for safe and effective delivery of antipsoriatic drugs. Constant progression in the development of newer formulations utilizing colloidal drug delivery systems has led to effective treatment of psoriasis. Colloidal carriers include vesicular and particulate systems like liposome, transferosome, niosomes, ethosomes, solid lipid nanoparticles, microspheres, micelles, dendrimers etc. have gained unique position as drug cargoes. Present review is an attempt to contemplate on psoriasis in terms of pathogenesis, role of cytokines, major hindrances in psoriasis treatment, currently available treatment options pertaining to mode of action, pharmacokinetics, marketed products, side effects of individual antipsoriatic drugs and recent developments in the delivery of various antipsoriatic drugs through novel colloidal drug carriers. PMID:23770117

  2. Oleuropein aglycone induces autophagy via the AMPK/mTOR signalling pathway: a mechanistic insight.

    PubMed

    Rigacci, Stefania; Miceli, Caterina; Nediani, Chiara; Berti, Andrea; Cascella, Roberta; Pantano, Daniela; Nardiello, Pamela; Luccarini, Ilaria; Casamenti, Fiorella; Stefani, Massimo

    2015-11-01

    The healthy effects of plant polyphenols, some of which characterize the so-called Mediterranean diet, have been shown to arise from epigenetic and biological modifications resulting, among others, in autophagy stimulation. Our previous work highlighted the beneficial effects of oleuropein aglycone (OLE), the main polyphenol found in the extra virgin olive oil, against neurodegeneration both in cultured cells and in model organisms, focusing, in particular, autophagy activation. In this study we investigated more in depth the molecular and cellular mechanisms of autophagy induction by OLE using cultured neuroblastoma cells and an OLE-fed mouse model of amylod beta (Aβ) deposition. We found that OLE triggers autophagy in cultured cells through the Ca2+-CAMKKβ-AMPK axis. In particular, in these cells OLE induces a rapid release of Ca2+ from the SR stores which, in turn, activates CAMKKβ, with subsequent phosphorylation and activation of AMPK. The link between AMPK activation and mTOR inhibition was shown in the OLE-fed animal model in which we found that decreased phospho-mTOR immunoreactivity and phosphorylated mTOR substrate p70 S6K levels match enhanced phospho-AMPK levels, supporting the idea that autophagy activation by OLE proceeds through mTOR inhibition. Our results agree with those reported for other plant polyphenols, suggesting a shared molecular mechanism underlying the healthy effects of these substances against ageing, neurodegeneration, cancer, diabetes and other diseases implying autophagy dysfunction. PMID:26474288

  3. Voltage-Gated Sodium Channels: Mechanistic Insights From Atomistic Molecular Dynamics Simulations.

    PubMed

    Oakes, V; Furini, S; Domene, C

    2016-01-01

    The permeation of ions and other molecules across biological membranes is an inherent requirement of all cellular organisms. Ion channels, in particular, are responsible for the conduction of charged species, hence modulating the propagation of electrical signals. Despite the universal physiological implications of this property, the molecular functioning of ion channels remains ambiguous. The combination of atomistic structural data with computational methodologies, such as molecular dynamics (MD) simulations, is now considered routine to investigate structure-function relationships in biological systems. A fuller understanding of conduction, selectivity, and gating, therefore, is steadily emerging due to the applicability of these techniques to ion channels. However, because their structure is known at atomic resolution, studies have consistently been biased toward K(+) channels, thus the molecular determinants of ionic selectivity, activation, and drug blockage in Na(+) channels are often overlooked. The recent increase of available crystallographic data has eminently encouraged the investigation of voltage-gated sodium (NaV) channels via computational methods. Here, we present an overview of simulation studies that have contributed to our understanding of key principles that underlie ionic conduction and selectivity in Na(+) channels, in comparison to the K(+) channel analogs. PMID:27586285

  4. Mechanistic Insights from Vascular and Cardiac Impairments in Rats Inhaling Diesel Exhaust Particles and Ozone

    EPA Science Inventory

    Although the causality has been established between air pollution and cardiovascular impairments, the molecular mechanisms are unknown. Moreover, cardiovascular effects of ozone have not been studied until recently. We hypothesize that vasculature and cardiac tissues are targets ...

  5. Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes

    SciTech Connect

    Arnett, Kelly L.; Hass, Matthew; McArthur, Debbie G.; Ilagan, Ma Xenia G.; Aster, Jon C.; Kopan, Raphael; Blacklow, Stephen C.

    2010-11-12

    Ligand-induced proteolysis of Notch produces an intracellular effector domain that transduces essential signals by regulating the transcription of target genes. This function relies on the formation of transcriptional activation complexes that include intracellular Notch, a Mastermind co-activator and the transcription factor CSL bound to cognate DNA. These complexes form higher-order assemblies on paired, head-to-head CSL recognition sites. Here we report the X-ray structure of a dimeric human Notch1 transcription complex loaded on the paired site from the human HES1 promoter. The small interface between the Notch ankyrin domains could accommodate DNA bending and untwisting to allow a range of spacer lengths between the two sites. Cooperative dimerization occurred on the human and mouse Hes5 promoters at a sequence that diverged from the CSL-binding consensus at one of the sites. These studies reveal how promoter organizational features control cooperativity and, thus, the responsiveness of different promoters to Notch signaling.

  6. Structural and mechanistic insight into DNA unwinding by Deinococcus radiodurans UvrD.

    PubMed

    Stelter, Meike; Acajjaoui, Samira; McSweeney, Sean; Timmins, Joanna

    2013-01-01

    DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3'-5' direction and shows ATP-dependent 3'-5', and surprisingly also, 5'-3' helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD's 3'-5' and 5'-3' helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5'-3' helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo. PMID:24143224

  7. Mechanistic insights into the formation of chloroform from natural organic matter using stable carbon isotope analysis

    NASA Astrophysics Data System (ADS)

    Breider, Florian; Hunkeler, Daniel

    2014-01-01

    Chloroform can be naturally formed in terrestrial environments (e.g. forest soils, peatland) by chlorination of natural organic matter (NOM). Recently, it was demonstrated that natural and anthropogenic chloroform have a distinctly different carbon isotope signature that makes it possible to identify its origin in soil and groundwater. In order to evaluate the contribution of different functional groups to chloroform production and factors controlling the isotopic composition of chloroform, carbon isotope trends during chlorination of model compounds, soil organic matter (SOM) and humic acids were evaluated, and apparent kinetic isotope effects (AKIEs) quantified. Phenol and propanone were selected as model compounds representing common functional groups in NOM. Chlorination was induced by hypochlorous acid to mimic natural chlorination. The pH ranged between 4 and 8 to cover typical soil conditions. For each model compound and pH, different AKIEs were observed. For phenol, the AKIE was normal at pH 4 (1.0156 ± 0.0012) and inverse at pH 8 (0.9900 ± 0.0007). For 2-propoanol, an opposite pH dependence was observed with an inverse AKIE at pH 4 (0.9935 ± 0.0007) and a normal AKIE at pH 8 (1.0189 ± 0.0016). The variations of the AKIE values suggest that the rate-limiting step of the reaction is either the re-hybridization of the carbon atom involved in chloroform formation or the hydrolysis of trichloroacetyl intermediates depending on the nature of functional group and pH. The chloroform formation from humic acid and SOM gives rise to small isotope variations. A comparison of the isotopic trends of chloroform formed from humic acid and SOM with those found for the model compounds suggest that opposed AKIE associated with the chlorination of phenolic and ketone moieties of NOM partly compensate each other during chlorination of NOM indicating that different types of functional groups contribute to chloroform formation.

  8. Mechanistic Insight into Trimethylamine N-Oxide Recognition by the Marine Bacterium Ruegeria pomeroyi DSS-3

    PubMed Central

    Li, Chun-Yang; Chen, Xiu-Lan; Shao, Xuan; Wei, Tian-Di; Wang, Peng; Xie, Bin-Bin; Qin, Qi-Long; Zhang, Xi-Ying; Su, Hai-Nan; Song, Xiao-Yan; Shi, Mei; Zhou, Bai-Cheng

    2015-01-01

    ABSTRACT Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria. Ruegeria pomeroyi DSS-3, a marine Roseobacter, has an ATP-binding cassette transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX of R. pomeroyi DSS-3 was characterized. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. A Ca2+ ion chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between “open” and “closed” states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-π interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The π-π stacking interactions between the side chains of Phe and Trp are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marine Roseobacter clade. This study sheds light on how marine microorganisms utilize TMAO. IMPORTANCE Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. The products of TMAO metabolized by bacteria are part of the precursors of the greenhouse gas nitrous oxide. It is unclear how TMAO is recognized and imported by bacteria. TmoX is the substrate-binding protein of a TMAO-specific transporter. Here, the substrate specificity of TmoX of Ruegeria pomeroyi DSS-3 was characterized. The TMAO-binding mechanism of TmoX was studied by biochemical, structural, and mutational analyses. Moreover, our results suggest that the TMAO-binding mechanism may have universal significance in marine bacteria. This study sheds light on how marine microorganisms utilize TMAO and should lead to a better understanding of marine nitrogen cycling. PMID:26283766

  9. (n-3) Fatty acids alleviate adipose tissue inflammation and insulin resistance: Mechanistic insights

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obesity is associated with the metabolic syndrome, a significant risk factor for developing type-2 diabetes and cardiovascular diseases. A chronic low-grade inflammation occurring in the adipose tissue of obese individuals is causally linked to the pathogenesis of insulin resistance and the metaboli...

  10. Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria

    PubMed Central

    Watanabe, Yasunori; Tamura, Yasushi; Kawano, Shin; Endo, Toshiya

    2015-01-01

    Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1–Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1–Mdm35 with and without PA. The Ups1–Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes. PMID:26235513

  11. Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata.

    PubMed

    Tran, Thi Kim Anh; MacFarlane, Geoff R; Kong, Richard Yuen Chong; O'Connor, Wayne A; Yu, Richard Man Kit

    2016-05-01

    Marine molluscs, such as oysters, respond to estrogenic compounds with the induction of the egg yolk protein precursor, vitellogenin (Vtg), availing a biomarker for estrogenic pollution. Despite this application, the precise molecular mechanism through which estrogens exert their action to induce molluscan vitellogenesis is unknown. As a first step to address this question, we cloned a gene encoding Vtg from the Sydney rock oyster Saccostrea glomerata (sgVtg). Using primers designed from a partial sgVtg cDNA sequence available in Genbank, a full-length sgVtg cDNA of 8498bp was obtained by 5'- and 3'-RACE. The open reading frame (ORF) of sgVtg was determined to be 7980bp, which is substantially longer than the orthologs of other oyster species. Its deduced protein sequence shares the highest homology at the N- and C-terminal regions with other molluscan Vtgs. The full-length genomic DNA sequence of sgVtg was obtained by genomic PCR and genome walking targeting the gene body and flanking regions, respectively. The genomic sequence spans 20kb and consists of 30 exons and 29 introns. Computer analysis identified three closely spaced half-estrogen responsive elements (EREs) in the promoter region and a 210-bp CpG island 62bp downstream of the transcription start site. Upregulation of sgVtg mRNA expression was observed in the ovaries following in vitro (explants) and in vivo (tank) exposure to 17β-estradiol (E2). Notably, treatment with an estrogen receptor (ER) antagonist in vitro abolished the upregulation, suggesting a requirement for an estrogen-dependent receptor for transcriptional activation. DNA methylation of the 5' CpG island was analysed using bisulfite genomic sequencing of the in vivo exposed ovaries. The CpG island was found to be hypomethylated (with 0-3% methylcytosines) in both control and E2-exposed oysters. However, no significant differential methylation or any correlation between methylation and sgVtg expression levels was observed. Overall, the results support the possible involvement of an ERE-containing promoter and an estrogen-activated receptor in estrogen signalling in marine molluscs. PMID:26963518

  12. Mechanistic insights on the hydrodesulfurization of biphenyl-2-thiol with nickel compounds.

    PubMed

    Torres-Nieto, Jorge; Brennessel, William W; Jones, William D; García, Juventino J

    2009-03-25

    The reactivity of the nickel(I) dimer [(dippe)Ni(mu-H)](2) (1) with biphenyl-2-thiol was explored with the aim of clarifying the key step of sulfur extrusion during the hydrodesulfurization process using dibenzothiophene (DBT). These reactions were monitored by variable temperature NMR experiments which allowed the complete characterization and isolation of [(dippe)(2)Ni(2)(mu-H)(mu-S-2-biphenyl)] (3). The latter compound evolves to the terminal nickel-hydride [(dippe)Ni (eta(1)-C-2-biphenyl)(H)] (4) and transient [(dippe)NiS] (5), to ultimately yield [(dippe)(2)Ni(2)(mu-S)] (2) and biphenyl as the resulting HDS products. The reactivity of 1 and biphenyl-2-thiol was examined using different ratios of reactants, which allowed preparation of [(dippe)Ni(eta(1)-S-biphenyl-2-thiolate)(2)] (6) when using an excess of this substrate. The reactivity of 6 with 1 was addressed, yielding compound 2 and an equivalent amount of biphenyl. PMID:19292493

  13. Mechanistic insight into marine bioluminescence: photochemistry of the chemiexcited Cypridina (sea firefly) lumophore.

    PubMed

    Ding, Bo-Wen; Naumov, Panče; Liu, Ya-Jun

    2015-02-10

    Cypridina hilgendorfii (sea firefly) is a bioluminescent crustacean whose bioluminescence (BL) reaction is archetypal for a number of marine organisms, notably other bioluminescent crustaceans and coelenterates. Unraveling the mechanism of its BL is paramount for future applications of its strongly emissive lumophore. Cypridina produces light in a three-step reaction: First, the cypridinid luciferin is activated by an enzyme to produce a peroxide intermediate, cypridinid dioxetanone (CDO), which then decomposes to generate excited oxyluciferin (OxyCLnH*). Finally, OxyCLnH* deexcites to its ground state along with emission of bright blue light. Unfortunately, the detailed mechanism of the critical step, the thermolysis of CDO, remains unknown, and it is unclear whether the light emitter is generated from a neutral form (CDOH) or anionic form (CDO(-)) of the CDO precursor. In this work, we investigated the key step in the process by modeling the thermal decompositions of both CDOH and CDO(-). The calculated results indicate that the decomposition of CDO(-) occurs via the gradually reversible charge transfer (CT)-initiated luminescence (GRCTIL) mechanism, whereas CDOH decomposes through an entropic trapping mechanism without an obvious CT process. The thermolysis of CDO(-) is sensitive to solvent effects and is energetically favorable in polar environments compared with the thermolysis of CDOH. The thermolysis of CDO(-) produces the excited oxyluciferin anion (OxyCLn(-)*), which combines with a proton from the environment to form OxyCLnH*, the actual light emitter for the natural system. PMID:26580916

  14. Heavy Metals and Human Health: Mechanistic Insight into Toxicity and Counter Defense System of Antioxidants

    PubMed Central

    Jan, Arif Tasleem; Azam, Mudsser; Siddiqui, Kehkashan; Ali, Arif; Choi, Inho; Haq, Qazi Mohd. Rizwanul

    2015-01-01

    Heavy metals, which have widespread environmental distribution and originate from natural and anthropogenic sources, are common environmental pollutants. In recent decades, their contamination has increased dramatically because of continuous discharge in sewage and untreated industrial effluents. Because they are non-degradable, they persist in the environment; accordingly, they have received a great deal of attention owing to their potential health and environmental risks. Although the toxic effects of metals depend on the forms and routes of exposure, interruptions of intracellular homeostasis include damage to lipids, proteins, enzymes and DNA via the production of free radicals. Following exposure to heavy metals, their metabolism and subsequent excretion from the body depends on the presence of antioxidants (glutathione, α-tocopherol, ascorbate, etc.) associated with the quenching of free radicals by suspending the activity of enzymes (catalase, peroxidase, and superoxide dismutase). Therefore, this review was written to provide a deep understanding of the mechanisms involved in eliciting their toxicity in order to highlight the necessity for development of strategies to decrease exposure to these metals, as well as to identify substances that contribute significantly to overcome their hazardous effects within the body of living organisms. PMID:26690422

  15. Curcumin-loaded colloidal carrier system: formulation optimization, mechanistic insight, ex vivo and in vivo evaluation.

    PubMed

    Naz, Zrien; Ahmad, Farhan Jalees

    2015-01-01

    The present work investigated the topical delivery potential of nanoemulsion gel loaded with curcumin (CR). CR nanoemulsion (CR-NE) was prepared by spontaneous emulsification method using oil (Labrafac PG/glyceryl triacetate), surfactant:cosurfactant (Smix) (tween 80/polyethylene glycol [PEG] 400) and water. The pseudo-ternary phase diagrams were constructed and thermodynamic stability testing was performed. Droplet size and zeta potential were evaluated using photon correlation spectroscopy and transmission electron spectroscopy. Six formulations selected with an average droplet size ≤70±2.72 nm showed a fourfold increase in skin permeation as compared to crude CR solution in oil. The formulation CR-NE4 having a flux of 117.04±2.32 µg/cm(2)/h and with maximum retention (42.87%) was selected, characterized (droplet size =41.13±3.34 nm and zeta potential =-33.1±1.45 mV), and incorporated into gel using carbopol-980 (1% w/v). Skin dynamics analyzed by confocal laser scanning microscopy showed maximum deposition of CR up to a depth of 86.98 µm and was in concordance with differential scanning calorimetry and Fourier transform infrared spectroscopy studies that confirmed lipid bilayer disruption, enhancing permeation. A 28-day anti-arthritic evaluation (body weight, paw edema, tibiotarsal joint thickness, TNF-α and IL-1β levels, and histopathology) on Freund's complete adjuvant induced arthritic rat model after topical application of CR-NE gel in Wistar rats demonstrated substantial reversal of arthritic symptoms. Thus, CR-NE gel possesses potential for therapeutic effects locally in inflammatory arthritic disorders with improved topical bioavailability. PMID:26170665

  16. Mechanistic Insights into the Palladium-Catalyzed Aziridination of Aliphatic Amines by C-H Activation.

    PubMed

    Smalley, Adam P; Gaunt, Matthew J

    2015-08-26

    Detailed kinetic studies and computational investigations have been performed to elucidate the mechanism of a palladium-catalyzed C-H activation aziridination. A theoretical rate law has been derived that matches with experimental observations and has led to an improvement in the reaction conditions. Acetic acid was found to be beneficial in controlling the formation of an off-cycle intermediate, allowing a decrease in catalyst loading and improved yields. Density functional theory (DFT) studies were performed to examine the selectivities observed in the reaction. Evidence for electronic-controlled regioselectivity for the cyclopalladation step was obtained by a distortion-interaction analysis, whereas the aziridination product was justified through dissociation of acetic acid from the palladium(IV) intermediate preceding the product-forming reductive elimination step. The understanding of this reaction mechanism under the synthesis conditions should provide valuable assistance in the comprehension and design of palladium-catalyzed reactions on similar systems. PMID:26247373

  17. Diatom-inspired skeletonisation of insulin - Mechanistic insights into crystallisation and extracellular bioactivity.

    PubMed

    Véliz, Diosángeles Soto; Alam, Catharina; Nietzel, Thiago; Wyborski, Rebecca; Rivero-Müller, Adolfo; Alam, Parvez

    2015-09-01

    In this paper, we encage insulin within calcium carbonate by means of a biomineralisation process. We find that both dogbone and crossbone morphologies develop during the crystallisation process. The crystals break down into small nanocrystals after prolonged immersion in phosphate buffer solution, which adhere extracellularly to mammalian cells without causing any observable damage or early cell-death. The mechanisms behind calcium carbonate encaging of single insulin monomers are detailed. This communication elucidates a novel, diatom-inspired approach to the mineral skeletonisation of insulin. PMID:26094146

  18. Mechanistic Insight into Nanomolar Binding of Multivalent Neoglycopeptides to Wheat Germ Agglutinin.

    PubMed

    Rohse, Philipp; Wittmann, Valentin

    2016-07-01

    Multivalent carbohydrate-protein interactions are frequently involved in essential biological recognition processes. Accordingly, multivalency is often also exploited for the design of high-affinity lectin ligands aimed at the inhibition of such processes. In a previous study (D. Schwefel et al., J. Am. Chem. Soc. 2010, 132, 8704-8719) we identified a tetravalent cyclopeptide-based ligand with nanomolar affinity to the model lectin wheat germ agglutinin (WGA). To unravel the structural features of this ligand required for high-affinity binding to WGA, we synthesized a series of cyclic and linear neoglycopeptides that differ in their conformational freedom as well as the number of GlcNAc residues. Combined evidence from isothermal titration calorimetry (ITC), enzyme-linked lectin assays (ELLA), and dynamic light scattering (DLS) revealed different binding modes of tetra- and divalent ligands and that conformational preorganization of the ligands by cyclization is not a prerequisite for achieving high binding affinities. The high affinities of the tetravalent ligands rather stem from their ability to form crosslinks between several WGA molecules. The results illustrate that binding affinities and mechanisms are strongly dependent on the used multivalent system which offers opportunities to tune and control binding processes. PMID:27273019

  19. Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics

    PubMed Central

    Kadoch, Cigall; Crabtree, Gerald R.

