Structural mechanism of Escherichia coli cyanase.

Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate-dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate-recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5-1.9 Šresolution using synchrotron X-rays and an X-ray free-electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate-binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate-binding and reaction process of cyanase.

Synthesis and antitumor activity of some cholesterol-based selenocyanate compounds.

The introduction of selenium-containing functional groups into steroids to study the biological activities of related derivatives is rarely reported in the literature. In the present study, using cholesterol as raw material, four cholesterol-3-selenocyanoates and eight B-norcholesterol selenocyanate derivatives were synthesized, respectively. The structures of the compounds were characterized by NMR and MS. The results of the in vitro antiproliferative activity test showed that the cholesterol-3-selenocyanoate derivatives did not exhibit obvious inhibitory on the tested tumor cell lines. However, the B-norcholesterol selenocyanate derivatives obtained by structural modification of cholesterol showed good inhibitory activity against the proliferation of tumor cell. Among them, compounds 9b-c, 9f and 12 showed similar inhibitory activity against tested tumor cells as positive control 2-methoxyestradiol, and better than Abiraterone. At the same time, these B-norcholesterol selenocyanate derivatives displayed a strong selective inhibitory against Sk-Ov-3 cell line. Except for compound 9g, the IC50 value of all B-norcholesterol selenocyanate compounds against Sk-Ov-3 cells was less than 10 µM, and compound 9d was 3.4 µM. In addition, Annexin V-FITC/PI double staining was used to analyze the cell death mechanism. The results showed that compound 9c could induce Sk-Ov-3 cells to enter programmed apoptosis in a dose-dependent manner. Furthermore, the in vivo antitumor experiments of compound 9f against zebrafish xenograft tumor showed that 9f displayed obvious inhibitory effect on the growth of human cervical cancer (HeLa) xenograft tumor in zebrafish. Our results provide new thinking for the study of such compounds as new antitumor drugs.

Biotinylated selenocyanates: Potent and selective cytostatic agents.

Most of the currently available cytotoxic agents for tackling cancer are devoid of selectivity, thus causing severe side-effects. This situation stimulated us to develop new antiproliferative agents with enhanced affinity towards tumour cells. We focused our attention on novel chalcogen-containing compounds (thiosemicarbazones, disulfides, selenoureas, thio- and selenocyanates), and particularly on selenium derivatives, as it has been documented that this kind of compounds might act as prodrugs releasing selenium-based reactive species on tumour cells. Particularly interesting in terms of potency and selectivity was a pharmacophore comprised by a selenocyanato-alkyl fragment connected to a p-phenylenediamine residue, where the nature of the second amino moiety (free, Boc-protected, enamine-protected) provided a wide variety of antiproliferative activities, ranging from the low micromolar to the nanomolar values. The optimized structure was in turn conjugated through a peptide linkage with biotin (vitamin B7), a cellular growth promoter, whose receptor is overexpressed in numerous cancer cells; the purpose was to develop a selective vector towards malignant cells. Such biotinylated derivative behaved as a very strong antiproliferative agent, achieving GI50 values in the low nM range for most of the tested cancer cells; moreover, it was featured with an outstanding selectivity, with GI50 > 100 µM against human fibroblasts. Mechanistic studies on the mode of inhibition of the biotinylated selenocyanate revealed (Annexin-V assay) a remarkable increase in the number of apoptotic cells compared to the control experiment; moreover, depolarization of the mitochondrial membrane was detected by flow cytometry analysis, and with fluorescent microscopy, what supports the apoptotic cell death. Prior to the apoptotic events, cytostatic effects were observed against SW1573 cells using label-free cell-living imaging; therefore, tumour cell division was prevented. Multidrug resistant cell lines exhibited a reduced sensitivity towards the biotinylated selenocyanate, probably due to its P-gp-mediated efflux. Remarkably, antiproliferative levels could be restored by co-administration with tariquidar, a P-gp inhibitor; this approach can, therefore, overcome multidrug resistance mediated by the P-gp efflux system.

