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Using a population of recombinant inbred lines (RILs) cowpea (Vigna unguiculata. L. Walp), we tested for co-linkages between lipid contents and chilling responses of photosynthesis. Under low temperature conditions (19°C/13°C, day/night), we observed co-linkages between quantitative trait loci (QTL) intervals for photosynthetic light reactions and specific fatty acids, most strikingly, the thylakoid-specific fatty acid 16:1 found exclusively in phosphatidylglycerol (PG 16:1t). By contrast, we did not observe co-associations with bulk polyunsaturated fatty acids or high-melting-point-PG (sum of PG 16:0, PG 18:0 PG 16:1t) previously thought to be involved in chilling sensitivity. These results suggest that in cowpea, chilling sensitivity is modulated by specific lipid interactions rather than bulk properties. We were able to recapitulate the predicted impact of PG 16:1t levels on photosynthetic responses at low temperature using mutants and transgenic Arabidopsis lines. Because PG 16:1t synthesis requires the activity of peroxiredoxin-Q, which is activated by HO and known to be involved in redox signaling, we hypothesize that the accumulation of PG 16:1t occurs as a result of upstream effects on photosynthesis that alter redox status and production of reactive oxygen species.

The work demonstrates the use of detailed, high-throughput phenotyping to generate and test mechanistic models to explain the genetic diversity of photosynthetic responses to abiotic stress. We assessed a population of recombinant inbred lines (RILs) of cowpea (Vigna unguiculata. (L.) Walp.) with significant differences in a range of photosynthetic responses to chilling. We found well-defined, colocalized (overlapping) QTL intervals for photosynthetic parameters, suggesting linkages among the redox states of Q, the thylakoid pmf, through effects on cyclic electron flow and photodamage to PSII. We propose that these genetic variations optimize photosynthesis in the tolerant lines under low temperatures, preventing recombination reactions within Photosystem II that can lead to deleterious O production. By contrast, we did not observe linkages to PSI redox state, PSI photodamage or ATP synthase activity, or nyctinastic (diurnally controlled) leaf movements, likely indicating that several proposed models likely do not contribute to the genetic control of photosynthesis at low temperature in our mapping panel. The identified QTL intervals include a range of potential causative genetic components, with direct applications to breeding of photosynthesis for more climate-resilient productivity.

When considering patients for a major hepatectomy, one must carefully consider the volume of liver to be left behind and if additional procedures are necessary to augment its volume. This review considers the optimal volume of the future liver remnant (FLR) and analyzes the techniques of augmenting this volume, the various growth parameters to assess adequate growth of the FLR, as well as further management when there has been inadequate growth of the FLR.

We present a new approach to more accurately and efficiently compute the absolute binding free energy for receptor-ligand complexes. Currently, the double decoupling method (DDM) and the potential of mean force method (PMF) are widely used to compute the absolute binding free energy of biomolecular complexes. DDM relies on alchemically decoupling the ligand from its environments, which can be computationally challenging for large ligands and charged ligands because of the large magnitude of the decoupling free energies involved. In contrast, the PMF method uses a physical pathway to directly transfer the ligand from solution to the receptor binding pocket and thus avoids some of the aforementioned problems in DDM. However, the PMF method has its own drawbacks: because of its reliance on a ligand binding/unbinding pathway that is free of steric obstructions from the receptor atoms, the method has difficulty treating ligands with buried atoms. To overcome the limitation in the standard PMF approach and enable buried ligands to be treated, here we develop a new method called AlchemPMF in which steric obstructions along the physical pathway for binding are alchemically removed. We have tested the new approach on two important drug targets involving charged ligands. One is HIV-1 integrase bound to an allosteric inhibitor; the other is the human telomeric DNA G-quadruplex in complex with a natural product protoberberine buried in the binding pocket. For both systems, the new approach leads to more reliable estimates of absolute binding free energies with smaller error bars and closer agreements with experiments compared with those obtained from the existing methods, demonstrating the effectiveness of the new method in overcoming the hysteresis often encountered in PMF binding free energy calculations of such systems. The new approach could also be used to improve the sampling of water equilibration and resolvation of the binding pocket as the ligand is extracted.