    2015-01-01

    Over the past 4 years, nearly 100 exome sequencing studies have revealed the high frequency of mutations in the genes encoding the subunits of ATP-dependent chromatin remodelers in human cancer. Most of these mutations are within the genes encoding subunits of the BAF (Brg/Brahma-associated factors) or mSWI/SNF complex, which is one of two dozen predicted ATP-dependent chromatin remodeling complexes in mammals. Considering BAF complexes as a single entity, the 15 subunits encoded by 29 genes are mutated in >20% of human cancer, across a broad range of tumor types. These observations demonstrate that there is little redundancy in the oncogenic function of BAF complexes with the other remodeling complexes, underscoring their unique roles. Several important conclusions emerge from these genomic data: specific subunits appear to be mutated in specific cancers, highlighting tissue-specific protective roles; mutations can function as tumor suppressors or oncogenes; mutations can be homozygous or, more commonly, heterozygous, implying their dosage-sensitive roles in an unknown yet fundamental process used to suppress the genesis of cancer. These new human genetic findings paired with biochemical studies are challenging old ideas on how chromatin remodeling complexes function, generating new hypotheses with respect to their normal and oncogenic mechanisms and highlighting potential avenues for therapeutic intervention in human cancer. PMID:26601204

  20. Mechanistic Insights from the Binding of Substrate and Carbocation Intermediate Analogues to Aristolochene Synthase

    PubMed Central

    Chen, Mengbin; Al-lami, Naeemah; Janvier, Marine; D'Antonio, Edward L.; Faraldos, Juan A.; Cane, David E.; Allemann, Rudolf K.; Christianson, David W.

    2013-01-01

    Aristolochene synthase, a metal-dependent sesquiterpene cyclase from Aspergillus terreus, catalyzes the ionization-dependent cyclization of farnesyl diphosphate (FPP) to form the bicyclic eremophilane (+)-aristolochene with perfect structural and stereochemical precision. Here, we report the X-ray crystal structure of aristolochene synthase complexed with three Mg2+ ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate (FSPP), showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions identical to those previously observed in the complex with three Mg2+ ions and inorganic pyrophosphate (PPi). Moreover, the binding conformation of FSPP directly mimics that expected for productively bound FPP, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 π system that would be required for C1-C10 bond formation in the first step of catalysis. We also report crystal structures of aristolochene synthase complexed with Mg2+3-PPi and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade. Various binding orientations are observed for these bicyclic analogues, and these orientations appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged PPi anion. Surprisingly, the active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry. Although this permissiveness in binding is unanticipated, based on the stereochemical precision of catalysis that leads exclusively to the (+)-aristolochene stereoisomer, it suggests the ability of the active site to enable controlled reorientation of intermediates during the cyclization cascade. Taken together, these structures illuminate important aspects of the catalytic mechanism. PMID:23905850

  1. Tryptophan Lyase (NosL): Mechanistic Insights from Substrate Analogues and Mutagenesis.

    PubMed

    Bhandari, Dhananjay M; Xu, Hui; Nicolet, Yvain; Fontecilla-Camps, Juan C; Begley, Tadhg P

    2015-08-11

    NosL is a member of a family of radical S-adenosylmethionine enzymes that catalyze the cleavage of the Cα-Cβ bond of aromatic amino acids. In this paper, we describe a set of experiments with substrate analogues and mutants for probing the early steps of the NosL mechanism. We provide biochemical evidence in support of the structural studies showing that the 5'-deoxyadenosyl radical abstracts a hydrogen atom from the amino group of tryptophan. We demonstrate that d-tryptophan is a substrate for NosL but shows relaxed regio control of the first β-scission reaction. Mutagenesis studies confirm that Arg323 is important for controlling the regiochemistry of the β-scission reaction and that Ser340 binds the substrate by hydrogen bonding to the indolic N1 atom. PMID:26204056

  2. Mechanistic Insights into Catalytic Ethanol Steam Reforming Using Isotope-Labeled Reactants.

    PubMed

    Crowley, Stephen; Castaldi, Marco J

    2016-08-26

    The low-temperature ethanol steam reforming (ESR) reaction mechanism over a supported Rh/Pt catalyst has been investigated using isotope-labeled EtOH and H2 O. Through strategic isotope labeling, all nonhydrogen atoms were distinct from one another, and allowed an unprecedented level of understanding of the dominant reaction pathways. All combinations of isotope- and non-isotope-labeled atoms were detected in the products, thus there are multiple pathways involved in H2 , CO, CO2 , CH4 , C2 H4 , and C2 H6 product formation. Both the recombination of C species on the surface of the catalyst and preservation of the C-C bond within ethanol are responsible for C2 product formation. Ethylene is not detected until conversion drops below 100 % at t=1.25 h. Also, quantitatively, 57 % of the observed ethylene is formed directly through ethanol dehydration. Finally there is clear evidence to show that oxygen in the SiO2 -ZrO2 support constitutes 10 % of the CO formed during the reaction. PMID:27487203

  3. Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination

    PubMed Central

    Misaghi, Shahram; Senger, Kate; Sai, Tao; Qu, Yan; Sun, Yonglian; Hamidzadeh, Kajal; Nguyen, Allen; Jin, Zhaoyu; Zhou, Meijuan; Yan, Donghong; Lin, Wei Yu; Lin, Zhonghua; Lorenzo, Maria N.; Sebrell, Andrew; Ding, Jiabing; Xu, Min; Caplazi, Patrick; Austin, Cary D.; Balazs, Mercedesz; Roose-Girma, Merone; DeForge, Laura; Warming, Søren; Lee, Wyne P.; Dixit, Vishva M.; Zarrin, Ali A.

    2013-01-01

    Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sμ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sμ. These results suggest that the sufficiency of Sμ to mediate IgH rearrangements may be influenced by context-dependent cues. PMID:24019479

  4. Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.

    PubMed

    Misaghi, Shahram; Senger, Kate; Sai, Tao; Qu, Yan; Sun, Yonglian; Hamidzadeh, Kajal; Nguyen, Allen; Jin, Zhaoyu; Zhou, Meijuan; Yan, Donghong; Lin, Wei Yu; Lin, Zhonghua; Lorenzo, Maria N; Sebrell, Andrew; Ding, Jiabing; Xu, Min; Caplazi, Patrick; Austin, Cary D; Balazs, Mercedesz; Roose-Girma, Merone; DeForge, Laura; Warming, Søren; Lee, Wyne P; Dixit, Vishva M; Zarrin, Ali A

    2013-09-24

    Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sμ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sμ. These results suggest that the sufficiency of Sμ to mediate IgH rearrangements may be influenced by context-dependent cues. PMID:24019479

  5. Structure-Based Mechanistic Insights into DNMT1-Mediated Maintenance DNA Methylation

    SciTech Connect

    Song, Jikui; Teplova, Marianna; Ishibe-Murakami, Satoko; Patel, Dinshaw J.

    2012-03-26

    DNMT1, the major maintenance DNA methyltransferase in animals, helps to regulate gene expression, genome imprinting, and X-chromosome inactivation. We report on the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex containing a central hemimethylated CpG site. The methyl group of methylcytosine is positioned within a shallow hydrophobic concave surface, whereas the cytosine on the target strand is looped out and covalently anchored within the catalytic pocket. The DNA is distorted at the hemimethylated CpG step, with side chains from catalytic and recognition loops inserting through both grooves to fill an intercalation-type cavity associated with a dual base flip-out on partner strands. Structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensures the high fidelity of DNMT1-mediated maintenance DNA methylation.

  6. Fast and tunable synthesis of ZrO2 nanocrystals: mechanistic insights into precursor dependence.

    PubMed

    De Keukeleere, Katrien; De Roo, Jonathan; Lommens, Petra; Martins, José C; Van Der Voort, Pascal; Van Driessche, Isabel

    2015-04-01

    In this work, ZrO2 nanocrystals (NCs) are synthesized via a solvothermal treatment in benzyl alcohol, which is an established method for the synthesis of many metal oxide nanocrystals. We found that the use of microwave heating allows for a reduction in reaction time from 2 days in the autoclave to merely 4 h in the microwave. Furthermore, we were able to tune the crystallographic phase from pure cubic to pure monoclinic zirconia by changing the reaction mechanism through the use of a different zirconium precursor. Via GC-MS measurements, we found that the release of a strong acid during synthesis controls the key mechanism behind the control over crystal phase formation. The as-synthesized ZrO2 NCs (cubic or monoclinic) are small in size (3-10 nm), yet aggregated. However, aggregate-free NCs are generated through a surface-functionalization with carboxylic acid ligands, providing stabilization in apolar solvents via steric hindrance. Solution (1)H NMR was used to study the details of this post-modification step and the surface chemistry of the resulting aggregate-free NCs. This led to the conclusion that not only a different crystal structure but also a different surface chemistry is obtained, depending on the precursor composition. PMID:25751155

  7. Mechanistic Insights into the Relationship between Lung and Vascular Response to Ambient Particulate Matter (PM)

    EPA Science Inventory

    The mechanisms by which pulmonary-encountered ambient PM induces vascular response are not well understood. We examined lung and aortic response of rats following intratracheal instillation of three ambient PM. Chemically characterized PM10 and PM2.5 from th...

  8. Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

    PubMed

    Khomane, Kailas S; Nandekar, Prajwal P; Wahlang, Banrida; Bagul, Pravin; Shaikh, Naeem; Pawar, Yogesh B; Meena, Chhuttan Lal; Sangamwar, Abhay T; Jain, Rahul; Tikoo, K; Bansal, Arvind K

    2012-09-01

    The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 μM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces. PMID:22779445

  9. Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity.

    PubMed

    Weigand, Julia E; Schmidtke, Sina R; Will, Tristan J; Duchardt-Ferner, Elke; Hammann, Christian; Wöhnert, Jens; Suess, Beatrix

    2011-04-01

    While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch. PMID:21149263

  10. Mechanistic insights into ozone-initiated oxidative degradation of saturated hydrocarbons and polymers.

    PubMed

    Lee, Richmond; Coote, Michelle L

    2016-09-21

    Accurate quantum chemical calculations were employed to investigate the mechanism of ozone-initiated oxidation of C-H bonds of saturated hydrocarbons and polymers. Step wise hydrogen atom abstraction generates the first resting state the trihydroxide -COOOH, which undergoes decomposition to produce the free radical species alkoxyl -CO˙ and peroxyl ˙OOH thereby setting off a complex chain of radical processes. The H transfer from peroxyl radical to alkoxyl allows formation of inactive alcohol and the singlet excited dioxygen. Other competitive processes include the self fragmentation or β-scission of the alkoxyl -CO˙ to give rise to a carbonyl (ketone or aldehyde) and a C-centred free radical species. Tertiary C-H bonds are most susceptible to O3 oxidation followed by secondary and primary. Among the polymers studied, poly(styrene) is the least resistant to C-H bond ozonation, followed by poly(propylene), poly(methacrylate), poly(methyl methacrylate) and poly(vinyl chloride). Calculations also reveal catalytic effects of water in promoting the C-H bond oxidation process in polymer systems without competing H-bond donor groups. PMID:27545312

  11. Socioeconomic status and the brain: mechanistic insights from human and animal research

    PubMed Central

    Hackman, Daniel A.; Farah, Martha J.; Meaney, Michael J.

    2010-01-01

    Human brain development occurs within a socioeconomic context and childhood socioeconomic status (SES) influences neural development — particularly of the systems that subserve language and executive function. Research in humans and in animal models has implicated prenatal factors, parent–child interactions and cognitive stimulation in the home environment in the effects of SES on neural development. These findings provide a unique opportunity for understanding how environmental factors can lead to individual differences in brain development, and for improving the programmes and policies that are designed to alleviate SES-related disparities in mental health and academic achievement. PMID:20725096

  12. Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

    NASA Astrophysics Data System (ADS)

    Goyal, Parveen; Krasteva, Petya V.; van Gerven, Nani; Gubellini, Francesca; van den Broeck, Imke; Troupiotis-Tsaïlaki, Anastassia; Jonckheere, Wim; Péhau-Arnaudet, Gérard; Pinkner, Jerome S.; Chapman, Matthew R.; Hultgren, Scott J.; Howorka, Stefan; Fronzes, Rémi; Remaut, Han

    2014-12-01

    Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å3 pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.

  13. Mechanistic insights into hypothermic ventricular fibrillation: the role of temperature and tissue size

    PubMed Central

    Filippi, Simonetta; Gizzi, Alessio; Cherubini, Christian; Luther, Stefan; Fenton, Flavio H.

    2014-01-01

    Aims Hypothermia is well known to be pro-arrhythmic, yet it has beneficial effects as a resuscitation therapy and valuable during intracardiac surgeries. Therefore, we aim to study the mechanisms that induce fibrillation during hypothermia. A better understanding of the complex spatiotemporal dynamics of heart tissue as a function of temperature will be useful in managing the benefits and risks of hypothermia. Methods and results We perform two-dimensional numerical simulations by using a minimal model of cardiac action potential propagation fine-tuned on experimental measurements. The model includes thermal factors acting on the ionic currents and the gating variables to correctly reproduce experimentally recorded restitution curves at different temperatures. Simulations are implemented using WebGL, which allows long simulations to be performed as they run close to real time. We describe (i) why fibrillation is easier to induce at low temperatures, (ii) that there is a minimum size required for fibrillation that depends on temperature, (iii) why the frequency of fibrillation decreases with decreasing temperature, and (iv) that regional cooling may be an anti-arrhythmic therapy for small tissue sizes however it may be pro-arrhythmic for large tissue sizes. Conclusion Using a mathematical cardiac cell model, we are able to reproduce experimental observations, quantitative experimental results, and discuss possible mechanisms and implications of electrophysiological changes during hypothermia. PMID:24569897

  14. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation

    PubMed Central

    Huo, Yanwu; Nam, Ki Hyun; Ding, Fang; Lee, Heejin; Wu, Lijie; Xiao, Yibei; Farchione, F. Daniel; Zhou, Sharleen; Rajashankar, Raj; Kurinov, Igor; Zhang, Rongguang; Ke, Ailong

    2014-01-01

    CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids using an RNA-mediated interference mechanism. Interference in Type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Cas3 bound to ss-DNA substrate and show that it is an obligated 3′-to-5′ ss-DNase preferentially accepting substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ss-DNA cleavage. ATP coordination and conformational flexibility are revealed for the SF2-type helicase moiety. Cas3 is specifically guided towards Cascade-bound target DNA with a correct PAM sequence, through physical interactions to both the non-target substrate strand and the CasA protein. The cascade of recognition events ensures a well-controlled DNA targeting and degradation of alien DNA by Cascade and Cas3. PMID:25132177

  15. Reactivity ratios, and mechanistic insight for anionic ring-opening copolymerization of epoxides.

    PubMed

    Lee, Bongjae F; Wolffs, Martin; Delaney, Kris T; Sprafke, Johannes K; Leibfarth, Frank A; Hawker, Craig J; Lynd, Nathaniel A

    2012-05-01

    Reactivity ratios were evaluated for anionic ring-opening copolymerizations of ethylene oxide (EO) with either allyl glycidyl ether (AGE) or ethylene glycol vinyl glycidyl ether (EGVGE) using a benzyl alkoxide initiator. The chemical shift for the benzylic protons of the initiator, as measured by (1)H NMR spectroscopy, were observed to be sensitive to the sequence of the first two monomers added to the initiator during polymer growth. Using a simple kinetic model for initiation and the first propagation step, reactivity ratios for the copolymerization of AGE and EGVGE with EO could be determined by analysis of the (1)H NMR spectroscopy for the resulting copolymer. For the copolymerization between EO and AGE, the reactivity ratios were determined to be r(AGE) = 1.31 ± 0.26 and r(EO) = 0.54 ± 0.03, while for EO and EGVGE, the reactivity ratios were r(EGVGE) = 3.50 ± 0.90 and r(EO) = 0.32 ± 0.10. These ratios were consistent with the compositional drift observed in the copolymerization between EO and EGVGE, with EGVGE being consumed early in the copolymerization. These experimental results, combined with density functional calculations, allowed a mechanism for oxyanionic ring-opening polymerization that begins with coordination of the Lewis-basic epoxide to the cation to be proposed. The calculated transition-state energies agree qualitatively with the observed relative rates for polymerization. PMID:23226879

  16. Mechanistic insights into a TIMP3-sensitive pathway constitutively engaged in the regulation of cerebral hemodynamics

    PubMed Central

    Capone, Carmen; Dabertrand, Fabrice; Baron-Menguy, Celine; Chalaris, Athena; Ghezali, Lamia; Domenga-Denier, Valérie; Schmidt, Stefanie; Huneau, Clément; Rose-John, Stefan; Nelson, Mark T; Joutel, Anne

    2016-01-01

    Cerebral small vessel disease (SVD) is a leading cause of stroke and dementia. CADASIL, an inherited SVD, alters cerebral artery function, compromising blood flow to the working brain. TIMP3 (tissue inhibitor of metalloproteinase 3) accumulation in the vascular extracellular matrix in CADASIL is a key contributor to cerebrovascular dysfunction. However, the linkage between elevated TIMP3 and compromised cerebral blood flow (CBF) remains unknown. Here, we show that TIMP3 acts through inhibition of the metalloprotease ADAM17 and HB-EGF to regulate cerebral arterial tone and blood flow responses. In a clinically relevant CADASIL mouse model, we show that exogenous ADAM17 or HB-EGF restores cerebral arterial tone and blood flow responses, and identify upregulated voltage-dependent potassium channel (KV) number in cerebral arterial myocytes as a heretofore-unrecognized downstream effector of TIMP3-induced deficits. These results support the concept that the balance of TIMP3 and ADAM17 activity modulates CBF through regulation of myocyte KV channel number. DOI: http://dx.doi.org/10.7554/eLife.17536.001 PMID:27476853

  17. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity

    PubMed Central

    Ahmed, Afsar U.; Williams, Bryan R. G.; Hannigan, Gregory E.

    2015-01-01

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding. PMID:26569329

  18. Mechanistic Insights into Hydride Transfer for Catalytic Hydrogenation of CO2 with Cobalt Complexes

    SciTech Connect

    Kumar, Neeraj; Camaioni, Donald M.; Dupuis, Michel; Raugei, Simone; Appel, Aaron M.

    2014-08-21

    The catalytic hydrogenation of CO2 to formate by Co(dmpe)2H can proceeds via direct hydride transfer or via CO2 coordination to Co followed by reductive elimination of formate. Both pathways have activation barriers consistent with experiment (~17.5 kcal/mol). Controlling the basicity of Co by ligand design is key to improve catalysis. The research by N.K., D.M.C. and A.M.A. was supported by the US Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences. The research by S.R. and M.D. was supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science. Pacific Northwest National Laboratory (PNNL) is a multiprogram national laboratory operated for the DOE by Battelle.