New Experimental Conditions for Diels-Alder and Friedel-Crafts Alquilation Reactions with Thiophene: A New Selenocyanate with Potent Activity against Cancer.

The reactivity of thiophene in Diels-Alder reactions is investigated with different maleimide derivatives. In this paper, we have synthesized for the first time the Diels-Alder adducts of thiophene at room temperature and atmospheric pressure. Maleimido-thiophene adducts were promoted by AlCl3. The effects of solvent, time, temperature and the use of different Lewis acids were studied, showing dramatic effects for solvent and Lewis acid. Furthermore, the catalysis with AlCl3 is highly stereoselective, preferably providing the exo form of the adduct. Additionally, we also discovered the ability of AlCl3 to catalyze the arylation of maleimides to yield 3-aryl succinimides in a straightforward manner following a Friedel-Crafts-type addition. The inclusion of a selenocyanate group contributes to the cytotoxic activity of the adduct. This derivatization (from compound 7 to compound 15) results in an average GI50 value of 1.98 µM in the DTP (NCI-60) cell panel, resulting in being especially active in renal cancer cells.

Carbamylation of elastic fibers is a molecular substratum of aortic stiffness.

Because of their long lifespan, matrix proteins of the vascular wall, such as elastin, are subjected to molecular aging characterized by non-enzymatic post-translational modifications, like carbamylation which results from the binding of cyanate (mainly derived from the dissociation of urea) to protein amino groups. While several studies have demonstrated a relationship between increased plasma concentrations of carbamylated proteins and the development of cardiovascular diseases, molecular mechanisms explaining the involvement of protein carbamylation in these pathological contexts remain to be fully elucidated. The aim of this work was to determine whether vascular elastic fibers could be carbamylated, and if so, what impact this phenomenon would have on the mechanical properties of the vascular wall. Our experiments showed that vascular elastin was carbamylated in vivo. Fiber morphology was unchanged after in vitro carbamylation, as well as its sensitivity to elastase degradation. In mice fed with cyanate-supplemented water in order to increase protein carbamylation within the aortic wall, an increased stiffness in elastic fibers was evidenced by atomic force microscopy, whereas no fragmentation of elastic fiber was observed. In addition, this increased stiffness was also associated with an increase in aortic pulse wave velocity in ApoE-/- mice. These results provide evidence for the carbamylation of elastic fibers which results in an increase in their stiffness at the molecular level. These alterations of vessel wall mechanical properties may contribute to aortic stiffness, suggesting a new role for carbamylation in cardiovascular diseases.

Benzimidazole- and Imidazole-Fused Selenazolium and Selenazinium Selenocyanates: Ionic Organoselenium Compounds with Efficient Peroxide Scavenging Activities.

Three new classes of ionic organoselenium compounds containing cationic benzimidazolium and imidazolium ring systems with selenocyanates as counterions are described. The cyclization of N,N'-disubstituted benzimidazolium and imidazolium bromides having N-(CH2)2-Br and N-(CH2)3-Br groups in the presence of potassium selenocyanate (KSeCN) led to formation of the corresponding selenazolium selenocyanates (21a, 21b, 22a, and 22b) and selenazinium selenocyanates (21c, 21d, 22c, and 22d). However, the open-chain selenocyanates with additional selenocyanate counterions (21e, 21f, 22e, and 22f) were formed from the N,N'-disubstituted benzimidazolium and imidazolium bromides having N-(CH2)6-Br groups. Mechanistic studies were carried out to understand the feasibility of such cyclization processes in the presence of KSeCN. The compounds were studied further for their potencies to catalytically reduce H2O2 in the presence of thiols. Interestingly, the cyclic selenazolium (21a, 21b, 22a, and 22b) and selenazinium compounds (21c, 21d, 22c, and 22d) exhibited significantly higher antioxidant activities than the corresponding acyclic selenocyanates (21f, 22e, and 22f). Selected compounds (22d and 22e) were further evaluated for their potencies in modulating the intracellular level of reactive oxygen species (ROS) in a representative macrophage cell line (RAW 264.7). Owing to the cationic nature of compounds, they may target and scavenge mitochondrial ROS in the cellular medium.

