The Importance of Stereochemically Active Lone Pairs For Influencing Pb II and As III Protein Binding

The toxicity of heavy metals, which is associated with the high affinity of the metals for thiolate rich proteins, constitutes a problem worldwide. However, despite this tremendous toxicity concern, the binding mode of As III and Pb II to proteins is poorly understood. To clarify the requirements for toxic metal binding to metalloregulatory sensor proteins such as As III in ArsR/ArsD and Pb II in PbrR or replacing Zn II in δ‐aminolevulinc acid dehydratase (ALAD), we have employed computational and experimental methods examining the binding of these heavy metals to designed peptide models. The computational results show that the mode of coordination of As III and Pb II is greatly influenced by the steric bulk within the second coordination environment of the metal. The proposed basis of this selectivity is the large size of the ion and, most important, the influence of the stereochemically active lone pair in hemidirected complexes of the metal ion as being crucial. The experimental data show that switching a bulky leucine layer above the metal binding site by a smaller alanine residue enhances the Pb II  binding affinity by a factor of five, thus supporting experimentally the hypothesis of lone pair steric hindrance. These complementary approaches demonstrate the potential importance of a stereochemically active lone pair as a metal recognition mode in proteins and, specifically, how the second coordination sphere environment affects the affinity and selectivity of protein targets by certain toxic ions. Experimental and computational methods have been employed to study the influence of the lone pair of As III and Pb II for the binding of these ions in proteins using designed peptide models. The results show that the mode of coordination of As III and Pb II is greatly influenced by the steric bulk within the second coordination environment of the metals (see figure).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90414/1/chem_201102786_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/90414/2/2040_ftp.pd

Mechanistic Analysis of Nucleophilic Substrates Oxidation by Functional Models of Vanadium-Dependent Haloperoxidases: A Density Functional Theory Study

Density functional theory has been used to investigate the structural, electronic, and reactivity properties of an established functional model for vanadium-dependent haloperoxidases, K[VO(O 2 )Hheida] (Hheida 2– = 2,2′-[(2-hydroxyethyl)imino]diacetate). Possible solution species were determined on the basis of potential exogenous donors present under the conditions necessary for reactivity. The energetically favored solution-state species is a 1:1 complex of Hheida and vanadium with a coordinated hydroxyethyl donor trans to the vanadium–oxido bond which is in agreement with the reported solid-state structure for K[VO(O 2 )Hheida]. Transition states of the oxidation reaction were located for four substrates: chloride, bromide, iodide, and dimethyl sulfide. The role of protonation and its effects on reactivity were examined for each substrate. Protonation of the peroxido moiety leads to a significant drop in the activation barrier for oxidation. In contrast no transition states could be located for an oxido-transfer process involving the oxido ligand. Barriers of activation calculated for halide oxidation were similar, providing support to the hypothesis that the p K a of the halide in acetonitrile is responsible for the decrease in reactivity between I – , Br – , and Cl – . The results presented herein provide a mechanistic correlation between a functional model and the enzyme, making K[VO(O 2 )Hheida] a “complete” functional model for vanadium-dependent haloperoxidase.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55972/1/515_ftp.pd

Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export

BACKGROUND: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Saccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase (LDH) gene. The LDH gene expression in a budding yeast cell introduces a novel and alternative pathway for the NAD(+ )regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading to a simultaneous accumulation of lactate and ethanol. RESULTS: Four different S. cerevisiae strains were transformed with six different wild type and one mutagenised LDH genes, in combination or not with the over-expression of a lactate transporter. The resulting yield values (grams of lactate produced per grams of glucose consumed) varied from as low as 0,0008 to as high as 0.52 g g(-1). In this respect, and to the best of our knowledge, higher redirections of the glycolysis flux have never been obtained before without any disruption and/or limitation of the competing biochemical pathways. CONCLUSION: In the present work it is shown that the redirection of the pathway towards the lactate production can be strongly modulated by the genetic background of the host cell, by the source of the heterologous Ldh enzyme, by improving its biochemical properties as well as by modulating the export of lactate in the culture media