Accurately predicting absolute binding free energies of protein-ligand complexes is important as a fundamental problem in both computational biophysics and pharmaceutical discovery. Calculating binding free energies for charged ligands is generally considered to be challenging because of the strong electrostatic interactions between the ligand and its environment in aqueous solution. In this work, we compare the performance of the potential of mean force (PMF) method and the double decoupling method (DDM) for computing absolute binding free energies for charged ligands. We first clarify an unresolved issue concerning the explicit use of the binding site volume to define the complexed state in DDM together with the use of harmonic restraints. We also provide an alternative derivation for the formula for absolute binding free energy using the PMF approach. We use these formulas to compute the binding free energy of charged ligands at an allosteric site of HIV-1 integrase, which has emerged in recent years as a promising target for developing antiviral therapy. As compared with the experimental results, the absolute binding free energies obtained by using the PMF approach show unsigned errors of 1.5-3.4 kcal mol-1, which are somewhat better than the results from DDM (unsigned errors of 1.6-4.3 kcal mol-1) using the same amount of CPU time. According to the DDM decomposition of the binding free energy, the ligand binding appears to be dominated by nonpolar interactions despite the presence of very large and favorable intermolecular ligand-receptor electrostatic interactions, which are almost completely cancelled out by the equally large free energy cost of desolvation of the charged moiety of the ligands in solution. We discuss the relative strengths of computing absolute binding free energies using the alchemical and physical pathway methods.

Background: The utilization of imaging procedures is under scrutiny due to high costs and radiation exposure to patients and staff associated with some radiologic procedures. Within our institution’s intensive care unit (ICU), it is common for patients to undergo chest radiography (CR) not only immediately following tracheostomy tube placement but also on a daily basis, irrespective of the patient’s clinical status. We hypothesize that the clinical utility of performing routine daily CR on patients with tracheostomy tubes is low and leads to unnecessary financial cost. Methods: A retrospective medical chart review was done on 761 CRs performed on 79 ICU patients with tracheostomy from April 2010 to July 2011. We searched the radiology reports of the 761 CRs for the presence of new radiographically detected complications and reviewed medical records to determine which complications were clinically suspected and which radiology reports led to changes in patient management. Results: Of the 761 CRs, only 18 (2.3%) radiographs revealed new complications. All complications were clinically suspected prior to imaging. Only 5 (0.7%) complications resulted in a management change. The most common management changes were a change in antibiotic regimen (0.3%) and ordering of diuretics (0.3%). Conclusions: Routine daily imaging of patients with tracheostomy in an ICU provides little clinical utility, and CR in this population should be performed selectively based on the patient’s clinical status.

Previously we demonstrated profound effects of dopamine transporter (DAT) SLC6A3 genotype on limbic responses to smoking cues (SCs). Probands carrying at least one copy of the 9-repeat allele (9-repeat carriers) had greater neural responses to SCs in the anatomically interconnected rostral ventral striatum/medial orbitofrontal cortex (VS/mOFC), compared with homozygotes for the 10-repeat allele (10/10-repeats). To test the reliability of the initial findings, we examined perfusion functional magnetic resonance images acquired during SC exposure in a new cohort of smokers (N=26) who were genotyped for the SLC6A3 polymorphism. In smokers overall, activity was enhanced in the VS/mOFC (t=3.77). Contrasts between allelic groups revealed that 9-repeat carriers had a greater response to SCs in the VS (t=3.12) and mOFC (t=3.19). In separate groups, 9-repeat carriers showed increased activity in the VS (t=5.47) and mOFC (T=4.96), while no increases were observed in 10-repeats. Subjective reports of craving correlated with increased activity in reward-related structures including the extended amygdala, insula and post-central gyrus, and decreased activity in the dorsolateral prefrontal cortex, and were DAT-genotype dependent (r=0.63-0.96). In secondary analyses, we found that The Fagerstrom Test for Nicotine Dependence scores correlated with enhanced SC-induced perfusion in 10/10-repeats in the insula, mOFC, medial temporal and superior frontal gyri (r=0.50-0.82), while correlations were absent in 9-repeat carriers. Despite heterogeneity introduced by a host of factors, including variance in other genes involved in smoking behavior, we confirm that DAT genotype predicts the direction and location of neural responses to SCs.