  19. Mechanistic insights aid the search for CFC substitutes: risk assessment of HCFC-123 as an example.

    PubMed

    Jarabek, A M; Fisher, J W; Rubenstein, R; Lipscomb, J C; Williams, R J; Vinegar, A; McDougal, J N

    1994-06-01

    An international consensus on the need to reduce the use of chlorofluorocarbons (CFCs) and other ozone-depleting gases such as the halons led to the adoptions of the 1987 Montreal Protocol and Title VI of the 1990 Clean Air Act Amendments, "Protecting Stratospheric Ozone." These agreements included major provisions for reducing and eventually phasing out production and use of CFCs and halons as well as advancing the development of replacement chemicals. Because of the ubiquitous use and benefits of CFCs and halons, an expeditious search for safe replacements to meet the legislative deadlines is of critical importance. Toxicity testing and health risk assessment programs were established to evaluate the health and environmental impact of these replacement chemicals. Development and implementation of these programs as well as the structural-activity relationships significant for the development of the replacement chemicals are described below. A dose-response evaluation for the health risk assessment of the replacement chemical HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane) is also presented to show an innovative use of physiologically based pharmacokinetic (PBPK) modeling. This is based on a parallelogram approach using data on the anesthetic gas halothane, a structural analog to HCFC-123. Halothane and HCFC-123 both form the same metabolite, trifluoroacetic acid (TFA), indicative of the same metabolic oxidative pathway attributed to hepatotoxicity. The parallelogram approach demonstrates the application of template model structures and shows how PBPK modeling, together with judicious experimental design, can be used to improve the accuracy of health risk assessment and to decrease the need for extensive laboratory animal testing. PMID:8029495

  20. Potential anticancer heterometallic Fe-Au and Fe-Pd agents: initial mechanistic insights.

    PubMed

    Lease, Nicholas; Vasilevski, Vadim; Carreira, Monica; de Almeida, Andreia; Sanaú, Mercedes; Hirva, Pipsa; Casini, Angela; Contel, María

    2013-07-25

    A series of gold(III) and palladium(II) heterometallic complexes with new iminophosphorane ligands derived from ferrocenylphosphanes [{Cp-P(Ph2)═N-Ph}2Fe] (1), [{Cp-P(Ph2)═N-CH2-2-NC5H4}2Fe] (2), and [{Cp-P(Ph2)═N-CH2-2-NC5H4}Fe(Cp)] (3) have been synthesized and structurally characterized. Ligands 2 and 3 afford stable coordination complexes [AuCl2(3)]ClO4, [{AuCl2}2(2)](ClO4)2, [PdCl2(3)], and [{PdCl2}2(2)]. The complexes have been evaluated for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), in human breast cancer cells (MCF7) and in a nontumorigenic human embryonic kidney cell line (HEK-293T). The highly cytotoxic trimetallic derivatives M2Fe (M = Au, Pd) are more cytotoxic to cancer cells than their corresponding monometallic fragments. Moreover, these complexes were significantly more cytotoxic than cisplatin in the resistant A2780R and the MCF7 cell lines. Studies of the interactions of the trimetallic compounds with DNA and the zinc-finger protein PARP-1 indicate that they exert anticancer effects in vitro based on different mechanisms of actions with respect to cisplatin. PMID:23786413

  1. Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

    PubMed Central

    Goyal, Parveen; Krasteva, Petya V.; Van Gerven, Nani; Gubellini, Francesca; Van den Broeck, Imke; Troupiotis-Tsaïlaki, Anastassia; Jonckheere, Wim; Péhau-Arnaudet, Gérard; Pinkner, Jerome S.; Chapman, Matthew R.; Hultgren, Scott J.; Howorka, Stefan; Fronzes, Rémi; Remaut, Han

    2014-01-01

    Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes)1–3. They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia1,4,5. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF6,7. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The trans-membrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å3 pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism. PMID:25219853

  2. Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structural and Mechanistic Insights

    PubMed Central

    Alqassim, Saif S.; Urquiza, Mauricio; Borgnia, Eitan; Nagib, Marc; Amzel, L. Mario; Bianchet, Mario A.

    2016-01-01

    MICALs (Molecule Interacting with CasL) are conserved multidomain enzymes essential for cytoskeletal reorganization in nerve development, endocytosis, and apoptosis. In these enzymes, a type-2 calponin homology (CH) domain always follows an N-terminal monooxygenase (MO) domain. Although the CH domain is required for MICAL-1 cellular localization and actin-associated function, its contribution to the modulation of MICAL activity towards actin remains unclear. Here, we present the structure of a fragment of MICAL-1 containing the MO and the CH domains—determined by X-ray crystallography and small angle scattering—as well as kinetics experiments designed to probe the contribution of the CH domain to the actin-modification activity. Our results suggest that the CH domain, which is loosely connected to the MO domain by a flexible linker and is far away from the catalytic site, couples F-actin to the enhancement of redox activity of MICALMO-CH by a cooperative mechanism involving a trans interaction between adjacently bound molecules. Binding cooperativity is also observed in other proteins regulating actin assembly/disassembly dynamics, such as ADF/Cofilins. PMID:26935886

  3. Mechanistic insights on petrosaspongiolide M inhibitory effects on immunoproteasome and autophagy.

    PubMed

    Monti, Maria Chiara; Margarucci, Luigi; Riccio, Raffaele; Bonfili, Laura; Mozzicafreddo, Matteo; Eleuteri, Anna Maria; Casapullo, Agostino

    2014-04-01

    The proteasome, a complex multimeric structure strictly implicated in cell protein degradation, has gained the status of privileged drug target since its functional involvement in relevant pathways ruling the cell life, such as cell cycle, transcription and protein quality control, and the recent marketing of bortezomib as proteasome inhibitor for anti-cancer therapy. The marine γ-hydroxybutenolide terpenoid petrosaspongiolide M has been recently discovered as new proteasome inhibitor through a chemical proteomic approach and in cell biological assays. In this study a deep investigation has been carried out on the molecular mechanism of interaction of petrosaspongiolide M with the immunoproteasome, a proteasomal variant mainly involved in the immune responses. The results define a picture in which petrosaspongiolide M exerts its inhibitory activity by binding the active sites in the inner core of the immunoproteasome and/or covalently linking a Lys residue at the proteasome core/11S activator particle interface. Moreover, petrosaspongiolide M is also able to impair autophagy, a complementary pathway involved in protein degradation and cross-talking with the proteasome system. On this basis, petrosaspongiolide M could represent an interesting molecule for its propensity to modulate intracellular proteolysis through a dual inhibition of the immunoproteasome and autophagy. PMID:24530967

  4. Mechanistic insights into light-driven graphene-induced peroxide decomposition: radical generation and disproportionation.

    PubMed

    Chu, Ya-Lan; Chen, Yen-An; Li, Wei-Chin; Chu, Jean-Ho; Chen, Chun-Hu; Chiang, Chao-Ming

    2016-07-28

    Interaction between adsorbed t-butyl peroxybenzoate and photoexcited graphene rendered trapped phenyl and t-butoxy radicals. Post-irradiation thermal desorption showed benzene, t-butanol, and isobutylene oxide as the end products. The required hydrogen atoms were obtained via the radical disproportionation. Graphene enabled radical species to be captured and their on-surface chemistry to be revealed. PMID:27366795

  5. Pyrolysis of Cyclopentadienone: Mechanistic Insights from a Direct Measurement of Product Branching Ratios.

    PubMed

    Ormond, Thomas K; Scheer, Adam M; Nimlos, Mark R; Robichaud, David J; Troy, Tyler P; Ahmed, Musahid; Daily, John W; Nguyen, Thanh Lam; Stanton, John F; Ellison, G Barney

    2015-07-16

    The thermal decomposition of cyclopentadienone (C5H4═O) has been studied in a flash pyrolysis continuous flow microreactor. Passing dilute samples of o-phenylene sulfite (C6H4O2SO) in He through the microreactor at elevated temperatures yields a relatively clean source of C5H4═O. The pyrolysis of C5H4═O was investigated over the temperature range 1000-2000 K. Below 1600 K, we have identified two decomposition channels: (1) C5H4═O (+ M) → CO + HC≡C-CH═CH2 and (2) C5H4═O (+ M) → CO + HC≡CH + HC≡CH. There is no evidence of radical or H atom chain reactions. To establish the thermochemistry for the pyrolysis of cyclopentadienone, ab initio electronic structure calculations (AE-CCSD(T)/aug-cc-pCVQZ//AE-CCSD(T)/cc-pVQZ and anharmonic FC-CCSD(T)/ANO1 ZPEs) were used to find ΔfH0(C5H4═O) to be 16 ± 1 kcal mol(-1) and ΔfH0(CH2═CH-C≡CH) to be 71 ± 1 kcal mol(-1). The calculations predict the reaction enthalpies ΔrxnH0(1) to be 28 ± 1 kcal mol(-1) (ΔrxnH298(1) is 30 ± 1 kcal mol(-1)) and ΔrxnH0(2) to be 66 ± 1 kcal mol(-1) (ΔrxnH298(2) is 69 ± 1 kcal mol(-1)). Following pyrolysis of C5H4═O, photoionization mass spectrometry was used to measure the relative concentrations of HCC-CHCH2 and HCCH. Reaction 1 dominates at low pyrolysis temperatures (1000-1400 K). At temperatures above 1400 K, reaction 2 becomes the dominant channel. We have used the product branching ratios over the temperature range 1000-1600 K to extract the ratios of unimolecular rate coefficients for reactions 1 and 2 . If Arrhenius expressions are used, the difference of activation energies for reactions 1 and 2 , E2 - E1, is found to be 16 ± 1 kcal mol(-1) and the ratio of the pre-exponential factors, A2/A1, is 7.0 ± 0.3. PMID:25608038

  6. Interstitial-matrix edema in burns: mechanistic insights from subatmospheric pressure treatment in vivo.

    PubMed

    McGee, Maria P; Morykwas, Michael; Campbell, Douglas; Hoge, Kathie; Argenta, Louis

    2014-01-01

    Thermal injury disrupts fluid homeostasis and hydration, affecting hemodynamics and local interstitial fluid-driving forces, leading rapidly to edema. This study explores local mechanisms in vivo, after deep partial-thickness burns in the dermal matrix. Heat-damaged skin was obtained from pig corpses, byproducts of unrelated burn treatment protocols approved by the Institutional Animal-Care-and-Use Committee. Hydration potential and flow rates were measured by osmotic stress techniques at 4 and 37 °C, and collagen folding/unfolding was examined by differential scanning calorimetry and diffusion tensor magnetic resonance imaging. Kinetic and equilibrium hydration parameters differed in heat-damaged and undamaged skin; the mean hydration potential and initial flow rates of damaged skin were negative at 37 but positive at 4 °C, in contrast to the positive mean at either temperature of explants taken from undamaged skin sites on the same animals. After subatmospheric pressure treatment (125 mmHg), parameters in damaged reversed to values similar to those of undamaged, whereas the proportion of folded collagen and unidirectional resistance to water diffusion increased. Together, results support interfacial rather than colloidosmotic fluid transfer mechanisms in burns and confirm in vivo the relevance of collagen folding/unfolding, further suggesting collagen structural transitions as potential therapeutic targets and models for engineered biomimetic materials. PMID:24393157

  7. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity.

    PubMed

    Ahmed, Afsar U; Williams, Bryan R G; Hannigan, Gregory E

    2015-01-01

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding. PMID:26569329

  8. Mechanistic Insight into the Copper-Catalyzed Regiodivergent Silacarboxylation of Allenes with CO2.

    PubMed

    Yuan, Ruming; Hu, Rong; Fu, Gang

    2016-08-01

    DFT calculations were performed to investigate the detailed reaction mechanisms in the copper-catalyzed regiodivergent silacarboxylation of allenes. According to our calculations, the catalysis would bifurcate at the allene silylcupration step, followed by CO2 insertion, eventually leading to the carboxylated vinylsilane or allylsilane products. The gaps between the two silylcupration barriers were predicted to be -2.3, -0.4, and 2.2 kcal mol(-1) when using (rac)-Me-DuPhos, dcpe, and PCy3 (+H2 O) as the ligands, which nicely accounted for the experimental vinylsilane/allylsilane ratios of 93:7, 50:50, and 15:85, respectively. By means of transition-state-energy decomposition, we found that the energy penalty of catalyst deformation into its transition-state geometry was the key factor in determining the direction of the reaction. The switchable regioselectivity by using different P ligands could be ascribed to structural changes of the Cu-Si and Cu-P bonds during the silylcupration process. PMID:27319319

  9. Copper-catalyzed aerobic oxidative C-H functionalizations: trends and mechanistic insights.

    PubMed

    Wendlandt, Alison E; Suess, Alison M; Stahl, Shannon S

    2011-11-18

    The selective oxidation of C-H bonds and the use of O(2) as a stoichiometric oxidant represent two prominent challenges in organic chemistry. Copper(II) is a versatile oxidant, capable of promoting a wide range of oxidative coupling reactions initiated by single-electron transfer (SET) from electron-rich organic molecules. Many of these reactions can be rendered catalytic in Cu by employing molecular oxygen as a stoichiometric oxidant to regenerate the active copper(II) catalyst. Meanwhile, numerous other recently reported Cu-catalyzed C-H oxidation reactions feature substrates that are electron-deficient or appear unlikely to undergo single-electron transfer to copper(II). In some of these cases, evidence has been obtained for the involvement of organocopper(III) intermediates in the reaction mechanism. Organometallic C-H oxidation reactions of this type represent important new opportunities for the field of Cu-catalyzed aerobic oxidations. PMID:22034061

  10. Curcumin-loaded colloidal carrier system: formulation optimization, mechanistic insight, ex vivo and in vivo evaluation

    PubMed Central

    Naz, Zrien; Ahmad, Farhan Jalees

    2015-01-01

    The present work investigated the topical delivery potential of nanoemulsion gel loaded with curcumin (CR). CR nanoemulsion (CR-NE) was prepared by spontaneous emulsification method using oil (Labrafac PG/glyceryl triacetate), surfactant:cosurfactant (Smix) (tween 80/polyethylene glycol [PEG] 400) and water. The pseudo-ternary phase diagrams were constructed and thermodynamic stability testing was performed. Droplet size and zeta potential were evaluated using photon correlation spectroscopy and transmission electron spectroscopy. Six formulations selected with an average droplet size ≤70±2.72 nm showed a fourfold increase in skin permeation as compared to crude CR solution in oil. The formulation CR-NE4 having a flux of 117.04±2.32 µg/cm2/h and with maximum retention (42.87%) was selected, characterized (droplet size =41.13±3.34 nm and zeta potential =−33.1±1.45 mV), and incorporated into gel using carbopol-980 (1% w/v). Skin dynamics analyzed by confocal laser scanning microscopy showed maximum deposition of CR up to a depth of 86.98 µm and was in concordance with differential scanning calorimetry and Fourier transform infrared spectroscopy studies that confirmed lipid bilayer disruption, enhancing permeation. A 28-day anti-arthritic evaluation (body weight, paw edema, tibiotarsal joint thickness, TNF-α and IL-1β levels, and histopathology) on Freund’s complete adjuvant induced arthritic rat model after topical application of CR-NE gel in Wistar rats demonstrated substantial reversal of arthritic symptoms. Thus, CR-NE gel possesses potential for therapeutic effects locally in inflammatory arthritic disorders with improved topical bioavailability. PMID:26170665

  11. Catalytic Kinetic Resolution of Disubstituted Piperidines by Enantioselective Acylation: Synthetic Utility and Mechanistic Insights.

    PubMed

    Wanner, Benedikt; Kreituss, Imants; Gutierrez, Osvaldo; Kozlowski, Marisa C; Bode, Jeffrey W

    2015-09-01

    The catalytic kinetic resolution of cyclic amines with achiral N-heterocyclic carbenes and chiral hydroxamic acids has emerged as a promising method to obtain enantio-enriched amines with high selectivity factors. In this report, we describe the catalytic kinetic resolution of disubstituted piperdines with practical selectivity factors (s, up to 52) in which we uncovered an unexpected and pronounced conformational effect resulting in disparate reactivity and selectivity between the cis- and trans-substituted piperidine isomers. Detailed experimental and computational studies of the kinetic resolution of various disubstituted piperidines revealed a strong preference for the acylation of conformers in which the α-substituent occupies the axial position. This work provides further experimental and computational support for the concerted 7-member transition state model for acyl transfer reagents and expands the scope and functional group tolerance of the secondary amine kinetic resolution. PMID:26308097

  12. Indolicidin targets duplex DNA: structural and mechanistic insight through a combination of spectroscopy and microscopy.

    PubMed

    Ghosh, Anirban; Kar, Rajiv Kumar; Jana, Jagannath; Saha, Abhijit; Jana, Batakrishna; Krishnamoorthy, Janarthanan; Kumar, Dinesh; Ghosh, Surajit; Chatterjee, Subhrangsu; Bhunia, Anirban

    2014-09-01

    Indolicidin (IR13), a 13-residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B-type DNA structures. Replacements of the central Trp-Trp pair with Ala-Ala, His-His, or Phe-Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high-resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds. PMID:25044630

  13. Ruthenium Catalyzed Intramolecular C-S Coupling Reactions: Synthetic Scope and Mechanistic Insight.

    PubMed

    Sharma, Shivani; Pathare, Ramdas S; Maurya, Antim K; Gopal, Kandasamy; Roy, Tapta Kanchan; Sawant, Devesh M; Pardasani, Ram T

    2016-02-01

    A ruthenium catalyzed intramolecular C-S coupling reaction of N-arylthioureas for the synthesis of 2-aminobenzothiazoles has been developed. Kinetic, isotope labeling, and computational studies reveal the involvement of an electrophilic ruthenation pathway instead of a direct C-H activation. Stereoelectronic effect of meta-substituents on the N-arylthiourea dictates the final regioselective outcome of the reaction. PMID:26761401

  14. Mechanistic insights of sulfur mustard-induced acute tracheal injury in rats.

    PubMed

    Zhu, Xiao-Ji; Xu, Rui; Meng, Xiao; Chu, Hai-Bo; Zhao, Chao; Lian, Cheng-Jin; Wang, Tao; Guo, Wen-Jun; Zhang, Sheng-Ming

    2014-01-01

    Sulfur mustard (SM) is believed to be a major threat to civilian populations because of the persistent asymmetric threat by nonstate actors, such as terrorist groups, the ease of synthesis and handling, and the risk of theft from stockpiles. The purpose of this study was to establish mechanisms of acute tracheal injury in rats induced by SM using histopathologic, immunohistochemical, and biochemical parameters. Male rats (Sprague-Dawley) were anesthetized, intratracheally intubated, and exposed to 2 mg/kg of SM. Animals were euthanized 6-, 24-, 48-, and 72-hour postexposure, and intracavitary blood samples from the heart and tracheal tissues were collected. Exposure of rats to SM resulted in rapid tracheal injury, including tracheal epithelial cell shedding, focal ulceration, and abundant lymphocyte invasion of the submucosa. There was also evidence of a large number of apoptotic cells in the epithelium and submucosa, the serum levels of tumor necrosis factor α, interleukin 1β (IL) 1β, IL-6, and γ-glutamyl transferase peaked at 24 hours, and the serum levels of lactate dehydrogenase, glutathione peroxidase, and thiobarbituric acid reactive substance peaked at 6 hours. The SM exposure also resulted in a loss of the cellular membrane, leakage of cytoplasm, fuzzy mitochondrial cristae, medullary changes in ciliated and goblet cells, and the nuclear chromatin appeared marginated in basal cells and fibroblasts. The results in the propylene glycol group were the same as the control group. These data demonstrated the histologic changes, inflammatory reactions, apoptosis, oxidative stress, and DNA damage following SM (2 mg/kg)-induced acute tracheal injury; the severity of changes was time dependent. PMID:25163474

  15. Controlled Cavitation to Augment SWL Stone Comminution: Mechanistic Insights In-Vitro

    PubMed Central

    Duryea, Alexander P.; Roberts, William W.; Cain, Charles A.; Hall, Timothy L.