Selenosteroids - promising hybrid compounds with pleiotropic biological activity: synthesis and biological aspects.

It is established that steroid based agents are an example of compounds obtained from natural patterns and are of great importance due to their application in the prevention and treatment of diseases. Selenosteroids are hybrids formed by attaching Se-moiety to a steroid molecule. In these types of hybrids, selenium can be present as selenide or as a part of selenosemicarbazones, isoselenocyanates, selenourea, etc. Attaching a Se-moiety to a biologically active steroid might enhance the biological properties of both fragments. Available literature indicates that these kinds of hybrids demonstrate significant anticancer activity, which renders them interesting in terms of medical use. In this review, we present various methods of synthesis and demonstrate that seleno-steroid compounds are promising molecules for further pharmaceutical application.

Synthesis of Novel Selenocyanates and Evaluation of Their Effect in Cultured Mouse Neurons Submitted to Oxidative Stress.

Herein, we report the synthesis of novel selenocyanates and assessment of their effect on the oxidative challenge elicited by hydrogen peroxide (H2O2) in cultured mouse neurons. First, α-methylene-ß-hydroxy esters were prepared as precursors of allylic bromides. A reaction involving the generated bromides and sodium selenocyanate was conducted to produce the desired selenocyanates (3a-f). We next prepared cultures of neurons from 7-day-old mice (n = 36). H2O2 (10-5 M) was added into the culture flasks as an oxidative stress inducer, alone or combined with one of each designed compounds. (PhSe)2 was used as a positive control. It was carried out assessment of lipid (thiobarbituric acid reactive species, 4-hydroxy-2'-nonenal, 8-isoprostane), DNA (8-hydroxy-2'-deoxyguanosine), and protein (carbonyl) modification parameters. Finally, catalase and superoxide dismutase activities were also evaluated. Among the compounds, 3b, 3d, and 3f exhibited the most pronounced pattern of antioxidant activity, similar to (PhSe)2. These novel aromatic selenocyanates could be promising to be tried in most sophisticated in vitro studies or even at the preclinical level.

Pre-clinical evidences of the antileishmanial effects of diselenides and selenocyanates.

A series of thirty-one selenocompounds covering a wide chemical space was assessed for in vitro leishmanicidal activities against Leishmania infantum amastigotes. The cytotoxicity of those compounds was also evaluated on human THP-1 cells. Interestingly most tested derivatives were active in the low micromolar range and seven of them (A.I.3, A.I.7, B.I.1, B.I.2, C.I.7 C.I.8 and C.II.8) stood out for selectivity indexes higher than the ones exhibited by reference compounds mitelfosine and edelfosine. These leader compounds were evaluated against infected macrophages and their trypanothione reductase (TryR) inhibition potency was measured to further approach the mechanism by which they caused their action. Among them diselenide tested structures were pointed out for their ability to reduce infection rates. Three of the leader compounds inhibited TryR effectively, therefore this enzyme may be implicated in the mechanism of action by which these compounds cause their leishmanicidal effect.

Design, synthesis and acaricidal activities of Cyflumetofen analogues based on carbon-silicon isosteric replacement.