We previously demonstrated differential activation of the mesocorticolimbic reward circuitry in response to cigarette cues independent of withdrawal. Despite robust effects, we noted considerable individual variability in brain and subjective responses. As dopamine (DA) is critical for reward and its predictive signals, genetically driven variation in DA transmission may account for the observed differences. Evidence suggests that a variable number of tandem repeats (VNTRs) polymorphism in the DA transporter (DAT) SLC6A3 gene may influence DA transport. Brain and behavioral responses may be enhanced in probands carrying the 9-repeat allele. To test this hypothesis, perfusion fMR images were acquired during cue exposure in 19 smokers genotyped for the 40 bp VNTR polymorphism in the SLC6A3 gene. Contrasts between groups revealed that 9-repeat (9-repeats) had a greater response to smoking (vs nonsmoking) cues than smokers homozygous for the 10-repeat allele (10/10-repeats) bilaterally in the interconnected ventral striatal/pallidal/orbitofrontal cortex regions (VS/VP/OFC). Activity was increased in 9-repeats and decreased in 10/10-repeats in the VS/VP/OFC (p

Aflatoxin B1 is a potent environmental carcinogen produced by certain strains of Aspergillus. Central to the biosynthesis of this mycotoxin is the reaction catalyzed by versicolorin B synthase (VBS) in which a racemic substrate, versiconal hemiacetal, is cyclized to an optically active product whose absolute configuration is crucial to the interaction of aflatoxin B1 with DNA. Attempted over-production of VBS in Escherichia coli led principally to protein aggregated into inclusion bodies but also small amounts of soluble but catalytically inactive enzyme. Comparisons to wild-type VBS by SDS-polyacrylamide gel electrophoresis and after N-glycosidase F treatment revealed that extensive glycosylation accounted for the mass discrepancy (7,000 ±; 1,500 Da) between the native and bacterially expressed proteins. Several over-expression systems in Saccharomyces cerevisiae were surveyed in which one that incorporated a secretion signal was found most successful. VBS of indistinguishable mass on SDS-polyacrylamide gel electrophoresis and kinetic properties from the wild-type enzyme could be obtained in 50-100-fold greater amounts and whose catalytic behavior has been examined. The translated protein sequence of VBS showed three potential N-glycosylation sites (Asn-Xaa-Ser/Thr) consistent with the modifications observed above and unexpectedly revealed extensive homology to the ADP-binding region prominently conserved in the glucose-methanol-choline (GMC) family of flavoenzymes. Over-production of VBS in yeast marks the first aflatoxin biosynthetic enzyme to be so obtained and opens the way to direct study of the enzymology of this complex biosynthetic pathway.

Versicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal (7) to the bisfuran ring system of (−)-versicolorin B (8), an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B1 (1). The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an amino-terminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbs-containing genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered.