    2013-01-01

    Stone comminution in shock wave lithotripsy (SWL) has been documented to result from mechanical stresses conferred directly to the stone, as well as the activity of cavitational microbubbles. Studies have demonstrated that the presence of this cavitation activity is crucial for stone subdivision; however, its exact role in the comminution process remains somewhat weakly defined, in part due to the fact that it is difficult to isolate the cavitational component from the shock waves themselves. In this study, we further explored the importance of cavitation in SWL stone comminution through the use of histotripsy ultrasound therapy. Histotripsy was utilized to target model stones designed to mimic the mid-range tensile fracture strength of naturally occurring cystine calculi with controlled cavitation at strategic time points in the SWL comminution process. All SWL was applied at a peak-positive pressure (p+) of 34 MPa and a peak-negative pressure (p−) of 8 MPa; a shock rate of 1 Hz was used. Histotripsy pulses had a p− of 33 MPa and were applied at a pulse repetition frequency (PRF) of 100 Hz. Ten model stones were sonicated in-vitro with each of five different treatment schemes: A. 10 minutes SWL (600 shocks) with 0.7 seconds of histotripsy interleaved between successive shocks (totaling to 42,000 pulses); B. 10 minutes SWL (600 shocks) followed by 10 minutes histotripsy applied in 0.7 second bursts (1 burst per second, totaling to 42,000 pulses); C. 10 minutes histotripsy applied in 0.7 second bursts (42,000 pulses) followed by 10 minutes SWL (600 shocks); D. 10 minutes SWL-only (600 shocks); E. 10 minutes histotripsy-only applied in 0.7 second bursts (42,000 pulses). Following sonication, debris was collected and sieved through 8, 6, 4, and 2 mm filters. It was found that SWL-only generated a broad range of fragment sizes, with an average of 14.9 ± 24.1% of the original stone mass remaining >8 mm. Histotripsy-only eroded the surface of stones to tiny particulate debris that was small enough to pass through the finest filter used in this study (<2 mm), leaving behind a single primary stone piece (>8 mm) with mass 85.1 ± 1.6% of the original following truncated sonication. The combination of SWL and histotripsy (schemes A, B, and C) resulted in a shift in the size distribution toward smaller fragments and complete elimination of debris >8 mm. When histotripsy controlled cavitation was applied following SWL (B), the increase in exposed stone surface area afforded by shock wave stone subdivision led to enhanced cavitation erosion. When histotripsy controlled cavitation was applied prior to SWL (C), it is likely that stone surface defects induced by cavitation erosion provided sights for crack nucleation and accelerated shock wave stone subdivision. Both these effects are likely at play in the interleaved therapy (A), although shielding of shock waves by remnant histotripsy microbubble nuclei may have limited the efficacy of this scheme. Nevertheless, these results demonstrate the important role played by cavitation in the stone comminution process, and suggest that the application of controlled cavitation at strategic time points can provide an adjunct to traditional SWL therapy. PMID:23357904

  16. Structural and mechanistic insights into phospholipid transfer by Ups1-Mdm35 in mitochondria

    NASA Astrophysics Data System (ADS)

    Watanabe, Yasunori; Tamura, Yasushi; Kawano, Shin; Endo, Toshiya

    2015-08-01

    Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1-Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1-Mdm35 with and without PA. The Ups1-Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes.

  17. Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acid

    SciTech Connect

    Miranda-Rojas, Sebastián; Toro-Labbé, Alejandro

    2015-05-21

    Fluoroacetate is a toxic compound whose environmental accumulation may represent an important contamination problem, its elimination is therefore a challenging issue. Fluoroacetate dehalogenase catalyzes its degradation through a two step process initiated by an S{sub N}2 reaction in which the aspartate residue performs a nucleophilic attack on the carbon bonded to the fluorine; the second step is hydrolysis that releases the product as glycolate. In this paper, we present a study based on density functional theory calculations of the S{sub N}2 initiation reaction modeled through the interaction between the substrate and the propionate anion as the nucleophile. Results are analyzed within the framework of the reaction force and using the reaction electronic flux to identify and characterize the electronic activity that drives the reaction. Our results reveal that the selective protonation of the substrate catalyzes the reaction by decreasing the resistance of the structural and electronic reorganization needed to reach the transition state. Finally, the reaction energy is modulated by the degree of stabilization of the fluoride anion formed after the S{sub N}2 reaction. In this way, a site-induced partial protonation acts as a chemical switch in a key process that determines the output of the reaction.

  18. Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging

    NASA Astrophysics Data System (ADS)

    Gao, Jing; Wang, Ye; Cai, Mingjun; Pan, Yangang; Xu, Haijiao; Jiang, Junguang; Ji, Hongbin; Wang, Hongda

    2015-01-01

    The clustering of membrane receptors such as EGFR is critical for various biological processes, for example cell signaling and tumorigenesis. However, the mechanism involved remains poorly understood. Here, we used a super resolution imaging technique, which has shattered the longstanding resolution barrier of light diffraction, to investigate the distribution of membrane EGFR on apical or basal surfaces of COS-7 cells and on the surface of suspended COS-7 cells. Our data show that more and larger EGFR clusters are detected on the apical surface in comparison with those on the basal surface and this difference is not affected by the EGFR activation state, whereas suspended COS-7 cells exhibit a moderate clustering state and a homogeneous distribution pattern, indicating that the external environment surrounding the cell membrane is the decisive factor in the EGFR clustering pattern. A dual-color dSTORM image reveals the significant colocalization of EGFR and lipid rafts; interestingly MβCD treatment leads to a dramatic decrease of the amount and size of EGFR clusters on both apical and basal surfaces, highlighting a key role of lipid rafts in EGFR cluster formation. Altogether, our results illustrate the distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance.The clustering of membrane receptors such as EGFR is critical for various biological processes, for example cell signaling and tumorigenesis. However, the mechanism involved remains poorly understood. Here, we used a super resolution imaging technique, which has shattered the longstanding resolution barrier of light diffraction, to investigate the distribution of membrane EGFR on apical or basal surfaces of COS-7 cells and on the surface of suspended COS-7 cells. Our data show that more and larger EGFR clusters are detected on the apical surface in comparison with those on the basal surface and this difference is not affected by the EGFR activation state, whereas suspended COS-7 cells exhibit a moderate clustering state and a homogeneous distribution pattern, indicating that the external environment surrounding the cell membrane is the decisive factor in the EGFR clustering pattern. A dual-color dSTORM image reveals the significant colocalization of EGFR and lipid rafts; interestingly MβCD treatment leads to a dramatic decrease of the amount and size of EGFR clusters on both apical and basal surfaces, highlighting a key role of lipid rafts in EGFR cluster formation. Altogether, our results illustrate the distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04962d

  19. Nanoscale Silicon as a Catalyst for Graphene Growth: Mechanistic Insight from in Situ Raman Spectroscopy

    DOE PAGESBeta

    Share, Keith; Carter, Rachel E.; Nikolaev, Pavel; Hooper, Daylong; Oakes, Landon; Cohn, Adam P.; Rao, Rahul; Puretzky, Alexander A.; Geohegan, David B.; Maruyama, Benji; et al

    2016-06-08

    Nanoscale carbons are typically synthesized by thermal decomposition of a hydrocarbon at the surface of a metal catalyst. Whereas the use of silicon as an alternative to metal catalysts could unlock new techniques to seamlessly couple carbon nanostructures and semiconductor materials, stable carbide formation renders bulk silicon incapable of the precipitation and growth of graphitic structures. In this article, we provide evidence supported by comprehensive in situ Raman experiments that indicates nanoscale grains of silicon in porous silicon (PSi) scaffolds act as catalysts for hydrocarbon decomposition and growth of few-layered graphene at temperatures as low as 700 K. Self-limiting growthmore » kinetics of graphene with activation energies measured between 0.32–0.37 eV elucidates the formation of highly reactive surface-bound Si radicals that aid in the decomposition of hydrocarbons. Nucleation and growth of graphitic layers on PSi exhibits striking similarity to catalytic growth on nickel surfaces, involving temperature dependent surface and subsurface diffusion of carbon. Lastly, this work elucidates how the nanoscale properties of silicon can be exploited to yield catalytic properties distinguished from bulk silicon, opening an important avenue to engineer catalytic interfaces combining the two most technologically important materials for modern applications—silicon and nanoscale carbons.« less

  20. Hollow alloy nanostructures templated by Au nanorods: synthesis, mechanistic insights, and electrocatalytic activity.

    PubMed

    Xue, Mengmeng; Tan, Yiwei

    2014-11-01

    A unique methodology having access to Au nanorods (AuNRs)-based hollow alloy nanostructures has been developed. The syntheses and characterization of the hollow Pt-Au nanoalloys with ellipsoidal and cylindrical shapes together with a rattle-type hollow Cu-Au nanoheterostructure are described. Unlike the conventional nanoscale Kirkendall process, the formation of these AuNRs-based hollow nanostructures occurs under extremely mild conditions, indicating a distinctive underlying mechanism. The key step for this present synthesis method is the incubation of AuNRs with CuCl2 at 60 °C in the presence of hexadecyltrimethylammonium bromide (CTAB) or hexadecyltrimethylammonium chloride (CTAC). The selective etching of the tips of AuNRs caused by Cu(2+) ions combined with the dissolved molecular oxygen promotes the generation of defects and vacancies, leading to a facile alloying reaction by the crystal fusion of AuNRs. Particularly, the results of the formation of the hollow nanoalloys in conjunction with various control experiments demonstrate that the halide ions that are specifically adsorbed on the AuNR surface afford sinks for vacancy accumulation and condensation during the unbalanced interdiffusion of alloying atoms, presumably because of the disproportion in the equilibrium concentration of vacancies. Thus, the void formation becomes kinetically favorable. The Pt-Au nanocages can provide modified surface electronic structures, resulting from their non-uniform crystalline structures and the surface segregation of Pt in the nanocages. These characteristics enable them to exhibit excellent electrocatalytic performance for the oxygen reduction reaction (ORR). PMID:25166262

  1. Nanoscale Silicon as a Catalyst for Graphene Growth: Mechanistic Insight from in Situ Raman Spectroscopy

    SciTech Connect

    Share, Keith; Carter, Rachel; Nikolaev, Pavel; Hooper, Daylong; Oakes, Landon; Cohn, Adam; Rao, Rahul; Puretzky, Alexander A; Geohegan, David; Maruyama,; Pint, Cary

    2016-01-01

    Nanoscale carbons are typically synthesized by thermal decomposition of a hydrocarbon at the surface of a metal catalyst.1,2 Whereas the use of silicon as an alternative to metal catalyst could unlock new techniques to seamlessly couple carbon nanostructures and semiconductor materials, stable carbide formation in bulk silicon prevents the precipitation and growth of graphitic structures.3,4 Here, we provide evidence supported by comprehensive in-situ Raman experiments that indicates nanoscale grains of silicon in porous silicon scaffolds act as catalysts for hydrocarbon decomposition and growth of few-layered graphene materials at temperatures as low as 700 K. Self-limiting growth kinetics of carbon with activation energies measured between 0.32 0.37 eV elucidates the formation of highly reactive surface-bound Si radicals that aid in the decomposition of hydrocarbons. Nucleation and growth of graphitic carbon layers on porous silicon exhibits striking similarity to catalytic growth on nickel surfaces, involving temperature dependent surface and subsurface diffusion of carbon. This work elucidates how the nanoscale properties of silicon can be exploited to yield catalytic properties distinguished from bulk materials, opening an important avenue to engineer catalytic interfaces combining the two most technologically-important materials for modern applications silicon and nanoscale carbons.

  2. Mechanistic insight into alkylation of the ethyl acetoacetate anion with different ethyl halides

    NASA Astrophysics Data System (ADS)

    Marković, S.; Đurđević, J.; Vukosavljević, M.; Petrović, Z.

    2013-12-01

    The alkylation reactions of the ambident ethyl acetoacetate anion with C2H5X (X = F, Cl, Br, and I) in the O2, C3, and O4 positions of the anion were investigated at the B3LYP/6-311+G( d,p) level of theory. It was found that the ethylation reaction does not occur in the position O4, as well as with ethyl fluoride in any position of the anion, due to very high activation energies and thermodynamic instability of the hypothetic products. The activation energies for the reactions in the position O2 are lower in comparison to the position C3, but the products of the reactions in the C3 position are more stable than those in the position O4, implying that the C/O products ratio is controlled by both thermodynamic and kinetic factors, leading to the O2-product with the chloride, and C3-product with the iodide as leaving group.

  3. Mechanistic insight into the hydrazine decomposition on Rh(111): effect of reaction intermediate on catalytic activity.

    PubMed

    Deng, Zhigang; Lu, Xiaoqing; Wen, Zengqiang; Wei, Shuxian; Liu, Yunjie; Fu, Dianling; Zhao, Lianming; Guo, Wenyue

    2013-10-14

    Periodic density functional theory (DFT) calculations have been performed to systematically investigate the effect of reaction intermediate on catalytic activity for hydrazine (N2H4) decomposition on Rh(111). Reaction mechanisms via intramolecular and NH2-assisted N2H4 decompositions are comparatively analyzed, including adsorption configuration, reaction energy and barrier of elementary step, and reaction network. Our results show that the most favorable N2H4 decomposition pathway starts with the initial N-N bond scission to the NH2 intermediate, followed by stepwise H stripping from adsorbed N2Hx (x = 1-4) species, and finally forms the N2 and NH3 products. Comparatively, the stepwise intramolecular dehydrogenation via N2H4→ N2H3→ N2H2→ N2H → N2, and N2H4→ NH2→ NH → N with or without NH2 promotion effect, are unfavorable due to higher energy barriers encountered. Energy barrier analysis, reaction rate constants, and electronic structures are used to identify the crucial competitive route. The promotion effect of the NH2 intermediate is structurally reflected in the weakening of the N-H bond and strengthening of the N-N bond in N2Hx in the coadsorption system; it results intrinsically from the less structural deformation of the adsorbate, and weakening of the interaction between dehydrogenated fragment and departing H in transition state. Our results highlight the crucial effect of reaction intermediate on catalytic activity and provide a theoretical approach to analyze the effect. PMID:23990024

  4. Mechanistic insights into the recognition of 5-methylcytosine oxidation derivatives by the SUVH5 SRA domain

    PubMed Central

    Rajakumara, Eerappa; Nakarakanti, Naveen Kumar; Nivya, M. Angel; Satish, Mutyala

    2016-01-01

    5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives. PMID:26841909

  5. Role of Glyoxalase 1 (Glo1) and methylglyoxal (MG) in behavior: recent advances and mechanistic insights

    PubMed Central

    Distler, Margaret G.; Palmer, Abraham A.

    2012-01-01

    Glyoxalase 1 (GLO1) is a ubiquitous cellular enzyme that participates in the detoxification of methylglyoxal (MG), a cytotoxic byproduct of glycolysis that induces protein modification (advanced glycation end-products, AGEs), oxidative stress, and apoptosis. The concentration of MG is elevated under high-glucose conditions, such as diabetes. As such, GLO1 and MG have been implicated in the pathogenesis of diabetic complications. Recently, findings have linked GLO1 to numerous behavioral phenotypes, including psychiatric diseases (anxiety, depression, schizophrenia, and autism) and pain. This review highlights GLO1's association with behavioral phenotypes, describes recent discoveries that have elucidated the underlying mechanisms, and identifies opportunities for future research. PMID:23181072

  6. Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission.

    PubMed

    Santiago, Julia; Brandt, Benjamin; Wildhagen, Mari; Hohmann, Ulrich; Hothorn, Ludwig A; Butenko, Melinka A; Hothorn, Michael

    2016-01-01

    Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. PMID:27058169

  7. Mechanistic and Structural Insights into the Prion-Disaggregase Activity of Hsp104.

    PubMed

    Sweeny, Elizabeth A; Shorter, James

    2016-05-01

    Hsp104 is a dynamic ring translocase and hexameric AAA+ protein found in yeast, which couples ATP hydrolysis to disassembly and reactivation of proteins trapped in soluble preamyloid oligomers, disordered protein aggregates, and stable amyloid or prion conformers. Here, we highlight advances in our structural understanding of Hsp104 and how Hsp104 deconstructs Sup35 prions. Although the atomic structure of Hsp104 hexamers remains uncertain, volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (ATP hydrolysis transition-state mimic), and ADP via small-angle x-ray scattering has revealed a peristaltic pumping motion upon ATP hydrolysis. This pumping motion likely drives directional substrate translocation across the central Hsp104 channel. Hsp104 initially engages Sup35 prions immediately C-terminal to their cross-β structure. Directional pulling by Hsp104 then resolves N-terminal cross-β structure in a stepwise manner. First, Hsp104 fragments the prion. Second, Hsp104 unfolds cross-β structure. Third, Hsp104 releases soluble Sup35. Deletion of the Hsp104 N-terminal domain yields a hypomorphic disaggregase, Hsp104(∆N), with an altered pumping mechanism. Hsp104(∆N) fragments Sup35 prions without unfolding cross-β structure or releasing soluble Sup35. Moreover, Hsp104(∆N) activity cannot be enhanced by mutations in the middle domain that potentiate disaggregase activity. Thus, the N-terminal domain is critical for the full repertoire of Hsp104 activities. PMID:26608812

  8. Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights.

    PubMed

    Melo, Tahira Souza; Gattass, Cerli Rocha; Soares, Deivid Costa; Cunha, Micael Rodrigues; Ferreira, Christian; Tavares, Maurício Temotheo; Saraiva, Elvira; Parise-Filho, Roberto; Braden, Hannah; Delorenzi, Jan Carlo

    2016-06-01

    Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC(50 L. braziliensis) 30.47 ± 6.35 μM; IC(50 L. amazonensis) 40.46 ± 14.21 μM; IC(50 L. infantum) 65.93 ± 15.12 μM) and amastigotes (IC(50 L. braziliensis) 68.75 ± 16.55 μM; IC(50 L. amazonensis) 38.45 ± 12.05 μM; IC(50 L. infantum) 64.08 ± 23.52 μM), with low cytotoxicity against mouse peritoneal macrophages (CC(50) 235.80 ± 36.95 μM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment. PMID:26772973

  9. Eukaryotic selenoprotein synthesis: mechanistic insight incorporating new factors and new functions for old factors.

    PubMed

    Squires, Jeffrey E; Berry, Marla J

    2008-04-01

    Selenium is an essential micronutrient that has been linked to various aspects of human health. Selenium exerts its biological activity through the incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of specific secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Herein, we discuss the various proteins known to function in eukaryotic selenoprotein biosynthesis, including several players whose roles have only been elucidated very recently. PMID:18344183

  10. Mechanistic insights into mode of action of novel natural cathepsin L inhibitors

    PubMed Central

    2013-01-01

    Background Development of a cancerous cell takes place when it ceases to respond to growth-inhibiting signals and multiplies uncontrollably and can detach and move to other parts of the body; the process called as metastasis. A particular set of cysteine proteases are very active during cancer metastasis, Cathepsins being one of them. They are involved in tumor growth and malignancy and have also been reported to be overexpressed in tumor cell lines. In the present study, a combinatorial approach comprising three-dimensional quantitative structure-activity relationship (3D QSAR), ligand-based pharmacophore modelling and search followed by cathepsin L structure-based high throughput screening was carried out using an initial set of 28 congeneric thiosemicarbazone derivatives as cathepsin L inhibitors. A 3D QSAR was derived using the alignment of a common thiosemicarbazone substructure. Essential structural features responsible for biological activity were taken into account for development of a pharmacophore model based on 29 congeneric thiosemicarbazone derivatives. This model was used to carry out an exhaustive search on a large dataset of natural compounds. A further cathepsin L structure-based screen identified two top scoring compounds as potent anti-cancer leads. Results The generated 3D QSAR model showed statistically significant results with an r2 value of 0.8267, cross-validated correlation coefficient q2 of 0.7232, and a pred_r2 (r2 value for test set) of 0.7460. Apart from these, a high F test value of 30.2078 suggested low probability of the model's failure. The pharmacophoric hypothesis chosen for searching the natural compound libraries was identified as DDHRR, where two Ds denote 2 hydrogen donors, H represents a hydrophobic group and two Rs represent aromatic rings, all of which are essential for the biological activity. We report two potential drug leads ZINC08764437 (NFP) and ZINC03846634 (APQ) obtained after a combined approach of pharmacophore-based search and structure-based virtual screen. These two compounds displayed extra precision docking scores of -7.972908 and -7.575686 respectively suggesting considerable binding affinity for cathepsin L. High activity values of 5.72 and 5.75 predicted using the 3D QSAR model further substantiated the inhibitory potential of these identified leads. Conclusion The present study attempts to correlate the structural features of thiosemicarbazone group with their biological activity by development of a robust 3D QSAR model. Being statistically valid, this model provides near accurate values of the activities predicted for the congeneric set on which it is based. These predicted activities are good for the test set compounds making it indeed a statistically sound 3D QSAR model. The identified pharmacophore model DDHRR.8 comprised of all the essential features required to interact with the catalytic triad of cathepsin L. A search for natural compounds based on this pharmacophore followed by docking studies further screened out two top scoring candidates: NFP and AFQ. The high binding affinity and presence of essential structural features in these two compounds make them ideal for consideration as natural anti-tumoral agents. Activity prediction using 3D QSAR model further validated their potential as worthy drug candidates against cathepsin L for treatment of cancer. PMID:24564425

  11. Computational Modeling of Human Paraoxonase 1: Preparation of Protein Models, Binding Studies, and Mechanistic Insights

    PubMed Central

    Sanan, Toby T.; Muthukrishnan, Sivaramakrishnan; Beck, Jeremy M.; Tao, Peng; Hayes, Carrigan J.; Otto, Tamara C.; Cerasoli, Douglas M.; Lenz, David E.; Hadad, Christopher M.