The application of a carbon-silicon bioisosteric replacement strategy to find new acaricides with improved properties led to the discovery of Sila-Cyflumetofen 6B, a novel and highly potent acaricide. The essential t-butyl group in the beta-ketonitrile acaricide Cyflumetofen 6A could be swapped with the bioisosteric trimethyl-silyl group with retention of high level acaricidal activity and favourable pharmacological properties. Sila-Cyflumetofen 6B was found to possess similar preferred energy-minimized conformation and electrostatic potential surface compare to Cyflumetofen 6A. Herein we also report the development and application of the first homology model of the spider mite mitochondrial electron transport complex II (succinate ubiquinone oxidoreductase; SQR) and demonstrated that the active metabolite AB-1 of Cyflumetofen 6A and its sila-analogue Sila-AB-1 bind to the Qp site in same binding pose and that both compounds form two H-bonds and a cation-π interaction with Trp 165, Tyr 433 and Arg 279, respectively. Furthermore, we also developed a new mode of action test for spider mite Complex II using cytochrome c as electron acceptor and blocking its re-oxidation by addition of KCN resulting in a sensitive and convenient colorimetric assay. This new method avoids the use of non-specific artificial electron acceptors and allows to measure SQR inhibition in crude extracts of Tetranychus urtice. In this assay Sila-AB-1, the intrinsically active metabolite of Sila-Cyflumetofen, 6A exhibited even a somewhat lower IC50 value than the metabolite of Cyflumetofen AB-1. Synthetic methodologies are described for the preparation of Sila-Cyflumetofen 6B and its active metabolite Sila-AB-1 which enable an efficient synthesis of these compounds in only 5 and 4 steps, respectively, from cheap commercial starting materials. Although the value of carbon-silicon bioisosteric replacements has already be demonstrated in the past it is to the best of our knowledge the first report of a successful application in crop protection research in the last two decades.

Synthesis, characterization and anticancer activity in vitro evaluation of novel dicyanoaurate (I)-based complexes.

Molecular structures containing gold, such as auranofin, have been extensively studied in the diagnosis and treatment of many diseases, including cancer treatment. The pharmacological properties of the newly synthesized unique gold-ligand structures have been reported for different cancer cell lines. However, findings on bishydeten-metal salt complexes with gold are rare. In this work, the synthesis of five novel cyanide-bridged coordination compounds having the closed formulae [Ni(bishydeten)][Au(CN)2]2 (1), [Cu(bishydeten)][Au(CN)2]2 (2), [Zn(bishydeten)2Au3(CN)4][Au2(CN)3] (3), [Cd(bishydeten)0,5]2[Au(CN)2]4.2H2O (4), and [Cd(bishydeten)2][Au(CN)2]2 (5) (where bisyhdeten = N,N-bis(2-hydroxyethyl)ethylene diamine), and their characterization by elemental, infrared, ESI-MS, X-ray (for 2) and thermic measurement methods were performed. Complexes 1 and 3 are thermally more stable than the other three complexes. For these, pharmacological adequacies were also tested. The nucleic acid and protein binding affinities of the Au (I) compounds were also estimated by spectroscopic and electrophoretic techniques. Au (I) complexes were identified as strong chemotherapeutic with mild cytotoxicity, and they demonstrated a dose-dependent inhibition on the growth of cancer cells with IC50 at 0.11 to 0.47 µM. Investigation of mechanisms of action on cells revealed that Au (I) compounds managed to inhibit cell migration and led to a decrease in cytoskeletal proteins such as CK7 and CK20. However, Au (I) compounds failed to inhibit DNA topoisomerase I. Overall, and we suggest that potent antiproliferative activity, mild cytotoxicity, good solubility, and micromolar dosage of Au (I) compounds containing bisyhdeten-metal derivatives render them the potential focus of further studies as chemotherapeutic agents.

Synthesis and Potential Anticancer Activity of Some Novel Selenocyanates and Diselenides.

In the present study, twenty-four selenocyanate and diselenide compounds were synthesized and characterized, and their anticancer activities against the human cancer cell lines Caco2, BGC-823, MCF-7 and PC-3 were determined. Interestingly, most of the new compounds were active in reducing the viability of different cancer cell lines. Two compounds exhibited higher promising activities than other derivatives. The most active compound showed the least IC50 values against the four cancer cell lines, particularly to PC-3 with IC50 values below 5 µm. Two compounds were selected to monitor the expression levels of Bcl-2, IL-2 and caspase-3 molecular biomarkers. Interestingly, the two compounds downregulated the Bcl-2 expression levels and upregulated the expression of IL-2 and caspase-3 in PC-3 cells compared to untreated cells. Moreover, most of the synthesized organoselenides exhibited good Gpx-like activities comparable to ebselen. These results appear that introduction of selenocyanate (-SeCN) or diselenides (-Se-Se-) moiety to some carboxy derivatives could serve as a promising launch point for the further design of this type of organic selenium anticancer agent.