Previously we demonstrated profound effects of dopamine transporter (DAT) SLC6A3 genotype on limbic responses to smoking cues (SCs). Probands carrying at least one copy of the 9-repeat allele (9-repeat carriers) had greater neural responses to SCs in the anatomically interconnected rostral ventral striatum/medial orbitofrontal cortex (VS/mOFC), compared with homozygotes for the 10-repeat allele (10/10-repeats). To test the reliability of the initial findings, we examined perfusion functional magnetic resonance images acquired during SC exposure in a new cohort of smokers (N = 26) who were genotyped for the SLC6A3 polymorphism. In smokers overall, activity was enhanced in the VS/mOFC (t = 3.77). Contrasts between allelic groups revealed that 9-repeat carriers had a greater response to SCs in the VS (t = 3.12) and mOFC (t = 3.19). In separate groups, 9-repeat carriers showed increased activity in the VS (t = 5.47) and mOFC (T = 4.96), while no increases were observed in 10-repeats. Subjective reports of craving correlated with increased activity in reward-related structures including the extended amygdala, insula and post-central gyrus, and decreased activity in the dorsolateral prefrontal cortex, and were DAT-genotype dependent (r = 0.63–0.96). In secondary analyses, we found that The Fagerström Test for Nicotine Dependence scores correlated with enhanced SC-induced perfusion in 10/10-repeats in the insula, mOFC, medial temporal and superior frontal gyri (r = 0.50–0.82), while correlations were absent in 9-repeat carriers. Despite heterogeneity introduced by a host of factors, including variance in other genes involved in smoking behavior, we confirm that DAT genotype predicts the direction and location of neural responses to SCs.

A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatography and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death.

The absolute configuration of the dihydrobisfuran ring system characteristic of aflatoxin B1 is essential to the covalent reaction of its metabolically activated form with double-stranded DNA. The biosynthesis of this potent mycotoxin proceeds through three configurationally labile intermediates to racemic versiconal hemiacetal. Subsequent enzymatic cyclization establishes the stereochemistry of this, critical ring fusion in (-)-versicolorin B and is catalyzed by versicolorin B synthase (VBS). The isolation and purification of VBS from Aspergillus parasiticus (SU-1, ATCC 56775) and its kinetic characterization and attempted inactivation are described. Initial purification trials were plagued both by a chromophoric impurity which was difficult to remove and by low recoveries of active protein. The discovery of a remarkably broad pH range of enzyme stability and catalytic activity led to an efficient procedure involving preparative isoelectric focusing and ion exchange FPLC chromatography. The enzyme behaved as a dimer upon gel filtration and migrated with M(r) 78000 Da during denaturing gel electrophoresis. The UV spectrum of pure VBS gave no evidence of a bound chromophore. Detailed kinetic analysis of VBS revealed that this protein selects from two equilibrating enantiomers of versiconal hemiacetal to cyclize the appropriate antipode to optically pure versicolorin B. By varying the amount of enzyme to a fixed concentration of substrate, the rate of enzymic cyclization could be limited by the intrinsic rate of enantiomerization of the substrate under the conditions of reaction. It was possible to quantitate the dynamics of this substrate enantiomerization/cyclization process, to establish the role played by VBS, and to evaluate the significance of each to the overall biosynthesis of aflatoxin. The potential role of an acidic residue of the enzyme in catalysis was supported by analysis of the pH-rate profile of VBS and chemical labeling studies. Successful demonstration of competitive inhibition of VBS by a simple substrate analogue led to the design and synthesis of a potential mechanism-based inactivator of the protein.