    2013-01-01

    The enzyme human paraoxonase 1 (huPON1) has demonstrated significant potential for use as a bioscavenger for treatment of exposure to organophosphorus (OP) nerve agents. Herein we report the development of protein models for the human isoform derived from a crystal structure of a chimeric version of the protein (pdb ID: 1V04) and a homology model derived from the related enzyme diisopropylfluorophosphatase (pdb ID: 1XHR). From these structural models, binding modes for OP substrates are predicted, and these poses are found to orient substrates in proximity to residues known to modulate specificity of the enzyme. Predictions are made with regard to the role that residues play in altering substrate binding and turnover, in particular with regard to the stereoselectivity of the enzyme, and the known differences in activity related to a natural polymorphism in the enzyme. Potential mechanisms of action of the protein for catalytic hydrolysis of OP substrates are also evaluated in light of the proposed binding modes. PMID:24077808

  12. Potential Anticancer Heterometallic Fe-Au and Fe-Pd Agents: Initial Mechanistic Insights

    PubMed Central

    Lease, Nicholas; Vasilevski, Vadim; Carreira, Monica; de Almeida, Andreia; Sanaú, Mercedes; Hirva, Pipsa; Casini, Angela; Contel, Maria

    2013-01-01

    A series of gold(III) and palladium(II) heterometallic complexes with new iminophosphorane ligands derived from ferrocenyl-phosphanes [{Cp-P(Ph2)=N-Ph}2Fe] (1), [{Cp-P(Ph2)=N-CH2-2-NC5H4}2Fe] (2) and [{Cp-P(Ph2)=N-CH2-2-NC5H4}Fe(Cp)] (3) have been synthesized and structurally characterized. Ligands 2 and 3 afford stable coordination complexes [AuCl2(3)]ClO4, [{AuCl2}2(2)](ClO4)2, [PdCl2(3)] and [{PdCl2}2(2)]. The complexes have been evaluated for their antripoliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), in human breast cancer cells (MCF7) and in a non-tumorigenic human embryonic kidney cell line (HEK-293T). The highly cytotoxic trimetallic derivatives M2Fe (M = Au, Pd) are more cytotoxic to cancer cells than their corresponding monometallic fragments. Moreover, these complexes were significantly more cytotoxic than cisplatin in the resistant A2780R and the MCF7 cell lines. Studies of the interactions of the trimetallic compounds with DNA and the zinc-finger protein PARP-1 indicate that they exert anticancer effects in vitro based on different mechanisms of actions with respect to cisplatin. PMID:23786413

  13. Nickel-Catalyzed Allylic Alkylation with Diarylmethane Pronucleophiles: Reaction Development and Mechanistic Insights.

    PubMed

    Sha, Sheng-Chun; Jiang, Hui; Mao, Jianyou; Bellomo, Ana; Jeong, Soo A; Walsh, Patrick J

    2016-01-18

    Palladium-catalyzed allylic substitution reactions are among the most efficient methods to construct C-C bonds between sp(3)-hybridized carbon atoms. In contrast, much less work has been done with nickel catalysts, perhaps because of the different mechanisms of the allylic substitution reactions. Palladium catalysts generally undergo substitution by a "soft"-nucleophile pathway, wherein the nucleophile attacks the allyl group externally. Nickel catalysts are usually paired with "hard" nucleophiles, which attack the metal before C-C bond formation. Introduced herein is a rare nickel-based catalyst which promotes substitution with diarylmethane pronucleophiles by the soft-nucleophile pathway. Preliminary studies on the asymmetric allylic alkylation are promising. PMID:26756444

  14. A Comprehensive Review on the Chemotherapeutic Potential of Piceatannol for Cancer Treatment, with Mechanistic Insights.

    PubMed

    Seyed, Mohamed Ali; Jantan, Ibrahim; Bukhari, Syed Nasir Abbas; Vijayaraghavan, Kavitha

    2016-02-01

    Cancer is a diverse class of diseases characterized by uncontrolled cell growth that constitutes the greatest cause of mortality and morbidity worldwide. Despite steady progress, the treatment modalities of cancer are still insufficient. Several new concepts have emerged for therapeutic intervention in malignant diseases with the goal of identifying specific targets and overcoming resistance against current cytotoxic therapies. Many studies have reported the remarkable and significant properties of dietary plant polyphenols such as curcumin, resveratrol, flavopiridol, indirubin, magnolol, piceatannol, parthenolide, epigallocatechin gallate, and cucurbitacin as anticancer agents known for their pleiotropic effects on cancer, immune cells, and inflammation. Piceatannol, an analogue and metabolite of resveratrol, is a natural stilbene commonly found in grape skins and wine. Compared to resveratrol, this molecule exhibits superior bioactivities as an inhibitor of COX-1/2 and the CSN-associated kinase. Piceatannol is thought to be a potent natural compound with many therapeutic effects, such as the prevention of hypercholesterolemia, arrhythmia, atherosclerosis, angiogenesis, and cardiovascular diseases. It also demonstrates vasorelaxation, antioxidant, and anticancer activities. This comprehensive review summarizes the current data regarding the mechanisms of action of piceatannol, its chemopreventive properties, and its possible therapeutic potential against various types of human cancer. PMID:26758628

  15. Mechanistic insight into the dermal delivery from nanoparticle-coated submicron O/W emulsions.

    PubMed

    Eskandar, Nasrin Ghouchi; Simovic, Spomenka; Prestidge, Clive A

    2010-02-01

    The influence of silica nanoparticle coating of negatively and positively charged submicron emulsion oil droplets on the dermal delivery of a lipophilic fluorescent probe, acridine orange 10-nonyl bromide (AONB) using an ex vivo porcine skin model is reported. The skin retention and depth of the penetration of AONB significantly increased (p

  16. Mechanistic Insights into the Structure-Dependent Selectivity of Catalytic Furfural Conversion on Platinum Catalysts

    SciTech Connect

    Cai, Qiuxia; Wang, Jianguo; Wang, Yang-Gang; Mei, Donghai

    2015-11-01

    The effects of structure and size on the selectivity of catalytic furfural conversion over supported Pt catalysts in the presence of hydrogen have been studied using first principles density functional theory (DFT) calculations and microkinetic modeling. Four Pt model systems, i.e., periodic Pt(111), Pt(211) surfaces, as well as small nanoclusters (Pt13 and Pt55) are chosen to represent the terrace, step, and corner sites of Pt nanoparticles. Our DFT results show that the reaction routes for furfural hydrogenation and decarbonylation are strongly dependent on the type of reactive sites, which lead to the different selectivity. On the basis of the size-dependent site distribution rule, we correlate the site distributions as a function of the Pt particle size. Our microkinetic results indicate the critical particle size that controls the furfural selectivity is about 1.0 nm, which is in good agreement with the reported experimental value under reaction conditions. This work was supported by National Basic Research Program of China (973 Program) (2013CB733501) and the National Natural Science Foundation of China (NSFC-21306169, 21176221, 21136001, 21101137 and 91334103). This work was also partially supported by the US Department of Energy (DOE), the Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences. Pacific Northwest National Laboratory (PNNL) is a multiprogram national laboratory operated for DOE by Battelle. Computing time was granted by the grand challenge of computational catalysis of the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL). EMSL is a national scientific user facility located at Pacific Northwest National Laboratory (PNNL) and sponsored by DOE’s Office of Biological and Environmental Research.

  17. Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA

    PubMed Central

    Song, Wooseok; Kim, Jin-Sik; Jiao, Li; Lee, Kangseok

    2015-01-01

    The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-Å resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site. PMID:26025888

  18. Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission

    PubMed Central

    Santiago, Julia; Brandt, Benjamin; Wildhagen, Mari; Hohmann, Ulrich; Hothorn, Ludwig A; Butenko, Melinka A; Hothorn, Michael

    2016-01-01

    Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. DOI: http://dx.doi.org/10.7554/eLife.15075.001 PMID:27058169

  19. Energetics of methanol and formic acid oxidation on Pt(111): Mechanistic insights from adsorption calorimetry

    NASA Astrophysics Data System (ADS)

    Silbaugh, Trent L.; Karp, Eric M.; Campbell, Charles T.

    2016-08-01

    The catalytic and electrocatalytic oxidation and reforming of methanol and formic acid have received intense interest due to potential use in direct fuel cells and as prototype models for understanding electrocatalysis. Consequently, the reaction energy diagram (energies of all the adsorbed intermediates and activation energies of all the elementary steps) have been estimated for these reactions on Pt(111) by density functional theory (DFT) in several studies. However, no experimental measurement of these energy diagrams have been reported, nor is there a consensus on the mechanisms. Here, we use energies of key intermediates on Pt(111) from single crystal adsorption calorimetry (SCAC) and temperature programmed desorption (TPD) to build a combined energy diagram for these reactions. It suggests a new pathway involving monodentate formate as a key intermediate, with bidentate formate only being a spectator species that slows the rate. This helps reconcile conflicting proposed mechanisms.

  20. Mechanistic insight of bivalent compound 21MO as potential neuroprotectant for Alzheimer’s disease

    PubMed Central

    Saathoff, John M.; Liu, Kai; Chojnacki, Jeremy E.; He, Liu; Chen, Qun; Lesnefsky, Edward J.; Zhang, Shijun

    2016-01-01

    We have recently developed a bivalent strategy to provide novel compounds that potentially target multiple risk factors involved in the development of Alzheimer’s disease (AD). Our previous studies employing a bivalent compound with a shorter spacer (17MN) implicated that this compound can localize into mitochondria and endoplasmic reticulum (ER), thus interfering with the change of mitochondria membrane potential (ΔΨm) and Ca2+ levels in MC65 cells upon removal of tetracycline (TC). In this report, we examined the effects by a bivalent compound with a longer spacer (21MO) in MC65 cells. Our results demonstrated that 21MO suppressed the change of ΔΨm, possibly via interaction with the mitochondrial complex I in MC65 cells. Interestingly, 21MO did not show any effects on the Ca2+ level upon TC removal in MC65 cells. Our previous studies suggested that the mobilization of Ca2+ in MC65 cells, upon withdraw of TC, is originated from ER, so the results implicated that 21MO may preferentially interact with mitochondria in MC65 cells under the current experimental conditions. Collectively, the results suggest that bivalent compounds with varied spacer length and cell membrane anchor moiety may exhibit neuroprotective activities via different mechanisms of action. PMID:27023508

  1. Mechanistic Insight into the Enzymatic Reduction of Truncated Hemoglobin N of Mycobacterium tuberculosis

    PubMed Central

    Singh, Sandeep; Thakur, Naveen; Oliveira, Ana; Petruk, Ariel A.; Hade, Mangesh Dattu; Sethi, Deepti; Bidon-Chanal, Axel; Martí, Marcelo A.; Datta, Himani; Parkesh, Raman; Estrin, Dario A.; Luque, F. Javier; Dikshit, Kanak L.

    2014-01-01

    Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 μm/min−1, which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly48 may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10−5 eV, which is lower than the value obtained for E. coli flavoHb (8.0 10−4 eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function. PMID:24928505

  2. Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake

    PubMed Central

    Wang, Lele; Yang, Xue; Li, Siwei; Wang, Zheng; Liu, Yu; Feng, Jianrong; Zhu, Yushan; Shen, Yuequan

    2014-01-01

    Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca2+ uniporter (MCU), which is the pore-forming subunit of a Ca2+ channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca2+-free and Ca2+-bound human MICU1. Our studies reveal that Ca2+-free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca2+ binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca2+ is approximately 15–20 μM. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake. PMID:24514027

  3. Mechanistic Insight with HBCH2CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases

    PubMed Central

    2015-01-01

    Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1–2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [3H]-saturated trimer-CoA ([3H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [3H]-sTCoA ([3H]-sT-CH2-CoA), saturated dimer-([3H]-sD-CO2H), and trimer-acid ([3H]-sT-CO2H), distinct from the expected methylene analogue of [3H]-saturated tetramer-CoA ([3H]-sTet-CH2-CoA). Detection of [3H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation. PMID:24896226

  4. Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights.

    PubMed

    Dong, Bamboo; Sánchez-Magraner, Lissete; Luecke, Hartmut

    2016-09-01

    Microbial rhodopsins are light-activated, seven-α-helical, retinylidene transmembrane proteins that have been identified in thousands of organisms across archaea, bacteria, fungi, and algae. Although they share a high degree of sequence identity and thus similarity in structure, many unique functions have been discovered and characterized among them. Some function as outward proton pumps, some as inward chloride pumps, whereas others function as light sensors or ion channels. Unique among the microbial rhodopsins characterized thus far, Anabaena sensory rhodopsin (ASR) is a photochromic sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is encoded on the same operon. The sensor itself stably interconverts between all-trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of illumination, although only the former participates in a photocycle with a signaling M intermediate. A mutation in the cytoplasmic half-channel of the protein, replacing Asp217 with Glu (D217E), results in the creation of a light-driven, single-photon, inward proton transporter. We present the 2.3 Å structure of dark-adapted D217E ASR, which reveals significant changes in the water network surrounding Glu217, as well as a shift in the carbon backbone near retinal-binding Lys210, illustrating a possible pathway leading to the protonation of Glu217 in the cytoplasmic half-channel, located 15 Å from the Schiff base. Crystallographic evidence for the protonation of nearby Glu36 is also discussed, which was described previously by Fourier transform infrared spectroscopy analysis. Finally, two histidine residues near the extracellular surface and their possible role in proton uptake are discussed. PMID:27602724

  5. Computational and Experimental Insights into the Mechanism of Substrate Recognition and Feedback Inhibition of Protoporphyrinogen Oxidase

    PubMed Central

    Yang, Sheng-Gang; Wang, Zhi-Fang; Zhan, Chang-Guo; Xi, Zhen; Yang, Guang-Fu

    2013-01-01

    Protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) is an essential enzyme catalyzing the last common step in the pathway leading to heme and chlorophyll biosynthesis. Great interest in PPO inhibitors arises from both its significance to agriculture and medicine. However, the discovery of PPO inhibitors with ultrahigh potency and selectivity is hampered due to lack of structural and mechanistic understanding about the substrate recognition, which remains a longstanding question central in porphyrin biology. To understand the mechanism, a novel binding model of protogen (protoporphyrinogen IX, the substrate) was developed through extensive computational simulations. Subsequently, amino acid residues that are critical for protogen binding identified by computational simulations were substituted by mutagenesis. Kinetic analyses of these mutants indicated that these residues were critical for protogen binding. In addition, the calculated free energies of protogen binding with these mutants correlated well with the experimental data, indicating the reasonability of the binding model. On the basis of this novel model, the fundamental mechanism of substrate recognition was investigated by performing potential of mean force (PMF) calculations, which provided an atomic level description of conformational changes and pathway intermediates. The free energy profile revealed a feedback inhibition mechanism of proto (protoporphyrin IX, the product), which was also in agreement with experimental evidence. The novel mechanistic insights obtained from this study present a new starting point for future rational design of more efficient PPO inhibitors based on the product-bound PPO structure. PMID:23935953

  6. Additional insights. Commentary on “the musical stroop effect: opening a new avenue to research on automatisms” by l. Grégoire, P. Perruchet, and B. Poulin-Charronnat (Experimental Psychology, 2013, vol. 60, pp. 269–278).

    PubMed

    Akiva-Kabiri, Lilach; Henik, Avishai

    2014-01-01

    In their paper "The Musical Stroop Effect: Opening a New Avenue to Research on Automatisms," Grégoire, Perruchet, and Poulin-Charronnat (2013) use a musical Stroop-like task to demonstrate the automaticity of musical note naming in musicians. In addition, the authors suggest that music training can serve as a tool in order to study the acquisition of automaticity. In the following commentary, we aim to address three main issues concerning the paper by Grégoire et al. (2013). First, we will suggest some additional interpretations of the results; specifically, we will relate to the association between music and space. Second, we will discuss a methodological issue dealing with interference, facilitation, and the role of the neutral condition. We suggest that the study by Grégoire et al. (2013) lacks a proper neutral condition and thus it is impossible to assert that the congruency effect is interference based. Third, we will discuss the authors' suggestion of using the musical Stroop effect as a tool for studying automatism. We consider the practical relevance of music training as a tool for studying the acquisition of automaticity by pointing out that music training is highly heterogeneous. PMID:24449650

  7. Warming will affect phytoplankton differently: evidence through a mechanistic approach

    PubMed Central

    Huertas, I. Emma; Rouco, Mónica; López-Rodas, Victoria; Costas, Eduardo

    2011-01-01

    Although the consequences of global warming in aquatic ecosystems are only beginning to be revealed, a key to forecasting the impact on aquatic communities is an understanding of individual species' vulnerability to increased temperature. Despite their microscopic size, phytoplankton support about half of the global primary production, drive essential biogeochemical cycles and represent the basis of the aquatic food web. At present, it is known that phytoplankton are important targets and, consequently, harbingers of climate change in aquatic systems. Therefore, investigating the capacity of phytoplankton to adapt to the predicted warming has become a relevant issue. However, considering the polyphyletic complexity of the phytoplankton community, different responses to increased temperature are expected. We experimentally tested the effects of warming on 12 species of phytoplankton isolated from a variety of environments by using a mechanistic approach able to assess evolutionary adaptation (the so-called ratchet technique). We found different degrees of tolerance to temperature rises and an interspecific capacity for genetic adaptation. The thermal resistance level reached by each species is discussed in relation to their respective original habitats. Our study additionally provides evidence on the most resistant phytoplankton groups in a future warming scenario. PMID:21508031

  8. A brief review of exercise, bipolar disorder, and mechanistic pathways

    PubMed Central

    Thomson, Daniel; Turner, Alyna; Lauder, Sue; Gigler, Margaret E.; Berk, Lesley; Singh, Ajeet B.; Pasco, Julie A.; Berk, Michael; Sylvia, Louisa

    2015-01-01

    Despite evidence that exercise has been found to be effective in the treatment of depression, it is unclear whether these data can be extrapolated to bipolar disorder. Available evidence for bipolar disorder is scant, with no existing randomized controlled trials having tested the impact of exercise on depressive, manic or hypomanic symptomatology. Although exercise is often recommended in bipolar disorder, this is based on extrapolation from the unipolar literature, theory and clinical expertise and not empirical evidence. In addition, there are currently no available empirical data on program variables, with practical implications on frequency, intensity and type of exercise derived from unipolar depression studies. The aim of the current paper is to explore the relationship between exercise and bipolar disorder and potential mechanistic pathways. Given the high rate of medical co-morbidities experienced by people with bipolar disorder, it is possible that exercise is a potentially useful and important intervention with regard to general health benefits; however, further research is required to elucidate the impact of exercise on mood symptomology. PMID:25788889

  9. Modeling Bird Migration under Climate Change: A Mechanistic Approach

    NASA Technical Reports Server (NTRS)

    Smith, James A.

    2009-01-01

    behavior can be maintained over increasing and sustained environmental change. Also, the problem is much more complex than described by the current processes captured in our model. We have taken some important and interesting steps, and our model does demonstrate how local scale information about individual stop-over sites can be linked into the migratory flyway as a whole. We are incorporating additional, species specific, mechanistic processes to better reflect different climate change scenarios

  10. Application of Mechanistic Toxicology Data to Ecological Risk Assessments

    EPA Science Inventory

    The ongoing evolution of knowledge and tools in the areas of molecular biology, bioinformatics, and systems biology holds significant promise for reducing uncertainties associated with ecological risk assessment. As our understanding of the mechanistic basis of responses of organ...

  11. MECHANISTIC DOSIMETRY MODELS OF NANOMATERIAL DEPOSITION IN THE RESPIRATORY TRACT

    EPA Science Inventory

    Accurate health risk assessments of inhalation exposure to nanomaterials will require dosimetry models that account for interspecies differences in dose delivered to the respiratory tract. Mechanistic models offer the advantage to interspecies extrapolation that physicochemica...

  12. Bridging paradigms: hybrid mechanistic-discriminative predictive models.

    PubMed

    Doyle, Orla M; Tsaneva-Atansaova, Krasimira; Harte, James; Tiffin, Paul A; Tino, Peter; Díaz-Zuccarini, Vanessa

    2013-03-01

    Many disease processes are extremely complex and characterized by multiple stochastic processes interacting simultaneously. Current analytical approaches have included mechanistic models and machine learning (ML), which are often treated as orthogonal viewpoints. However, to facilitate truly personalized medicine, new perspectives may be required. This paper reviews the use of both mechanistic models and ML in healthcare as well as emerging hybrid methods, which are an exciting and promising approach for biologically based, yet data-driven advanced intelligent systems. PMID:23392334

  13. Catalytic Control in Cyclizations: From Computational Mechanistic Understanding to Selectivity Prediction.

    PubMed

    Peng, Qian; Paton, Robert S

    2016-05-17

    catalyst structures for enantioselective cycloisomerizations. Calculations have revealed that stepwise C-C bond formation and proton transfer dictate the exclusive endo diastereoselectivity of the intramolecular Michael addition to form 2-azabicyclo[3.3.1]nonane skeletons catalyzed by primary amines. These insights have led to development of a highly enantioselective catalyst with higher atom economy than previous generations. This Account also explores transition-metal-catalyzed cycloisomerizations, where our theoretical investigations have uncovered an unexpected reaction pathway in the [5 + 2] cycloisomerization of ynamides. This has led to the design of new phosphoramidite ligands to enable double-stereodifferentiating cycloisomerizations in both matched and mismatched catalyst-substrate settings. Computational understanding of the factors responsible for the regio-, enantio-, and diasterocontrol is shown to generate tangible predictions leading to an acceleration of catalyst development for selective cyclizations. PMID:27137131

  14. In Vitro Multitissue Interface Model Supports Rapid Vasculogenesis and Mechanistic Study of Vascularization across Tissue Compartments.