Cyanate Induces Oxidative Stress Injury and Abnormal Lipid Metabolism in Liver through Nrf2/HO-1.

Chronic kidney disease (CKD) is problem that has become one of the major issues affecting public health. Extensive clinical data suggests that the prevalence of hyperlipidemia in CKD patients is significantly higher than in the general population. Lipid metabolism disorders can damage the renal parenchyma and promote the occurrence of cardiovascular disease (CVD). Cyanate is a uremic toxin that has attracted widespread attention in recent years. Usually, 0.8% of the molar concentration of urea is converted into cyanate, while myeloperoxidase (MPO) catalyzes the oxidation of thiocyanate to produce cyanate at the site of inflammation during smoking, inflammation, or exposure to environmental pollution. One of the important physiological functions of cyanate is protein carbonylation, a non-enzymatic post-translational protein modification. Carbamylation reactions on proteins are capable of irreversibly changing protein structure and function, resulting in pathologic molecular and cellular responses. In addition, recent studies have shown that cyanate can directly damage vascular tissue by producing large amounts of reactive oxygen species (ROS). Oxidative stress leads to the disorder of liver lipid metabolism, which is also an important mechanism leading to cirrhosis and liver fibrosis. However, the influence of cyanate on liver has remained unclear. In this research, we explored the effects of cyanate on the oxidative stress injury and abnormal lipid metabolism in mice and HL-7702 cells. In results, cyanate induced hyperlipidemia and oxidative stress by influencing the content of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), superoxide dismutase (SOD), catalase (CAT) in liver. Cyanate inhibited NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and the phosphorylation of adenosine 5'monophosphate-activated protein kinase (AMPK), activated the mTOR pathway. Oxidative stress on the cells reduced significantly by treating with TBHQ, an antioxidant, which is also an activator of Nrf2. The activity of Nrf2 was rehabilitated and phosphorylation of mTOR decreased. In conclusion, cyanate could induce oxidative stress damage and lipid deposition by inhibiting Nrf2/HO-1 pathway, which was rescued by inhibitor of Nrf2.

Proteasome-dependent degradation of intracellular carbamylated proteins.

Carbamylation, which corresponds to the binding of isocyanic acid to the amino groups of proteins, is a nonenzymatic post-translational modification responsible for alterations of protein structural and functional properties. Tissue accumulation of carbamylation-derived products and their role in pathological processes such as atherosclerosis or chronic renal failure have been previously documented. However, few studies have focused on the carbamylation of intracellular proteins and their subsequent role in cellular aging. This study aimed to determine the extent of intracellular protein carbamylation, its impact on cell functions and the ability of cells to degrade these modified proteins. Fibroblasts were incubated with cyanate or urea and the carbamylation level was evaluated by immunostaining and homocitrulline quantification. The results showed that carbamylated proteins accumulated intracellularly and that all proteins were susceptible. The presence of intracellular carbamylated proteins did not modify cell proliferation or type I collagen synthesis nor did it induce cell senescence, but it significantly decreased cell motility. Fibroblasts were able to degrade carbamylated proteins through the ubiquitin-proteasome system. In conclusion, intracellular proteins are susceptible to carbamylation but their accumulation does not seem to deeply affect cell function, owing largely to their elimination by the ubiquitin-proteasome system.

Interaction of cyanate uptake by rice seedlings with nitrate assimilation: gene expression analysis.