Numerous factors are involved in the motivation to smoke and are associated with relapse, including stress, peer pressure, availability, menstrual cycle phase, and even weight management.1–5 However, smoking cue–induced and withdrawal-induced cravings are 2 of the major contributors to relapse.6–9 Inability to combat withdrawal-induced craving, which declines within a month,10 plays a role in early relapse. Nevertheless, smokers report that smoking cues (eg, seeing a pack of cigarettes, socializing with others who smoke, and even internal mood states repeatedly associated with smoking) can trigger relapse months or even years after quitting. Some smokers who are thought to possess high “cue re-activity” are especially vulnerable and have an increased probability of relapse initiated by exposure to smoking cues.11,12 Therefore, treatments that target cue reactivity are important, particularly for cue-vulnerable individuals, but the effect of existing smoking cessation medications on smoking cue reactivity has not been thoroughly investigated. Thus far, research has focused on reduction of withdrawal and nicotine reward, which are known mechanisms underlying the effectiveness of first-line smoking cessation agents, such as varenicline, nicotine replacement therapy, and bupropion hydrochloride.13–15 Varenicline is a first-line smoking cessation agent16,17 that acts as a partial agonist at α4β2 acetylcholine nicotinic receptors, with indirect agonist and antagonist actions on the mesolimbic dopamine system. During the absence of nicotine, as in a quit attempt, it acts as an agonist to mildly increase dopaminergic tone and reduce withdrawal-induced craving. When nicotine is available, as in a relapse, it acts as an antagonist, preventing nicotine-evoked dopamine release and effectively blocking the reward usually received from nicotine while smoking.15 It is thought that the dual agonist-antagonist properties of varenicline are key mechanisms underlying its clinical effectiveness. Various imaging modalities have observed a consistent neural substrate for cocaine, heroin, cigarette, and sexual cues.3,18–21 In studies of smoking cue reactivity, we characterized a neuroanatomical brain signature in response to exposure to smoking cues, independent of withdrawal, wherein the most profound effects were found in the interconnected ventral striatum (VS) and medial orbitofrontal cortex (mOFC).22–24 Our findings are in accordance with the substantial preclinical literature.25–27 Based on the evidence supporting a role for the medial ventral aspects of the mesolimbic system in drug cue reactivity, and varenicline’s actions to manipulate dopamine release, we hypothesized that chronic varenicline administration would suppress these responses; specifically, we hypothesized that varenicline would modulate activity in the mOFC, which is involved in sensory integration (representing the affective value of reinforcers) and decision making (for emotional rewards).28,29 Furthermore, we suspected that varenicline might diminish ventral striatal responses to cues because this region exerts strong control over emotional and motivational behavior, including craving.30,31 Preliminary data from our laboratory showed that varenicline selectively activated the lateral OFC (LOFC) in the brain at rest. Based on our data and the literature demonstrating that lateral pre-frontal regions are involved in regulating impulses, in reevaluating previously rewarded behavior, and in modulating downstream limbic regions involved in motivated behavior,29,32,33 we suspected that varenicline might enhance activity in lateral prefrontal regions. We predicted that varenicline-induced activation of the LOFC in the brain at rest would correlate with diminished neural responses during exposure to smoking cues. To quantify the effects of long-term administration of varenicline on the resting brain and on the brain’s responses during exposure to smoking cues, we implemented a laboratory model of conditioned responding and the technique of continuous arterial spin-labeled (CASL) perfusion functional magnetic resonance imaging (fMRI) to image nonabstinent smokers before and after a 3-week double-blind randomized placebo-controlled medication regimen. The importance of using nontreatment-seeking smokers in our paradigm is 2-fold. First, our goal was to determine if and how varenicline affected smoking cue reactivity independent of withdrawal (which can persist for up to a month) because it has been shown that withdrawal itself can affect brain activity.34 Second, it was important that varenicline-treated and placebo-treated groups had similar smoking characteristics because differences in smoking behavior modulate brain activity.35 Thus, issues related to withdrawal and quitting smoking, which might obviate accurate interpretation of the effects of varenicline on exposure to smoking cues, were minimized. Similar to positron emission tomography, perfusion fMRI is quantitative, providing a measure of cerebral blood flow in milliliters of blood per 100 g of tissue per minute,36 which facilitates the measurement of medication-induced neural modifications in the brain in response to tasks (cue exposure)23 and in the brain in the resting condition (without provocation)37 at successive time points. A pharmacological manipulation can have profound effects on the brain that cannot be observed using a relative measure such as blood oxygen level–dependent fMRI, which can only accurately examine changes that occur within a scanning session during a task or other provocation. Perfusion fMRI is reliable and reproducible following intervals as long as 7 weeks and is therefore ideal for longitudinal studies examining brain modifications induced by pharmacological agents.37