    PubMed

    Buno, Kevin P; Chen, Xuemei; Weibel, Justin A; Thiede, Stephanie N; Garimella, Suresh V; Yoder, Mervin C; Voytik-Harbin, Sherry L

    2016-08-31

    , are critical determinants of vascularization within a given tissue compartment and across tissue interfaces. This new in vitro multitissue interface model and the associated mechanistic insights it yields provide guiding principles for the design and optimization of multitissue vascularization strategies for research and clinical applications. PMID:27136321

  15. Mechanistic Studies of Combustion and Structure Formation During Synthesis of Advanced Materials

    NASA Technical Reports Server (NTRS)

    Varma, A.; Lau, C.; Mukasyan, A. S.

    2001-01-01

    reactants occurs within each particle, and mixtures of elemental powders, where interparticle contacts are important for the reaction; and 3) Mechanistic Studies of Phase Separation in Combustion of Thermite Systems. Studies are devoted to experiments on thermite systems (metal oxide-reducing metal) where phase separation processes occur to produce alloys with tailored compositions and properties. The separation may be either gravity-driven or due to surface forces, and systematic studies to elucidate the true mechanism are being conducted. The knowledge obtained will be used to find the most promising ways of controlling the microstructure and properties of combustion-synthesized materials. Low-gravity experiments are essential to create idealized an environment for insights into the physics and chemistry of advanced material synthesis processes.

  16. A mechanistic model of the cysteine synthase complex.

    PubMed

    Feldman-Salit, Anna; Wirtz, Markus; Hell, Ruediger; Wade, Rebecca C

    2009-02-13

    OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation. PMID:18801369

  17. A translational preclinical model of interstitial pulmonary fibrosis and pulmonary hypertension: mechanistic pathways driving disease pathophysiology

    PubMed Central

    Jarman, Elizabeth R.; Khambata, Valerie S.; Yun Ye, Li; Cheung, Kenneth; Thomas, Matthew; Duggan, Nicholas; Jarai, Gabor

    2014-01-01

    Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease, in which a decline in patient prognosis is frequently associated with the onset of pulmonary hypertension (PH). Animal models exhibiting principle pathophysiological features of IPF and PH could provide greater insight into mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches for intervention. Here, we describe an in vivo disease model, in which animals develop progressive interstitial pulmonary fibrosis and associated PH, as defined by the presence of fibrotic foci adjacent to areas of alveolar injury and remodeling of the pulmonary vasculature. Associated changes in physiological parameters included a decline in lung function and increase in mean pulmonary arterial pressure (mPAP) >25 mmHg. The early fibrotic pathology is associated with a profibrogenic microenvironment, elevated levels of the matrix metalloproteases, MMP‐2, MMP‐7, and MMP‐12, TIMP‐1, the chemoattractant and mitogen, PDGF‐β, and the chemokines CCL2 and CXCL12, that are associated with the recruitment of macrophages, mast cells, and fibrocytes. Principle mechanistic pathways associated with disease pathogenesis are upregulated in the lungs and pulmonary arteries, with sustained increases in gene transcripts for the profibrotic mediator TGF‐β1 and components of the TGF‐β signaling pathway; PAI‐1, Nox‐4, and HIF‐1α. Therapeutic treatment with the ALK‐5/TGF‐β RI inhibitor SB‐525334 reversed established pulmonary fibrosis and associated vascular remodeling, leading to normalization in clinically translatable physiological parameters including lung function and hemodynamic measurements of mPAP. These studies highlight the application of this model in validating potential approaches for targeting common mechanistic pathways driving disease pathogenesis. PMID:25214520

  18. Hard to swallow: Developmental biological insights into pediatric dysphagia.

    PubMed

    LaMantia, Anthony-Samuel; Moody, Sally A; Maynard, Thomas M; Karpinski, Beverly A; Zohn, Irene E; Mendelowitz, David; Lee, Norman H; Popratiloff, Anastas

    2016-01-15

    Pediatric dysphagia-feeding and swallowing difficulties that begin at birth, last throughout childhood, and continue into maturity--is one of the most common, least understood complications in children with developmental disorders. We argue that a major cause of pediatric dysphagia is altered hindbrain patterning during pre-natal development. Such changes can compromise craniofacial structures including oropharyngeal muscles and skeletal elements as well as motor and sensory circuits necessary for normal feeding and swallowing. Animal models of developmental disorders that include pediatric dysphagia in their phenotypic spectrum can provide mechanistic insight into pathogenesis of feeding and swallowing difficulties. A fairly common human genetic developmental disorder, DiGeorge/22q11.2 Deletion Syndrome (22q11DS) includes a substantial incidence of pediatric dysphagia in its phenotypic spectrum. Infant mice carrying a parallel deletion to 22q11DS patients have feeding and swallowing difficulties that approximate those seen in pediatric dysphagia. Altered hindbrain patterning, craniofacial malformations, and changes in cranial nerve growth prefigure these difficulties. Thus, in addition to craniofacial and pharyngeal anomalies that arise independently of altered neural development, pediatric dysphagia may result from disrupted hindbrain patterning and its impact on peripheral and central neural circuit development critical for feeding and swallowing. The mechanisms that disrupt hindbrain patterning and circuitry may provide a foundation to develop novel therapeutic approaches for improved clinical management of pediatric dysphagia. PMID:26554723

  19. Mechanistic Study of Manganese-Substituted Glycerol Dehydrogenase Using a Kinetic and Thermodynamic Analysis

    PubMed Central

    Fang, Baishan; Niu, Jin; Ren, Hong; Guo, Yingxia; Wang, Shizhen

    2014-01-01

    Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated. PMID:24896258

  20. Mechismo: predicting the mechanistic impact of mutations and modifications on molecular interactions

    PubMed Central

    Betts, Matthew J.; Lu, Qianhao; Jiang, YingYing; Drusko, Armin; Wichmann, Oliver; Utz, Mathias; Valtierra-Gutiérrez, Ilse A.; Schlesner, Matthias; Jaeger, Natalie; Jones, David T.; Pfister, Stefan; Lichter, Peter; Eils, Roland; Siebert, Reiner; Bork, Peer; Apic, Gordana; Gavin, Anne-Claude; Russell, Robert B.

    2015-01-01

    Systematic interrogation of mutation or protein modification data is important to identify sites with functional consequences and to deduce global consequences from large data sets. Mechismo (mechismo.russellab.org) enables simultaneous consideration of thousands of 3D structures and biomolecular interactions to predict rapidly mechanistic consequences for mutations and modifications. As useful functional information often only comes from homologous proteins, we benchmarked the accuracy of predictions as a function of protein/structure sequence similarity, which permits the use of relatively weak sequence similarities with an appropriate confidence measure. For protein–protein, protein–nucleic acid and a subset of protein–chemical interactions, we also developed and benchmarked a measure of whether modifications are likely to enhance or diminish the interactions, which can assist the detection of modifications with specific effects. Analysis of high-throughput sequencing data shows that the approach can identify interesting differences between cancers, and application to proteomics data finds potential mechanistic insights for how post-translational modifications can alter biomolecular interactions. PMID:25392414

  1. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    DOE PAGESBeta

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, whichmore » coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.« less

  2. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    SciTech Connect

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  3. Radical Cyclisation of α-Halo Aluminium Acetals: A Mechanistic Study.

    PubMed

    Bénéteau, Romain; Boussonnière, Anne; Rouaud, Jean-Christophe; Lebreton, Jacques; Graton, Jérôme; Jacquemin, Denis; Sebban, Muriel; Oulyadi, Hassan; Hamdoun, Ghanem; Hancock, Amber N; Schiesser, Carl H; Dénès, Fabrice

    2016-03-24

    α-Bromo aluminium acetals are suitable substrates for Ueno-Stork-like radical cyclisations affording γ-lactols and acid-sensitive methylene-γ-lactols in high yields. The mechanistic study herein sets the scope and limitation of this reaction. The influence of the halide (or chalcogenide) atom X (X=Cl, Br, I, SPh, SePh) in the precursors α-haloesters, as well as influence of the solvent and temperature was studied. The structure of the aluminium acetal intermediates resulting from the reduction of the corresponding α-haloesters has been investigated by low-temperature (13) C-INEPT diffusion-ordered NMR spectroscopy (DOSY) experiments and quantum calculations, providing new insights into the structures of these thermally labile intermediates. Oxygen-bridged dimeric structures with a planar Al2 O2 ring are proposed for the least hindered aluminium acetals, while monomeric structures seem to prevail for the most hindered species. A comparison against the radical cyclisation of aluminium acetals derived from allyl and propargyl alcohols with the parent Ueno-Stork has been made at the BHandHLYP/6-311++G(d,p) level of theory, highlighting mechanistic similarities and differences. PMID:26890896

  4. Structural and Mechanistic Roles of Novel Chemical Ligands on the SdiA Quorum-Sensing Transcription Regulator

    PubMed Central

    Nguyen, Y; Nguyen, Nam X.; Rogers, Jamie L.; Liao, Jun; MacMillan, John B.

    2015-01-01

    ABSTRACT Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not in an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment. PMID:25827420

  5. Mechanistic Basis of Desmosome-Targeted Diseases

    PubMed Central

    Al-Jassar, Caezar; Bikker, Hennie; Overduin, Michael; Chidgey, Martyn

    2013-01-01

    Desmosomes are dynamic junctions between cells that maintain the structural integrity of skin and heart tissues by withstanding shear forces. Mutations in component genes cause life-threatening conditions including arrhythmogenic right ventricular cardiomyopathy, and desmosomal proteins are targeted by pathogenic autoantibodies in skin blistering diseases such as pemphigus. Here, we review a set of newly discovered pathogenic alterations and discuss the structural repercussions of debilitating mutations on desmosomal proteins. The architectures of native desmosomal assemblies have been visualized by cryo-electron microscopy and cryo-electron tomography, and the network of protein domain interactions is becoming apparent. Plakophilin and desmoplakin mutations have been discovered to alter binding interfaces, structures, and stabilities of folded domains that have been resolved by X-ray crystallography and NMR spectroscopy. The flexibility within desmoplakin has been revealed by small-angle X-ray scattering and fluorescence assays, explaining how mechanical stresses are accommodated. These studies have shown that the structural and functional consequences of desmosomal mutations can now begin to be understood at multiple levels of spatial and temporal resolution. This review discusses the recent structural insights and raises the possibility of using modeling for mechanism-based diagnosis of how deleterious mutations alter the integrity of solid tissues. PMID:23911551

  6. Mechanistic Studies on the Copper-Catalyzed N-Arylation of Amides

    PubMed Central

    Strieter, Eric R.; Bhayana, Brijesh; Buchwald, Stephen L.

    2009-01-01

    The copper-catalyzed N-arylation of amides, i.e., the Goldberg reaction, is an efficient method for the construction of products relevant to both industry and academic settings. Herein, we present mechanistic details concerning the catalytic and stoichiometric N-arylation of amides. In the context of the catalytic reaction, our findings reveal the importance of chelating diamine ligands in controlling the concentration of the active catalytic species. The consistency between the catalytic and stoichiometric results suggest that the activation of aryl halides occurs through a 1,2-diamine-ligated copper(I) amidate complex. Kinetic studies on the stoichiometric N-arylation of aryl iodides using 1,2-diamine ligated Cu(I) amidates also provide insights into the mechanism of aryl halide activation. PMID:19072233

  7. From formamide to purine: an energetically viable mechanistic reaction pathway.

    PubMed

    Wang, Jing; Gu, Jiande; Nguyen, Minh Tho; Springsteen, Greg; Leszczynski, Jerzy

    2013-02-28

    A step-by-step mechanistic pathway following the transformation of formamide to purine through a five-membered ring intermediate has been explored by density functional theory computations. The highlight of the mechanistic route detailed here is that the proposed pathway represents the simplest reaction pathway. All necessary reactants are generated from a single starting compound, formamide, through energetically viable reactions. Several important reaction steps are involved in this mechanistic route: formylation-dehydration, Leuckart reduction, five- and six-membered ring-closure, and deamination. On the basis of the study of noncatalytic pathways, catalytic water has been found to provide energetically viable step-by-step mechanistic pathways. Among these reaction steps, five-member ring-closure is the rate-determining step. The energy barrier (ca. 42 kcal/mol) of this rate-control step is somewhat lower than the rate-determining step (ca. 44 kcal/mol) for a pyrimidine-based pathway reported previously. The mechanistic pathway reported herein is less energetically demanding than for previously proposed routes to adenine. PMID:23347082

  8. Explanation and inference: mechanistic and functional explanations guide property generalization

    PubMed Central

    Lombrozo, Tania; Gwynne, Nicholas Z.

    2014-01-01

    The ability to generalize from the known to the unknown is central to learning and inference. Two experiments explore the relationship between how a property is explained and how that property is generalized to novel species and artifacts. The experiments contrast the consequences of explaining a property mechanistically, by appeal to parts and processes, with the consequences of explaining the property functionally, by appeal to functions and goals. The findings suggest that properties that are explained functionally are more likely to be generalized on the basis of shared functions, with a weaker relationship between mechanistic explanations and generalization on the basis of shared parts and processes. The influence of explanation type on generalization holds even though all participants are provided with the same mechanistic and functional information, and whether an explanation type is freely generated (Experiment 1), experimentally provided (Experiment 2), or experimentally induced (Experiment 2). The experiments also demonstrate that explanations and generalizations of a particular type (mechanistic or functional) can be experimentally induced by providing sample explanations of that type, with a comparable effect when the sample explanations come from the same domain or from a different domains. These results suggest that explanations serve as a guide to generalization, and contribute to a growing body of work supporting the value of distinguishing mechanistic and functional explanations. PMID:25309384

  9. Concurrent esterification and N-acetylation of amino acids with orthoesters: A useful reaction with interesting mechanistic implications.

    PubMed

    Gibson, Sarah; Romero, Dickie; Jacobs, Hollie K; Gopalan, Aravamudan S

    2010-12-22

    The concurrent esterification and N-acetylation of amino acids has been studied with triethyl orthoacetate (TEOA) and triethyl orthoformate (TEOF). In a surprising finding, only one equivalent of TEOA in refluxing toluene was necessary to convert L-proline and L-phenylalanine to the corresponding N-acetyl ethyl esters in good yield. The same transformation using TEOF was not effective. Stereochemical outcome and stoichiometric studies as well as structural variation of the amino acids in this reaction provided unexpected mechanistic insight. PMID:21286246

  10. Concurrent esterification and N-acetylation of amino acids with orthoesters: A useful reaction with interesting mechanistic implications

    PubMed Central

    Gibson, Sarah; Romero, Dickie; Jacobs, Hollie K.; Gopalan, Aravamudan S.

    2010-01-01

    The concurrent esterification and N-acetylation of amino acids has been studied with triethyl orthoacetate (TEOA) and triethyl orthoformate (TEOF). In a surprising finding, only one equivalent of TEOA in refluxing toluene was necessary to convert L-proline and L-phenylalanine to the corresponding N-acetyl ethyl esters in good yield. The same transformation using TEOF was not effective. Stereochemical outcome and stoichiometric studies as well as structural variation of the amino acids in this reaction provided unexpected mechanistic insight. PMID:21286246

  11. Network-based discovery through mechanistic systems biology. Implications for applications--SMEs and drug discovery: where the action is.

    PubMed

    Benson, Neil

    2015-08-01

    Phase II attrition remains the most important challenge for drug discovery. Tackling the problem requires improved understanding of the complexity of disease biology. Systems biology approaches to this problem can, in principle, deliver this. This article reviews the reports of the application of mechanistic systems models to drug discovery questions and discusses the added value. Although we are on the journey to the virtual human, the length, path and rate of learning from this remain an open question. Success will be dependent on the will to invest and make the most of the insight generated along the way. PMID:26464089

  12. Vitamins C and E: Beneficial effects from a mechanistic perspective

    PubMed Central

    Traber, Maret G.; Stevens, Jan F.

    2011-01-01

    The mechanistic properties of two dietary antioxidants that are required by humans, vitamins C and E, are discussed relative to their biological effects. Vitamin C (ascorbic acid) is an essential cofactor for α-ketoglutarate-dependent dioxygenases. Examples are prolyl hydroxylases, which play a role in the biosynthesis of collagen and in down-regulation of hypoxia-inducible factor (HIF)-1, a transcription factor that regulates many genes responsible for tumor growth, energy metabolism, and neutrophil function and apoptosis. Vitamin C-dependent inhibition of the HIF pathway may provide alternative or additional approaches for controlling tumor progression, infections and inflammation. Vitamin E (α-tocopherol) functions as an essential lipid soluble antioxidant, scavenging hydroperoxyl radicals in lipid milieu. Human symptoms of vitamin E deficiency suggest that its antioxidant properties play a major role in protecting erythrocyte membranes and nervous tissues. As an antioxidant, vitamin C provides protection against oxidative stress-induced cellular damage by scavenging of reactive oxygen species, vitamin E-dependent neutralization of lipid hydroperoxyl radicals, and by protecting proteins from alkylation by electrophilic lipid peroxidation products. These bioactivities bear relevance to inflammatory disorders. Vitamin C plays also a role in the function of endothelial nitric oxide synthase (eNOS) by recycling the eNOS cofactor, tetrahydrobiopterin, which is relevant to arterial elasticity and blood pressure regulation. Evidence from plants supports a role for vitamin C in the formation of covalent adducts with electrophilic secondary metabolites. Mechanism-based effects of vitamin C and E supplementation on biomarkers and on clinical outcomes from randomized, placebo-controlled trials are emphasized in this review. PMID:21664268

  13. Additional geological insight brought by 3-D seismic data

    NASA Astrophysics Data System (ADS)

    Liu, Y.; Milton, J.

    2002-12-01

    3-D seismic data allows geo-scientists to study the earth at a level that is much more detailed than ever. It is shown in this case study that 3-D seismic can not only be used to identify different types of oil/gas reservoirs, significantly reduce the risk of misinterpretation, but also help to understand geological history and establish paleogeography at different geological times. In the study of Southeast Maricopa Seismic Survey in southern San Joaquin Valley, two types of potential hydrocarbon traps are interpreted: stratigraphical traps due to turbidite channels, and structural traps due to faulting. The distinctive characteristics of two types of channels indicate different depositional environments. With 3-D visualization tools, it is found that localized faults had been leaking during certain geological times, resulting in structural traps of oil/gas. A geological history of the local area can be estimated by building a series of pseudo-paleogeographic maps using 3-D seismic data, which further reconfirms the existence of different depositional systems indicated by two distinctive types of channels.

  14. Copper/TEMPO-Catalyzed Aerobic Alcohol Oxidation: Mechanistic Assessment of Different Catalyst Systems.

    PubMed

    Hoover, Jessica M; Ryland, Bradford L; Stahl, Shannon S

    2013-11-01

    Combinations of homogeneous Cu salts and TEMPO have emerged as practical and efficient catalysts for the aerobic oxidation of alcohols. Several closely related catalyst systems have been reported, which differ in the identity of the solvent, the presence of 2,2'-bipyridine as a ligand, the identity of basic additives, and the oxidation state of the Cu source. These changes have a significant influence on the reaction rates, yields, and substrate scope. In this report, we probe the mechanistic basis for differences among four different Cu/TEMPO catalyst systems and elucidate the features that contribute to efficient oxidation of aliphatic alcohols. PMID:24558634

  15. Parameterization of phosphine ligands reveals mechanistic pathways and predicts reaction outcomes

    NASA Astrophysics Data System (ADS)

    Niemeyer, Zachary L.; Milo, Anat; Hickey, David P.; Sigman, Matthew S.