Plakilactones G and H from a marine sponge. Stereochemical determination of highly flexible systems by quantitative NMR-derived interproton distances combined with quantum mechanical calculations of C-13 chemical shifts

In this paper the stereostructural investigation of two new oxygenated polyketides, plakilactones G and H, isolated from the marine sponge Plakinastrella mamillaris collected at Fiji Islands, is reported. The stereostructural studies began on plakilactone H by applying an integrated approach of the NOE-based protocol and quantum mechanical calculations of (13)C chemical shifts. In particular, plakilactone H was used as a template to extend the application of NMR-derived interproton distances to a highly flexible molecular system with simultaneous assignment of four non-contiguous stereocenters. Chemical derivatization and quantum mechanical calculations of (13)C on plakilactone G along with a plausible biogenetic interconversion between plakilactone G and plakilactone H allowed us to determine the absolute configuration in this two new oxygenated polyketides

Analysis of cadence/heart rate (RPM/HR) versus power output (PO) during incremental test in cyclists

Studies on cycling cadence have been mainly focussed on the optimisation of cycling efficiency [1-3]. In order to obtain new insight into physiological indicators of performance, the present study aimed to evaluate the relationship between freely chosen cadence (FCC), heart rate (HR) and power output (PO) profiles in members of professional and non professional cycling teams during an incremental test (10W 30 sec). Heterogeneous group of 25 male cyclists performed a maximal incremental test on SRM powermeter (SRM Training Systems, Jülich, Germany) and FCC/HR vs PO was measured. In all subjects the FCC/HR vs PO obtained showed a linear phase followed by a curvilinear phase starting at comparable FCC/HR value in all subjects (0,62 ± 0,06 SD; ES: 0,01, IC95%: 0,60 - 0,65, IC99%: 0.59 - 0.65). Whether the observed deflection point corresponds to anaerobic threshold deserves future investigation

Comparing the prognostic value of stress myocardial perfusion imaging by conventional and cadmium-zinc telluride single-photon emission computed tomography through a machine learning approach

We compared the prognostic value of myocardial perfusion imaging (MPI) by conventional- (C-) single-photon emission computed tomography (SPECT) and cadmium-zinc-telluride- (CZT-) SPECT in a cohort of patients with suspected or known coronary artery disease (CAD) using machine learning (ML) algorithms. A total of 453 consecutive patients underwent stress MPI by both C-SPECT and CZT-SPECT. The outcome was a composite end point of all-cause death, cardiac death, nonfatal myocardial infarction, or coronary revascularization procedures whichever occurred first. ML analysis performed through the implementation of random forest (RF) and k-nearest neighbors (KNN) algorithms proved that CZT-SPECT has greater accuracy than C-SPECT in detecting CAD. For both algorithms, the sensitivity of CZT-SPECT (96% for RF and 60% for KNN) was greater than that of C-SPECT (88% for RF and 53% for KNN). A preliminary univariate analysis was performed through Mann-Whitney tests separately on the features of each camera in order to understand which ones could distinguish patients who will experience an adverse event from those who will not. Then, a machine learning analysis was performed by using Matlab (v. 2019b). Tree, KNN, support vector machine (SVM), Naïve Bayes, and RF were implemented twice: first, the analysis was performed on the as-is dataset; then, since the dataset was imbalanced (patients experiencing an adverse event were lower than the others), the analysis was performed again after balancing the classes through the Synthetic Minority Oversampling Technique. According to KNN and SVM with and without balancing the classes, the accuracy (p value = 0.02 and p value = 0.01) and recall (p value = 0.001 and p value = 0.03) of the CZT-SPECT were greater than those obtained by C-SPECT in a statistically significant way. ML approach showed that although the prognostic value of stress MPI by C-SPECT and CZT-SPECT is comparable, CZT-SPECT seems to have higher accuracy and recall