Cyanate (CNO-) has been produced in the environment through either natural or anthropogenic sources. However, due to industrialization, it has been led more over-loads. In this study, interaction of CNO- uptake by rice seedlings with nitrate assimilation was investigated using gene expression analysis after an acute phytotoxicity assay. Our results showed that CNO- exposure caused inhibition on relative growth rates of plants. CNO- analysis demonstrated that rice seedlings had higher potential for CNO- uptake and the removal rates showed a zero-order kinetic. PCR analysis exposed that OsCYN transcript was not significantly induced by CNO- treatments in rice tissues and CNO- exposure also repressed gene expression of the collaborative enzyme carbonic anhydrase (CA), suggesting that assimilation of CNO- initiated by the enzyme cyanase (CYN) in rice seedlings was an enzyme-limitation reaction. Gene expression of other enzymes involved in nitrate metabolism was tissue-specific under CNO- exposure, suggesting that rice seedlings were able to trigger its intrinsic regulative and responsive mechanisms to cope up with uneven N conditions. Significant upregulation of three OsGDH isogenes, except for OsGDH1 in roots, was detected in both rice materials with enhancing CNO- concentrations, suggesting that GDH may play a primary role to maintain the balance of C and N in plants under CNO- exposure. In conclusion, because the innate pool of CYN activity was non-sufficient to degrade exogenous CNO- by rice seedlings, CNO-derived ammonium only can serve as a supporting N source to support growth of rice seedling under non-effective doses of CNO- exposure.

Comparison of thiocyanate and selenocyanate for potentiation of antimicrobial photodynamic therapy.

We have previously shown that antimicrobial photodynamic therapy (aPDT) mediated by different photosensitizers (PS) can be potentiated by a variety of inorganic salts. Potassium thiocyanate (KSCN) potentiated aPDT mediated by methylene blue (MB), while potassium selenocyanate (KSeCN) potentiated aPDT mediated by MB, Rose Bengal and the anionic porphyrin 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin dihydrochloride. However, the mechanisms of action that were proposed were fundamentally different. In the present study, we compare these two salts (KSCN and KSeCN) with different light-activated PS and different oxidative reactions for killing gram-positive and gram-negative bacteria. Overall KSeCN was more powerful than KSCN, and worked with a wider range of PS, while KSCN only worked with phenothiazinium salts. KSeCN produced killing when cells were added after light suggesting production of a semistable species called selenocyanogen (SeCN)2 . We tested three different oxidative reactions that can all potentially kill bacteria: lead tetraacetate (Pb[OAc]4 ); Fenton reagent (hydrogen peroxide [H2 O2 ] and ferrous sulfate) H2 O2 and horseradish peroxidase (HRP). In every case, KSeCN was substantially more effective (several logs) than KSCN in potentiating the bacterial killing. We conclude that (SeCN)2 is the mediator for aPDT using KSeCN, while sulfur trioxide radical anion is the mediator for KSCN using phenothiaziums. For H2 O2 /HRP with KSCN, hypothiocyanite is proposed to be the antibacterial agent in the literature, while hyposelenocyanite is said not to exist. Pb[OAc]4 is known to produce (SeCN)2 from KSeCN as well as the analogous (SCN)2 from KSCN. The mediators from Fenton reaction are unclear (pseudohalogen radical ions?) Both KSCN (which occurs naturally in the human body) and KSeCN may be clinically applicable.

Antimicrobial photodynamic inactivation is potentiated by the addition of selenocyanate: Possible involvement of selenocyanogen?

We previously showed that antimicrobial photodynamic inactivation (aPDI) of Gram-positive and Gram-negative bacteria mediated by the phenothiazinium dye, methylene blue (MB), was potentiated by the addition of potassium thiocyanate (10 mM). The mechanism was suggested to involve a singlet oxygen-mediated reaction with SCN to form sulfite and cyanide and then to produce sulfur trioxide radical anion. We now report that potassium selenocyanate (concentrations up to 100 mM) can also potentiate (up to 6 logs of killing) aPDI mediated by a number of different photosensitizers (PS): MB, rose bengal and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin dihydrochloride (as low as 200 nM). When a mixture of selenocyanate with these PS in solution was illuminated and then bacteria were added after the light, there was up to 6 logs of killing (Gram-negative > Gram-positive) but the antibacterial species decayed rapidly (by 20 minutes). Our hypothesis to explain this antibacterial activity is the formation of selenocyanogen (SeCN)2 by reaction with singlet oxygen (1 O2 ) as shown by quenching of 1 O2 by SeCN and increased photoconsumption of oxygen. The fact that lead tetraacetate reacted with SeCN (literature preparation of (SeCN)2 ) also produced a short-lived antibacterial species supports this hypothesis.