    2016-06-01

    The mechanistic foundation behind the identity of a phosphine ligand that best promotes a desired reaction outcome is often non-intuitive, and thus has been addressed in numerous experimental and theoretical studies. In this work, multivariate correlations of reaction outcomes using 38 different phosphine ligands were combined with classic potentiometric analyses to study a Suzuki reaction, for which the site selectivity of oxidative addition is highly dependent on the nature of the phosphine. These studies shed light on the generality of hypotheses regarding the structural influence of different classes of phosphine ligands on the reaction mechanism(s), and deliver a methodology that should prove useful in future studies of phosphine ligands.

  16. Copper/TEMPO-Catalyzed Aerobic Alcohol Oxidation: Mechanistic Assessment of Different Catalyst Systems

    PubMed Central

    Hoover, Jessica M.; Ryland, Bradford L.; Stahl, Shannon S.

    2013-01-01

    Combinations of homogeneous Cu salts and TEMPO have emerged as practical and efficient catalysts for the aerobic oxidation of alcohols. Several closely related catalyst systems have been reported, which differ in the identity of the solvent, the presence of 2,2′-bipyridine as a ligand, the identity of basic additives, and the oxidation state of the Cu source. These changes have a significant influence on the reaction rates, yields, and substrate scope. In this report, we probe the mechanistic basis for differences among four different Cu/TEMPO catalyst systems and elucidate the features that contribute to efficient oxidation of aliphatic alcohols. PMID:24558634

  17. When higher working memory capacity hinders insight.

    PubMed

    DeCaro, Marci S; Van Stockum, Charles A; Wieth, Mareike B

    2016-01-01

    Higher working memory capacity (WMC) improves performance on a range of cognitive and academic tasks. However, a greater ability to control attention sometimes leads individuals with higher WMC to persist in using complex, attention-demanding approaches that are suboptimal for a given task. We examined whether higher WMC would hinder insight problem solving, which is thought to rely on associative processes that operate largely outside of close attentional control. In addition, we examined whether characteristics of the insight problems influence whether this negative relationship will be revealed. In Experiment 1, participants completed matchstick arithmetic problems, which require a similar initial problem representation for all problems. Higher WMC was associated with less accurate insight problem solving. In Experiment 2, participants completed insight word problems, which require substantially different representations for each problem. Higher WMC was again negatively associated with insight, but only after statistically controlling for shared variance between insight and incremental problem-solving accuracy. These findings suggest that WMC may benefit performance on fundamental processes common to both incremental and insight problem solving (e.g., initial problem representation), but hinder performance on the processes that are unique to insight (e.g., solution and restructuring). By considering the WMC of the individual, and the nature of the insight task, we may better understand the process of insight and how to best support it. (PsycINFO Database Record PMID:26120772

  18. Key to Opening Kidney for In Vitro-In Vivo Extrapolation Entrance in Health and Disease: Part II: Mechanistic Models and In Vitro-In Vivo Extrapolation.

    PubMed

    Scotcher, Daniel; Jones, Christopher; Posada, Maria; Galetin, Aleksandra; Rostami-Hodjegan, Amin

    2016-09-01

    It is envisaged that application of mechanistic models will improve prediction of changes in renal disposition due to drug-drug interactions, genetic polymorphism in enzymes and transporters and/or renal impairment. However, developing and validating mechanistic kidney models is challenging due to the number of processes that may occur (filtration, secretion, reabsorption and metabolism) in this complex organ. Prediction of human renal drug disposition from preclinical species may be hampered by species differences in the expression and activity of drug metabolising enzymes and transporters. A proposed solution is bottom-up prediction of pharmacokinetic parameters based on in vitro-in vivo extrapolation (IVIVE), mediated by recent advances in in vitro experimental techniques and application of relevant scaling factors. This review is a follow-up to the Part I of the report from the 2015 AAPS Annual Meeting and Exhibition (Orlando, FL; 25th-29th October 2015) which focuses on IVIVE and mechanistic prediction of renal drug disposition. It describes the various mechanistic kidney models that may be used to investigate renal drug disposition. Particular attention is given to efforts that have attempted to incorporate elements of IVIVE. In addition, the use of mechanistic models in prediction of renal drug-drug interactions and potential for application in determining suitable adjustment of dose in kidney disease are discussed. The need for suitable clinical pharmacokinetics data for the purposes of delineating mechanistic aspects of kidney models in various scenarios is highlighted. PMID:27506526

  19. The Role of {Ca2+ / CO32-} Ratio in Calcite Dissolution and Growth: Implications for Mechanistic Control of Biomineralization

    NASA Astrophysics Data System (ADS)

    Arvidson, R. S.; Davis, K. J.; Luttge, A.

    2003-12-01

    The hypothesis that secular variations in the Mg/Ca ratio of seawater have exerted a fundamental control over the mineralogy and abundance of both skeletal and nonskeletal carbonates has received substantial support from both experimental and field data. In this context, ongoing efforts directed at understanding the mechanistic basis for interaction of Mg and Sr with carbonate mineral surfaces during growth (e.g., Davis et al. 2000) are of obvious importance. However, a growing body of experimental dissolution data records additional site-specific interactions between the surface and dissolved free carbon species and carbonate complexes. We suggest these data may provide additional insight into mechanisms by which organisms maintain skeletal integrity under variable conditions, including the possible development of surface precursors of mixed carbonate phases. For example, recent data have shown that kink dynamics along the fast, obtuse (+) step directions are highly sensitive to the ratio of magnesium to carbonate ion. We have used these observations as the basis for exploration of the relationship between the simple ratio of dissolved calcium to carbonate ion and surface dynamics. In sets of carefully designed experiments, we sought to maintain (1) a constant distance from equilibrium by varying {Ca2+ / CO32-} ratio at constant IAP, (2) constant {Ca2+ / CO32-} at variable IAP, (3) all under conditions of both over- and undersaturation ranging from far to close to equilibrium. Using an integrated approach, observations were made over a wide range of space and time scales using both AFM and VSI (vertical scanning interferometry). These coupled observations provide resolution of the relationship between the overall rate of reaction (total change in surface topography) and detailed observations of characteristic step dynamics developed during both dissolution and growth. Our preliminary results confirm a strong sensitivity of the conventional fast step direction to

  20. Kohler's Insight Revisited.

    ERIC Educational Resources Information Center

    Windholtz, George

    1985-01-01

    Psychology textbooks frequently present Wolfgang Kohler's two-stick experiment with chimpanzees as having demonstrated insight in learning. Studies that replicated Kohler's work support his findings but not his interpretation in terms of insightful solution. The uncritical inclusion of Kohler's insight interpretation in texts is not warranted in…

  1. Proofs that Develop Insight

    ERIC Educational Resources Information Center

    Weber, Keith

    2010-01-01

    Many mathematics educators have noted that mathematicians do not only read proofs to gain conviction but also to obtain insight. The goal of this article is to discuss what this insight is from mathematicians' perspective. Based on interviews with nine research-active mathematicians, two sources of insight are discussed. The first is reading a…

  2. DEVELOPMENT AND VALIDATION OF A MECHANISTIC GROUND SPRAYER MODEL

    EPA Science Inventory

    In the last ten years the Spray Drift Task Force (SDTF), U.S. Environmental Protection Agency (EPA), USDA Agricultural Research Service, and USDA Forest Service cooperated in the refinement and evaluation of a mechanistically-based aerial spray model (contained within AGDISP and ...

  3. MECHANISTIC AND SOURCE UNDERSTANDING OF PCDD/F FORMATION

    EPA Science Inventory

    The paper discusses mechanistic and source understanding of polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/F) formation. (NOTE: Considerable research effort has been expended over the last 15-plus years to understand how combustion sources result in formation of PCDDs/F...

  4. MECHANISTIC-BASED DISINFECTION AND DISINFECTION BYPRODUCT MODELS

    EPA Science Inventory

    We propose developing a mechanistic-based numerical model for chlorine decay and regulated DBP (THM and HAA) formation derived from (free) chlorination; the model framework will allow future modifications for other DBPs and chloramination. Predicted chlorine residual and DBP r...

  5. Does Mechanistic Thinking Improve Student Success in Organic Chemistry?

    ERIC Educational Resources Information Center

    Grove, Nathaniel P.; Cooper, Melanie M.; Cox, Elizabeth L.

    2012-01-01

    The use of the curved-arrow notation to depict electron flow during mechanistic processes is one of the most important representational conventions in the organic chemistry curriculum. Our previous research documented a disturbing trend: when asked to predict the products of a series of reactions, many students do not spontaneously engage in…

  6. Mechanistically Consistent Reduced Models of Synthetic Gene Networks

    PubMed Central

    Mier-y-Terán-Romero, Luis; Silber, Mary; Hatzimanikatis, Vassily

    2013-01-01

    Designing genetic networks with desired functionalities requires an accurate mathematical framework that accounts for the essential mechanistic details of the system. Here, we formulate a time-delay model of protein translation and mRNA degradation by systematically reducing a detailed mechanistic model that explicitly accounts for the ribosomal dynamics and the cleaving of mRNA by endonucleases. We exploit various technical and conceptual advantages that our time-delay model offers over the mechanistic model to probe the behavior of a self-repressing gene over wide regions of parameter space. We show that a heuristic time-delay model of protein synthesis of a commonly used form yields a notably different prediction for the parameter region where sustained oscillations occur. This suggests that such heuristics can lead to erroneous results. The functional forms that arise from our systematic reduction can be used for every system that involves transcription and translation and they could replace the commonly used heuristic time-delay models for these processes. The results from our analysis have important implications for the design of synthetic gene networks and stress that such design must be guided by a combination of heuristic models and mechanistic models that include all relevant details of the process. PMID:23663853

  7. THYROID FOLLICULAR CELL CARCINOGENESIS: MECHANISTIC AND SCIENCE POLICY CONSIDERATIONS

    EPA Science Inventory

    EPA's Guidelines for Carcinogen Risk Assessment call for use of mechanistic and other relevant information in making choices about the models to be used in extrapolating hazard estimates from high to low exposures. The Forum report on thyroid neoplasia proposes that, under clearl...

  8. Rearrangements of Allylic Sulfinates to Sulfones: A Mechanistic Study

    ERIC Educational Resources Information Center

    Ball, David B.; Mollard, Paul; Voigtritter, Karl R.; Ball, Jenelle L.

    2010-01-01

    Most current organic chemistry textbooks are organized by functional groups and those of us who teach organic chemistry use functional-group organization in our courses but ask students to learn organic chemistry from a mechanistic approach. To enrich and extend the chemical understanding and knowledge of pericyclic-type reactions for chemistry…

  9. Insights into the function of ion channels by computational electrophysiology simulations.

    PubMed

    Kutzner, Carsten; Köpfer, David A; Machtens, Jan-Philipp; de Groot, Bert L; Song, Chen; Zachariae, Ulrich

    2016-07-01

    Ion channels are of universal importance for all cell types and play key roles in cellular physiology and pathology. Increased insight into their functional mechanisms is crucial to enable drug design on this important class of membrane proteins, and to enhance our understanding of some of the fundamental features of cells. This review presents the concepts behind the recently developed simulation protocol Computational Electrophysiology (CompEL), which facilitates the atomistic simulation of ion channels in action. In addition, the review provides guidelines for its application in conjunction with the molecular dynamics software package GROMACS. We first lay out the rationale for designing CompEL as a method that models the driving force for ion permeation through channels the way it is established in cells, i.e., by electrochemical ion gradients across the membrane. This is followed by an outline of its implementation and a description of key settings and parameters helpful to users wishing to set up and conduct such simulations. In recent years, key mechanistic and biophysical insights have been obtained by employing the CompEL protocol to address a wide range of questions on ion channels and permeation. We summarize these recent findings on membrane proteins, which span a spectrum from highly ion-selective, narrow channels to wide diffusion pores. Finally we discuss the future potential of CompEL in light of its limitations and strengths. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26874204

  10. Pharmacogenomics in diabetes mellitus: insights into drug action and drug discovery.

    PubMed

    Zhou, Kaixin; Pedersen, Helle Krogh; Dawed, Adem Y; Pearson, Ewan R

    2016-06-01

    Genomic studies have greatly advanced our understanding of the multifactorial aetiology of type 2 diabetes mellitus (T2DM) as well as the multiple subtypes of monogenic diabetes mellitus. In this Review, we discuss the existing pharmacogenetic evidence in both monogenic diabetes mellitus and T2DM. We highlight mechanistic insights from the study of adverse effects and the efficacy of antidiabetic drugs. The identification of extreme sulfonylurea sensitivity in patients with diabetes mellitus owing to heterozygous mutations in HNF1A represents a clear example of how pharmacogenetics can direct patient care. However, pharmacogenomic studies of response to antidiabetic drugs in T2DM has yet to be translated into clinical practice, although some moderate genetic effects have now been described that merit follow-up in trials in which patients are selected according to genotype. We also discuss how future pharmacogenomic findings could provide insights into treatment response in diabetes mellitus that, in addition to other areas of human genetics, facilitates drug discovery and drug development for T2DM. PMID:27062931

  11. Structural insights into the interaction and disease mechanism of neurodegenerative disease-associated optineurin and TBK1 proteins.

    PubMed

    Li, Faxiang; Xie, Xingqiao; Wang, Yingli; Liu, Jianping; Cheng, Xiaofang; Guo, Yujiao; Gong, Yukang; Hu, Shichen; Pan, Lifeng

    2016-01-01

    Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients. PMID:27620379

  12. Elusive transmetalation intermediate in copper-catalyzed conjugate additions: direct NMR detection of an ethyl group attached to a binuclear phosphoramidite copper complex.

    PubMed

    von Rekowski, Felicitas; Koch, Carina; Gschwind, Ruth M

    2014-08-13

    Copper-catalyzed asymmetric conjugate addition reactions are a very powerful and widely applied method for enantioselective carbon-carbon bond formation. However, structural and mechanistic insight into these famous reactions has been very limited so far. In this article, the first direct experimental detection of transmetalation intermediates in copper-catalyzed reactions is presented. Special combinations of (1)H,(31)P HMBC spectra allow for the identification of complexes with chemical bonds between the alkyl groups and the copper complexes. For the structural characterization of these transmetalation intermediates, a special approach is applied, in which samples using enantiopure ligands are compared with samples using enantiomeric mixtures of ligands. It is experimentally proven, for the first time, that the dimeric copper complex structure is retained upon transmetalation, providing an intermediate with mixed trigonal/tetrahedral coordination on the copper atoms. In addition, monomeric intermediates with one ligand, but no intermediates with two ligands, are detected. These experimental results, in combination with the well-known optimal ligand-to-copper ratio of 2:1 in synthetic applications, allow us to propose that a binuclear transmetalation intermediate is the reactive species in copper-catalyzed asymmetric conjugate addition reactions. This first direct experimental insight into the structure of the transmetalation intermediate is expected to support the mechanistic and theoretical understanding of this important class of reactions and to enable their further synthetic development. In addition, the special NMR approach presented here for the identification and characterization of intermediates below the detection limit of (1)H NMR spectra can be applied also to other classes of catalyses. PMID:25072403

  13. A mechanistic view of drying suspension droplets.

    PubMed

    van der Kooij, Hanne M; van de Kerkhof, Gea T; Sprakel, Joris

    2016-03-21

    When a dispersion droplet dries, a rich variety of spatial and temporal heterogeneities emerge. Controlling these phenomena is essential for many applications yet requires a thorough understanding of the underlying mechanisms. Although the process of film formation from initially dispersed polymer particles is well documented and is known to involve three main stages - evaporation, particle deformation and coalescence - it is impossible to fully disentangle the effects of particle deformation and coalescence, as these stages are closely linked. We circumvent this problem by studying suspensions of colloidal rubber particles that are incapable of coalescing. Varying the crosslink density allows us to tune the particle deformability in a controlled manner. We develop a theoretical framework of the main regimes and stresses in drying droplets of these suspensions, and validate this framework experimentally. Specifically, we show that changing the particle modulus by less than an order of magnitude can completely alter the stress development and resulting instabilities. Scanning electron microscopy reveals that particle deformability is a key factor in stress mitigation. Our model is the suspension equivalent of the widely used Routh-Russel model for film formation in drying dispersions, with additional focus on lateral nonuniformities such as cracking and wrinkling inherent to the droplet geometry, thus adding a new dimension to the conventional view of particle deformation. PMID:26843025

  14. Horseradish peroxidase catalyzed hydroxylations: mechanistic studies.

    PubMed

    Dordick, J S; Klibanov, A M; Marletta, M A

    1986-05-20

    The hydroxylation of phenol to hydroquinone and catechol in the presence of dihydroxyfumaric acid and oxygen catalyzed by horseradish peroxidase was studied under conditions where the product yield was high and the side reactions were minimal. The reaction is partially uncoupled with a molar ratio of dihydroxyfumaric acid consumed to hydroxylated products of 12:1. Hydrogen peroxide does not participate in the reaction as evidenced by the lack of effect of catalase and by the direct addition of hydrogen peroxide. Conversely, superoxide and hydroxyl radicals are involved as their scavengers are potent inhibitors. Experiments were all consistent with the involvement of compound III (oxygenated ferrous complex) of peroxidase in the reaction. Compound III is stable in the presence of phenol alone but decomposes rapidly in the presence of both phenol and dihydroxyfumaric acid with the concomitant formation of product. Therefore, phenol and dihydroxyfumaric acid must be present with compound III in order for the hydroxylation reaction to occur. A mechanism consistent with the experimental results is proposed. PMID:3718931

  15. Mechanistic investigation of a hemostatic keratin biomaterial

    NASA Astrophysics Data System (ADS)

    Rahmany, Maria Bahawdory

    Traumatic injury leads to more productive years lost than heart disease, cancer and stroke combined. Trauma is often accompanied and complicated by uncontrolled bleeding. Human hair keratin biomaterials have demonstrated efficacy in controlling hemorrhage in both small and large animal models; however little is known about the mechanism by which these proteins aid in blood clotting. Inspection of the amino acid sequence of known keratins shows the presence of several cellular binding motifs, suggesting a possible mechanism and potentially eliminating the need to functionalize the material's surface for cellular interaction. In addition to small animal studies, the hemostatic activity of keratin hydrogels was explored through porcine hemorrhage models representing both a high flow and low flow bleed. In both studies, keratin hydrogels appeared to lead to a significant reduction in blood loss. The promising results from these in vivo studies provided the motivation for this project. The objective of this dissertation work was to assess the mechanism of action of a hemostatic keratin biomaterial, and more broadly assess the biomaterial-cellular interaction(s). It is our hypothesis that keratin biomaterials have the capacity to specifically interact with cells and lead to propagation of intracellular signaling pathway, specifically contributing to hemostasis. Through application of biochemical and molecular tools, we demonstrate here that keratin biomaterials contribute to hemostasis through two probable mechanisms; integrin mediated platelet adhesion and increased fibrin polymerization. Platelets are the major cell type involved in coagulation both by acting as a catalytic surface for the clotting cascade and adhering to extracellular matrix (ECM) proteins providing a soft platelet plug. Because keratin biomaterials have structural and biochemical characteristics similar to ECM proteins, we utilized several adhesion assays to investigate platelet adhesion to keratin

  16. Mechanistic Studies at the Interface Between Organometallic Chemistry and Homogeneous Catalysis

    SciTech Connect

    Casey, Charles P

    2012-11-14

    Mechanistic Studies at the Interface Between Organometallic Chemistry and Homogeneous Catalysis Charles P. Casey, Principal Investigator Department of Chemistry, University of Wisconsin - Madison, Madison, Wisconsin 53706 Phone 608-262-0584 FAX: 608-262-7144 Email: casey@chem.wisc.edu http://www.chem.wisc.edu/main/people/faculty/casey.html Executive Summary. Our goal was to learn the intimate mechanistic details of reactions involved in homogeneous catalysis and to use the insight we gain to develop new and improved catalysts. Our work centered on the hydrogenation of polar functional groups such as aldehydes and ketones and on hydroformylation. Specifically, we concentrated on catalysts capable of simultaneously transferring hydride from a metal center and a proton from an acidic oxygen or nitrogen center to an aldehyde or ketone. An economical iron based catalyst was developed and patented. Better understanding of fundamental organometallic reactions and catalytic processes enabled design of energy and material efficient chemical processes. Our work contributed to the development of catalysts for the selective and mild hydrogenation of ketones and aldehydes; this will provide a modern green alternative to reductions by LiAlH4 and NaBH4, which require extensive work-up procedures and produce waste streams. (C5R4OH)Ru(CO)2H Hydrogenation Catalysts. Youval Shvo described a remarkable catalytic system in which the key intermediate (C5R4OH)Ru(CO)2H (1) has an electronically coupled acidic OH unit and a hydridic RuH unit. Our efforts centered on understanding and improving upon this important catalyst for reduction of aldehydes and ketones. Our mechanistic studies established that the reduction of aldehydes by 1 to produce alcohols and a diruthenium bridging hydride species occurs much more rapidly than regeneration of the ruthenium hydride from the diruthenium bridging hydride species. Our mechanistic studies require simultaneous transfer of hydride from ruthenium to

  17. Insights into enzymatic halogenation from computational studies

    NASA Astrophysics Data System (ADS)

    Senn, Hans

    2014-11-01

    The halogenases are a group of enzymes that have only come to the fore over the last ten years thanks to the discovery and characterization of several of novel representatives. They have re-vealed the fascinating variety of distinct chemical mechanisms that nature utilizes to activate and introduce halogens into organic substrates. Computational studies using a range of approaches have already elucidated many details of the mechanisms of these enzymes, often in synergistic combination with experiment. This Review summarizes the main insights gained from these stud-ies. It also seeks to identify open questions that are amenable to computational investigations. The studies discussed herein also serve to illustrate some of the limitations of the current computa-tional approaches and the challenges encountered in computational mechanistic enzymology.