Imaging techniques for assessment of coronary flow reserve

The assessment of coronary flow reserve (CFR) may be useful for the functional evaluation of coronary artery disease (CAD). Invasive techniques, such as intracoronary Doppler ultrasound and pressure-derived method, directly assess CFR velocity and fractional flow reserve. Positron emission tomography (PET) has emerged as an accurate noninvasive technique to quantify CFR. Nevertheless, this approach has not been applied to routine studies because of its high cost and complexity. Recently, attempts to estimate CFR with single-photon emission computed tomography (SPECT) tracers have been made in order to obtain, with noninvasive methods, data for quantitative functional assessment of CAD. This review analyzes the relative merit and limitations of CFR measurements by cardiac imaging techniques and describes the potential clinical applications

Discovery That Theonellasterol a Marine Sponge Sterol Is a Highly Selective FXR Antagonist That Protects against Liver Injury in Cholestasis

Background: The farnesoid-x-receptor (FXR) is a bile acid sensor expressed in the liver and gastrointestinal tract. Despite FXR ligands are under investigation for treatment of cholestasis, a biochemical condition occurring in a number of liver diseases for which available therapies are poorly effective, mice harboring a disrupted FXR are protected against liver injury caused by bile acid overload in rodent models of cholestasis. Theonellasterol is a 4-methylene-24-ethylsteroid isolated from the marine sponge Theonella swinhoei. Here, we have characterized the activity of this theonellasterol on FXR-regulated genes and biological functions. Principal Findings: Interrogation of HepG2 cells, a human hepatocyte cell line, by microarray analysis and transactivation assay shows that theonellasterol is a selective FXR antagonist, devoid of any agonistic or antagonistic activity on a number of human nuclear receptors including the vitamin D receptor, PPARs, PXR, LXRs, progesterone, estrogen, glucorticoid and thyroid receptors, among others. Exposure of HepG2 cells to theonellasterol antagonizes the effect of natural and synthetic FXR agonists on FXR-regulated genes, including SHP, OSTa, BSEP and MRP4. A proof-of-concept study carried out to investigate whether FXR antagonism rescues mice from liver injury caused by the ligation of the common bile duct, a model of obstructive cholestasis, demonstrated that theonellasterol attenuates injury caused by bile duct ligation as measured by assessing serum alanine aminostrasferase levels and extent of liver necrosis at histopathology. Analysis of genes involved in bile acid uptake and excretion by hepatocytes revealed that theonellasterol increases the liver expression of MRP4, a basolateral transporter that is negatively regulated by FXR. Administering bile duct ligated mice with an FXR agonist failed to rescue from liver injury and downregulated the expression of MRP4. Conclusions: FXR antagonism in vivo results in a positive modulation of MRP4 expression in the liver and is a feasible strategy to target obstructive cholestasis

A zinc, copper and citric acid biocomplex shows promise for control of Xylella fastidiosa subsp. pauca in olive trees in Apulia region (southern Italy)

The bacterium Xylella fastidiosa subsp. pauca is associated with the “olive quick decline syndrome” in the Apulia region of southern Italy. To investigate control of this phytopathogen, a compound containing zinc and copper complexed with citric-acid hydracids (Dentamet®) was evaluated for in vitro and in planta bactericidal activity. Confocal laser scanning microscopy, fluorescent quantification and atomic emission spectroscopy were then used to determine if the compound reached the xylem networks of leaves, twigs and branches of olive, to release zinc and copper within the xylem. A 3-year field trial in an olive orchard containing mature Cellina di Nardò and Ogliarola salentina olive trees, and officially declared infected by X. fastidiosa subsp. pauca,was also carried out o to determine if the compound affected severity of the disease. Each year, from early April to October (excluding July and August), six spray treatments of 0.5% (v:v) Dentamet® were applied on the olive tree crowns. The compound reduced severity of symptoms in both cultivars. Most untreated trees died by the end of the trial, whereas all treated trees survived with good vegetative status as assessed by a normalized difference vegetation index. Quantitative real-time PCR was performed from June 2016 to September 2017, following the official procedures established by the European and Mediterranean Plant Protection Organization. The analysis revealed a statistically significant reduction of X. fastidiosa cell densities within the leaves of treated trees. These promising results suggest that integrated management to reduce severity of X. fastidiosa that includes regular pruning and soil harrowing with spring and summer spray treatments with Dentamet®, is likely to effectively control the disease.