Cyanate improves insulin sensitivity and hepatic steatosis in normal and high fat-fed mice: Anorexic and antioxidative effects.

Obesity is an important contributing factor to progression of chronic kidney disease. Cyanate, known as uremic toxin, is an electrophile produced spontaneously from urea or by myeloperoxidase-catalyzed oxidation of thiocyanate. Herein, we explored metabolic effects of cyanate in normal chow diet (NCD)- and high fat diet (HFD)-fed mice. Mice were treated with cyanate (1 mg/mL in drinking water) and fed NCD or HFD. Peritoneal glucose tolerance test (PGTT) and insulin tolerance test (ITT) were performed. Blood urea nitrogen (BUN) and creatinine concentrations were determined. Kidney and liver tissues were analyzed for reactive oxygen species (ROS) and lipid accumulations. Human albumin was carbamylated and evaluated for ROS scavenging activities. Contrary to our expectations, we found that cyanate treatment improved increased insulin sensitivity and alleviated hepatic steatosis in NCD- and HFD-fed mice. PGTT and ITT revealed faster and immediate glucose clearance in cyanate-treated NCD- and HFD-fed mice. Histological analysis of kidney and serum levels of BUN and creatinine showed no significant differences between cyanate-treated and control mice groups. Cyanate treatment reduced appetite and body weight in both NCD- and HFD-fed mice groups. Cyanate also decreased lipid peroxidation levels in the sera and the kidney, attenuated ROS levels in the kidney, which lead us to the findings that cAlb significantly reduced ROS levels compared to Alb in Caki-1 kidney and human umbilical vein endothelial cells. The results in this study may indicate that cyanate improves insulin sensitivity and hepatic steatosis possibly via exerting anorexic and antioxidative effects.

Top-down modulation of olfactory-guided behaviours by the anterior olfactory nucleus pars medialis and ventral hippocampus.

Olfactory processing is thought to be actively modulated by the top-down input from cortical regions, but the behavioural function of these signals remains unclear. Here we find that cortical feedback from the anterior olfactory nucleus pars medialis (mAON) bidirectionally modulates olfactory sensitivity and olfaction-dependent behaviours. To identify a limbic input that tunes this mAON switch, we further demonstrate that optogenetic stimulation of ventral hippocampal inputs to the mAON is sufficient to alter olfaction-dependent behaviours.

Cyanate-Impaired Angiogenesis: Association With Poor Coronary Collateral Growth in Patients With Stable Angina and Chronic Total Occlusion.

BACKGROUND: Cyanate has recently gained attention for its role in the pathogenesis of vascular injury. Nonetheless, the effect of cyanate on angiogenesis remains unclear. METHODS AND RESULTS: In this study, we demonstrated that oral administration of cyanate impaired blood perfusion recovery in a mouse hind-limb ischemia model. A reduction in blood perfusion recovery at day 21 was observed in the ischemic tissue of cyanate-treated mice. Likewise, there were fewer capillaries in the ischemic hind-limb tissue of cyanate-exposed mice. Our in vitro study showed that cyanate, together with its carbamylated products, inhibited the migration, proliferation, and tube-formation abilities of endothelial cells. Further research revealed that cyanate regulated angiogenesis partly by interrupting the vascular endothelial growth factor receptor 2/phosphatidylinositol 3-kinase/Akt pathway. The serum concentrations of homocitrulline, a marker of cyanate exposure, were determined in 117 patients with stable angina and chronic total occlusion. Consistent with the antiangiogenic role of cyanate, homocitrulline levels were increased in patients with poor coronary collateralization (n=58) compared with those with high collateralization (n=59; 21.09±13.08 versus 15.54±9.02 ng/mL, P=0.009). In addition, elevated homocitrulline concentration was a strong predictor of poor coronary collateral growth. CONCLUSIONS: Impaired angiogenesis induced by cyanate might contribute to poor coronary collateral growth.