  18. Insights into enzymatic halogenation from computational studies

    PubMed Central

    Senn, Hans M.

    2014-01-01

    The halogenases are a group of enzymes that have only come to the fore over the last 10 years thanks to the discovery and characterization of several novel representatives. They have revealed the fascinating variety of distinct chemical mechanisms that nature utilizes to activate halogens and introduce them into organic substrates. Computational studies using a range of approaches have already elucidated many details of the mechanisms of these enzymes, often in synergistic combination with experiment. This Review summarizes the main insights gained from these studies. It also seeks to identify open questions that are amenable to computational investigations. The studies discussed herein serve to illustrate some of the limitations of the current computational approaches and the challenges encountered in computational mechanistic enzymology. PMID:25426489

  19. Emerging structural insights into the function of ionotropic glutamate receptors

    PubMed Central

    Karakas, Erkan; Regan, Michael C.; Furukawa, Hiro

    2015-01-01

    Summary Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission crucial for brain development and function including learning and memory formation. Recently a wealth of structural studies on iGluRs, including AMPA receptors (AMPARs), kainate receptors, and NMDA receptors (NMDARs) became available.. These studies showed structures of non-NMDARs including AMPAR and kainate receptor in various functional states, thereby providing the first visual sense of how non-NMDAR iGluRs may function in the context of homotetramers. Furthermore, they provided the first view of heterotetrameric NMDAR ion channels, which illuminated the similarities with and differences from non-NMDARs, thus raising a mechanistic distinction between the two groups of iGluRs. Here we review mechanistic insights into iGluR functions gained through structural studies of multiple groups. PMID:25941168

  20. Mechanistic modelling of fluidized bed drying processes of wet porous granules: a review.

    PubMed

    Mortier, Séverine Thérèse F C; De Beer, Thomas; Gernaey, Krist V; Remon, Jean Paul; Vervaet, Chris; Nopens, Ingmar

    2011-10-01

    Fluidized bed dryers are frequently used in industrial applications and also in the pharmaceutical industry. The general incentives to develop mechanistic models for pharmaceutical processes are listed, and our vision on how this can particularly be done for fluidized bed drying processes of wet granules is given. This review provides a basis for future mechanistic model development for the drying process of wet granules in pharmaceutical processes. It is intended for a broad audience with a varying level of knowledge on pharmaceutical processes and mathematical modelling. Mathematical models are powerful tools to gain process insight and eventually develop well-controlled processes. The level of detail embedded in such a model depends on the goal of the model. Several models have therefore been proposed in the literature and are reviewed here. The drying behaviour of one single granule, a porous particle, can be described using the continuum approach, the pore network modelling method and the shrinkage of the diameter of the wet core approach. As several granules dry at a drying rate dependent on the gas temperature, gas velocity, porosity, etc., the moisture content of a batch of granules will reside in a certain interval. Population Balance Model (ling) (PBM) offers a tool to describe the distribution of particle properties which can be of interest for the application. PBM formulation and solution methods are therefore reviewed. In a fluidized bed, the granules show a fluidization pattern depending on the geometry of the gas inlet, the gas velocity, characteristics of the particles, the dryer design, etc. Computational Fluid Dynamics (CFD) allows to model this behaviour. Moreover, turbulence can be modelled using several approaches: Reynolds-averaged Navier-Stokes Equations (RANS) or Large Eddy Simulation (LES). Another important aspect of CFD is the choice between the Eulerian-Lagrangian and the Eulerian-Eulerian approach. Finally, the PBM and CFD frameworks

  1. Validating Mechanistic Sorption Model Parameters and Processes for Reactive Transport in Alluvium

    SciTech Connect

    Zavarin, M; Roberts, S K; Rose, T P; Phinney, D L

    2002-05-02

    The laboratory batch and flow-through experiments presented in this report provide a basis for validating the mechanistic surface complexation and ion exchange model we use in our hydrologic source term (HST) simulations. Batch sorption experiments were used to examine the effect of solution composition on sorption. Flow-through experiments provided for an analysis of the transport behavior of sorbing elements and tracers which includes dispersion and fluid accessibility effects. Analysis of downstream flow-through column fluids allowed for evaluation of weakly-sorbing element transport. Secondary Ion Mass Spectrometry (SIMS) analysis of the core after completion of the flow-through experiments permitted the evaluation of transport of strongly sorbing elements. A comparison between these data and model predictions provides additional constraints to our model and improves our confidence in near-field HST model parameters. In general, cesium, strontium, samarium, europium, neptunium, and uranium behavior could be accurately predicted using our mechanistic approach but only after some adjustment was made to the model parameters. The required adjustments included a reduction in strontium affinity for smectite, an increase in cesium affinity for smectite and illite, a reduction in iron oxide and calcite reactive surface area, and a change in clinoptilolite reaction constants to reflect a more recently published set of data. In general, these adjustments are justifiable because they fall within a range consistent with our understanding of the parameter uncertainties. These modeling results suggest that the uncertainty in the sorption model parameters must be accounted for to validate the mechanistic approach. The uncertainties in predicting the sorptive behavior of U-1a and UE-5n alluvium also suggest that these uncertainties must be propagated to nearfield HST and large-scale corrective action unit (CAU) models.

  2. A reductionist mechanistic model for bioconcentration of neutral and weakly polar organic compounds in fish.

    PubMed

    Kuo, Dave T F; Di Toro, Dominic M

    2013-09-01

    The bioconcentration factor (BCF) of neutral and weakly polar organic chemicals in fish is modeled using independently calibrated models of chemical partitioning (freely dissolved fraction of chemical in the aqueous phase [φsys ] and wet-weight fish-water partition coefficient [KFW ]), respiratory exchange (respiratory update rate constant [k1 ], and respiratory elimination rate constant [k2  = k1 /KFW ]), and biotransformation (whole-body biotransformation rate constant [kM ]) as BCF = φsys KFW /(1 + kM /k2 ). Existing k1 models tend to overestimate for chemicals with log KOW  < 3.5, which constituted 30% to 50% of the examined chemicals. A revised k1 model covering a wider log KOW range (0-8.5) is presented k1  = (5.46 × 10(-6) MW + 0.261/KOW )(-1) , where MW is the molecular weight. The biotransformation rate constant kM is modeled using biota internal partitioning and Abraham parameters as reactivity descriptors. The reductionist model was tested using 3 different BCF data sets (US Environmental Protection Agency's Estimation Programs Interface [EPI], n = 548; Hertfordshire, n = 210; Arnot-Gobas, n = 1855) and compared with the following 3 state-of-the-art models: 1) the EPI Suite BCFBAF module, 2) the European Commision's Computer Assisted Evaluation of industrial chemical Substances According to Regulations (CAESAR), and 3) the EPI/Arnot mechanistic kinetic model. The reductionist model performed comparably with the alternative models (root mean square errors [RMSEs] = 0.72-0.77), with only 5 fitting parameters and no training against experimental BCFs. Respiratory elimination and biotransformation dominate the total depuration (i.e., [k2  + kM ]/kT  ≥ 0.8) for approximately 98% of the data entries, thus validating the reductionist approximation. Mechanistic models provide greater insights into bioaccumulation and are more sensitive to biological variation. All three BCF data sets and relevant

  3. Mechanistic model for catalytic recombination during aerobraking maneuvers

    NASA Technical Reports Server (NTRS)

    Willey, Ronald J.

    1989-01-01

    Several mechanistic models are developed to predict recombination coefficients for use in heat shield design for reusable surface insulation (RSI) on aerobraking vehicles such as space shuttles. The models are applied over a temperature range of 300 to 1800 K and a stagnation pressure range of 0 to 3,000 Pa. A four parameter model in temperature was found to work best; however, several models (including those with atom concentrations at the surface) were also investigated. Mechanistic models developed with atom concentration terms may be applicable when sufficient data becomes available. The requirement is shown for recombination experiments in the 300 to 1000 K and 1500 to 1850 K temperature range, with deliberate concentration variations.

  4. Insight in bipolar disorder.

    PubMed

    Látalová, Klára

    2012-09-01

    Although there has been interest in insight in bipolar disorder, research has not been as developed as in schizophrenia. The Medline, Embase, and PsychInfo data bases were searched. The key words used in the search were "bipolar", "mania", "manic", "awareness", and "insight". Books, editorials, letters, and reports on pediatric subjects were excluded. Abstracts or full texts were screened for relevance. Better insight is associated with better adherence to treatment and better outcomes. Impairments of executive functions and memory, as well as higher severity of psychotic symptoms, are associated with impairments of insight. Insight is more impaired during an illness episode than during remission, in mixed than in pure manic episodes, in bipolar II than in bipolar I patients, in pure mania than in bipolar or unipolar depression. Psychosocial treatments improve insight and outcomes. There is a need for integration of quantitative assessment methods and their introduction into research and clinical practice. PMID:22101737

  5. Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

    PubMed Central

    Krishna, Katla Sai; Biswas, Sanchita; Navin, Chelliah V.; Yamane, Dawit G.; Miller, Jeffrey T.; Kumar, Challa S.S.R.

    2013-01-01

    Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst. PMID:24327099

  6. Millifluidics for chemical synthesis and time-resolved mechanistic studies.

    PubMed

    Krishna, Katla Sai; Biswas, Sanchita; Navin, Chelliah V; Yamane, Dawit G; Miller, Jeffrey T; Kumar, Challa S S R

    2013-01-01

    Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst. PMID:24327099

  7. Underreliance on mechanistic models: Comment on Ferguson (2015).

    PubMed

    Tryon, Warren W

    2016-09-01

    Ferguson (see record ) proposed that our overreliance on mechanistic models is responsible for the public's negative view of psychology. On the contrary, I claim that our explanations do not actually explain because they lack mechanism information and that is why the public has a negative view of psychology. Some of the mechanism information required to move from interpretations to explanations can be found in parallel distributed processing connectionist neural network models of psychology and behavior. (PsycINFO Database Record PMID:27571530

  8. Mechanistic model and analysis of doxorubicin release from liposomal formulations.

    PubMed

    Fugit, Kyle D; Xiang, Tian-Xiang; Choi, Du H; Kangarlou, Sogol; Csuhai, Eva; Bummer, Paul M; Anderson, Bradley D

    2015-11-10

    Reliable and predictive models of drug release kinetics in vitro and in vivo are still lacking for liposomal formulations. Developing robust, predictive release models requires systematic, quantitative characterization of these complex drug delivery systems with respect to the physicochemical properties governing the driving force for release. These models must also incorporate changes in release due to the dissolution media and methods employed to monitor release. This paper demonstrates the successful development and application of a mathematical mechanistic model capable of predicting doxorubicin (DXR) release kinetics from liposomal formulations resembling the FDA-approved nanoformulation DOXIL® using dynamic dialysis. The model accounts for DXR equilibria (e.g. self-association, precipitation, ionization), the change in intravesicular pH due to ammonia release, and dialysis membrane transport of DXR. The model was tested using a Box-Behnken experimental design in which release conditions including extravesicular pH, ammonia concentration in the release medium, and the dilution of the formulation (i.e. suspension concentration) were varied. Mechanistic model predictions agreed with observed DXR release up to 19h. The predictions were similar to a computer fit of the release data using an empirical model often employed for analyzing data generated from this type of experimental design. Unlike the empirical model, the mechanistic model was also able to provide reasonable predictions of release outside the tested design space. These results illustrate the usefulness of mechanistic modeling to predict drug release from liposomal formulations in vitro and its potential for future development of in vitro - in vivo correlations for complex nanoformulations. PMID:26310713

  9. Oedipus and insight.

    PubMed

    Michels, R

    1986-10-01

    Insight is a core concept in psychoanalytic theory. The Oedipus myth has been a central metaphor in the evolution of psychoanalytic theory, particularly the psychoanalytic theory of development. Similarly, Sophocles' drama, its relation to the myth, and its repeated reinterpretation throughout the ages provide a valuable metaphor for our understanding of the role of insight in psychoanalysis and in development. We may have underestimated the importance of insight in normal development while oversimplifying its significance as an agent of therapeutic change. PMID:3797556

  10. Home range analysis using a mechanistic home range model

    SciTech Connect

    Moorcroft, P.R. . Dept. of Ecology and Evolutionary Biology); Lewis, M.A. . Dept. of Mathematics) Crabtree, R.L. . Dept. of Fish and Wildlife Resources)

    1999-07-01

    The traditional models used to characterize animal home ranges have no mechanistic basis underlying their descriptions of space use, and as a result, the analysis of animal home ranges has primarily been a descriptive endeavor. In this paper, the authors characterize coyote (Canis latrans) home range patterns using partial differential equations for expected space use that are formally derived from underlying descriptions of individual movement behavior. To the authors' knowledge, this is the first time that mechanistic models have been used to characterize animal home ranges. The results provide empirical support for a model formulation of movement response to scent marks, and suggest that having relocation data for individuals in adjacent groups is necessary to capture the spatial arrangement of home range boundaries. The authors then show how the model fits can be used to obtain predictions for individual movement and scent marking behavior and to predict changes in home range patterns. More generally, the findings illustrate how mechanistic models permit the development of a predictive theory for the relationship between movement behavior and animal spatial distribution.

  11. a Mechanistic View of the Ion/solid Interaction Using Laser Postionization, Time-Of Mass Spectrometry, and Molecular Dynamics Computer Simulations

    NASA Astrophysics Data System (ADS)

    Rosencrance, Scott William

    1995-01-01

    Simultaneous collection of energy and angle resolved neutral (EARN) distributions resulting from keV ion bombardment of various materials is performed using a novel position sensitive detection scheme in tandem with multiphoton laser ionization of desorbed neutral species. This enables experimental investigation of desorption processes at the atomic level. Insight into long-standing mechanistic uncertainties surrounding ion/solid interactions is set forth. When these results are viewed collectively a coherent picture of the desorption event emerges. In the first study, experimentally obtained EARN distributions for ground state Ni and Rh atoms desorbed from the { 001} crystal face by 5 keV Ar^+ bombardment are compared with molecular dynamics (MD) simulations. These calculations utilize the molecular dynamics/Monte Carlo -corrected effective medium (MD/MC-CEM) interaction potential. The similarity of the mechanistically resolved quantities reinforces the view that inherent registry in the crystal lattice dictates microscopic events leading to the observed angular distributions of particles ejected subsequent to keV ion bombardment of solid surfaces. Additional evaluation of quantum state resolved energy distributions and populations of atoms desorbed from rm Ni{ 001} reveals that particles ejecting in the rm a^3F_{4,3,2} states have kinetic energy distributions which peak at 3 eV while particles ejecting in the rm a^3D_{3,2 } or rm a^1D_2 states have distributions which peak at 4.3 eV. Furthermore, the population in the ^3D_ {3,2} states is larger than the ground F state. These are the first results demonstrating that the band structure of the solid dictates desorption energetics and populations. Further investigation into desorption processes utilizes MD simulations that incorporate sample temperature. These calculations reproduce experimentally observed temperature dependence in ejection and suggests that the effect predominately results from surface quenching

  12. A tissue-engineered gastric cancer model for mechanistic study of anti-tumor drugs.

    PubMed

    Gao, Ming; Cai, Yiting; Wu, Wei; Shi, Yazhou; Fei, Zhewei

    2013-08-01

    The use of the traditional xenograft subcutaneous tumor model has been contested because of its limitations, such as a slow tumorigenesis, inconsistent chemotherapeutic results, etc. In light of these challenges, we aim to revamp the traditional model by employing an electrospun scaffold composed of polydioxanone, gelatin and elastin to boost the tumorigenesis. The scaffold featured a highly porous microstructure and successfully supported the growth of tumor cells in vitro without provoking apoptosis. In vivo studies showed that in the scaffold model the tumor volume increased by 43.27% and the weight by 75.58%, respectively, within a 12-week period. In addition, the scaffold model saw an increase of CD24(+) and CD44(+) cells in the tumor mass by 42% and 313%, respectively. The scaffolding materials did not lead to phenotypic changes during the tumorigenesis. Thereafter, in the scaffold model, we found that the chemotherapeutic regimen of docetaxel, cisplatin and fluorouracil unleashed a stronger capability than the regimen comprising cisplatin and fluorouracil to deplete the CD44(+) subpopulation. This discovery sheds mechanistic lights on the role of docetaxel for its future chemotherapeutic applications. This revamped model affords cancer scientists a convenient and reliable platform to mechanistically investigate the chemotherapeutic drugs on gastric cancer stem cells. PMID:23715169

  13. Isocyano Enones: Addition-Cyclization Cascade to Oxazoles.

    PubMed

    Chao, Allen; Lujan-Montelongo, J Armando; Fleming, Fraser F

    2016-07-01

    Copper iodide catalyzes the conjugate addition of organometallic and heteroatom nucleophiles to isocyano enones to afford oxazoles. A range of enolates, metalated nitriles, amines, and thiols undergo catalyzed conjugate addition to cyclic and acyclic oxoalkene isocyanides. Mechanistic studies suggest that copper complexation facilitates the nucleophilic attack on the isocyano enone to generate an enolate that cyclizes onto the isocyanide leading to a variety of substituted acyclic or ring-fused oxazoles. PMID:27282173

  14. What Are We Putting in Our Food That Is Making Us Fat? Food Additives, Contaminants, and Other Putative Contributors to Obesity

    PubMed Central

    Simmons, Amber L.; Schlezinger, Jennifer J.; Corkey, Barbara E.

    2014-01-01

    The “chemical obesogen” hypothesis conjectures that synthetic, environmental contaminants are contributing to the global epidemic of obesity. In fact, intentional food additives (e.g., artificial sweeteners and colors, emulsifiers) and unintentional compounds (e.g., bisphenol A, pesticides) are largely unstudied in regard to their effects on overall metabolic homeostasis. With that said, many of these contaminants have been found to dysregulate endocrine function, insulin signaling, and/or adipocyte function. Although momentum for the chemical obesogen hypothesis is growing, supportive, evidence-based research is lacking. In order to identify noxious synthetic compounds in the environment out of the thousands of chemicals that are currently in use, tools and models from toxicology should be adopted (e.g., functional high throughput screening methods, zebrafish-based assays). Finally, mechanistic insight into obesogen-induced effects will be helpful in elucidating their role in the obesity epidemic as well as preventing and reversing their effects. PMID:25045594

  15. In Search of Insight.

    ERIC Educational Resources Information Center

    Kaplan, Craig A.; Simon, Herbert A.

    1990-01-01

    Attaining the insight needed to solve the Mutilated Checkerboard problem, which requires discovery of an effective problem representation (EPR), is described. Performance on insight problems can be predicted from the availability of generators and constraints in the search for an EPR. Data for 23 undergraduates were analyzed. (TJH)

  16. Native Speaker Insight

    ERIC Educational Resources Information Center

    Broughton, Geoffrey

    1978-01-01

    Defines the concept of native speaker insight and suggests that, for the purpose of teaching English as a second language, the goal should not be native speaker insight (NSI) but NS Type 1, a reduced, adequate and attainable goal for foreign learners. (CFM)

  17. Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid

    PubMed Central

    York, Royce L.; Yousefi, Payam A.; Bogdanoff, Walter; Haile, Sara; Tripathi, Sarvind

    2015-01-01

    ABSTRACT Human astroviruses (HAstVs) are nonenveloped, positive-sense, single-stranded RNA viruses that are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we re