Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycinruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycinruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycinruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycinruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

Synthesis of the two isomers of the potential sex pheromone of Thaumetopoea pityocampa (Lepidoptera, Notodontidae) and related model compounds

The synthesis of the major component of the sex pheromone secretion of the processionary moth, Tkawnztopoeja pltyocampa (Denis and Schiff.) (Lepidoptera, Notodontidae), (Z)-13-hexadecen-ll-ynyl acetate (1), the corresponding (E)-isomer (2) and the four structurally related model compounds (Z⁄E,Z,Z)-5,9,13-hexadecatrienyl acetate (3), (Z⁄E,Z,Z)-3,7,ll-hexadecatrienyl acetate (4), (Z⁄E,E,Z)-7,9,13-hexadecatrienyl acetate (5) and (Z)-7-hexadecen-5-ynyl acetate (6) is described.

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

Metabolic origin of δ15N values in nitrogenous compounds from Brassica napus L. leaves

Nitrogen isotope composition (δ15N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ15N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ15N. Here, we investigated the δ15N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ15N between nitrogenous compounds (up to 30 ) and the content in (15N enriched) nitrate had a clear influence on leaf δ15N. Using a simple steady-state model of day metabolism, we suggest that the δ15N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme-catalysed reactions in primary nitrogen metabolism. δ15N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ15N value of leaf nitrogenous compounds

Bupivacaine 0.5 % versus articaine 4 % for the removal of lower third molars. A crossover randomized controlled trial

Objective: To compare the anesthetic action of 0.5% bupivacaine in relation to 4% articaine, both with 1:200,000 epinephrine, in the surgical removal of lower third molars. As a secondary objective hemodynamic changes using both anesthetics were analyzed. Study Design: Triple-blind crossover randomized clinical trial. Eighteen patients underwent bilateral removal of impacted lower third molars using 0.5% bupivacaine or 4% articaine in two different appointments. Preoperative, intraoperative and postoperative variables were recorded. Differences were assessed with McNemar tests and repeated measures ANOVA tests. Results: Both solutions exhibited similar latency times and intraoperative efficacy. Statistical significant lower pain levels were observed with bupivacaine between the fifth (p=0.011) and the ninth (p=0.007) postoperative hours. Bupivacaine provided significantly longer lasting soft tissue anesthesia (p<0.05). Systolic blood pressure and heart rate values were significantly higher with articaine. Conclusions: Bupivacaine could be a valid alternative to articaine especially due to its early postoperative pain prevention ability.

Synthesis of Janus compounds for the recognition of G-U mismatched nucleobase pairs

The design and synthesis of two Janus-type heterocycles with the capacity to simultaneously recognize guanine and uracyl in G-U mismatched pairs through complementary hydrogen bond pairing is described. Both compounds were conveniently functionalized with a carboxylic function and efficiently attached to a tripeptide sequence by using solid-phase methodologies. Ligands based on the derivatization of such Janus compounds with a small aminoglycoside, neamine, and its guanidinylated analogue have been synthesized, and their interaction with Tau RNA has been investigated by using several biophysical techniques, including UV-monitored melting curves, fluorescence titration experiments, and 1H NMR. The overall results indicated that Janus-neamine/guanidinoneamine showed some preference for the +3 mutated RNA sequence associated with the development of some tauopathies, although preliminary NMR studies have not confirmed binding to G-U pairs. Moreover, a good correlation has been found between the RNA binding affinity of such Janus-containing ligands and their ability to stabilize this secondary structure upon complexation.

Differential effects of polyphenols and alcohol of red wine on the expression of adhesion molecules and inflammatory cytokines related to atherosclerosis: a randomized clinical trial

Background: Few clinical studies have focused on the alcoholindependent cardiovascular effects of the phenolic compounds of red wine (RW). Objective: We aimed to evaluate the effects of ethanol and phenolic compounds of RW on the expression of inflammatory biomarkers related to atherosclerosis in subjects at high risk of cardiovascular disease. Design: Sixty-seven high-risk, male volunteers were included in a randomized, crossover consumption trial. After a washout period, all subjects received RW (30 g alcohol/d), the equivalent amount of dealcoholized red wine (DRW), or gin (30 g alcohol/d) for 4 wk. Before and after each intervention period, 7 cellular and 18 serum inflammatory biomarkers were evaluated. Results: Alcohol increased IL-10 and decreased macrophage-derived chemokine concentrations, whereas the phenolic compounds of RW decreased serum concentrations of intercellular adhesion molecule- 1, E-selectin, and IL-6 and inhibited the expression of lymphocyte function-associated antigen 1 in T lymphocytes and macrophage-1 receptor, Sialil-Lewis X, and C-C chemokine receptor type 2 expression in monocytes. Both ethanol and phenolic compounds of RW downregulated serum concentrations of CD40 antigen, CD40 ligand, IL-16, monocyte chemotactic protein-1, and vascular cell adhesion molecule-1. Conclusion: The results suggest that the phenolic content of RW may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of RW may modulate soluble inflammatory mediators in high-risk patients. The trial was registered in the International Standard Randomized Controlled Trial Number Register at http://www. isrctn.org/ as ISRCTN88720134

Differential effects of polyphenols and alcohol of red wine on the expression of adhesion molecules and inflammatory cytokines related to atherosclerosis: a randomized clinical trial

Background: Few clinical studies have focused on the alcoholindependent cardiovascular effects of the phenolic compounds of red wine (RW). Objective: We aimed to evaluate the effects of ethanol and phenolic compounds of RW on the expression of inflammatory biomarkers related to atherosclerosis in subjects at high risk of cardiovascular disease. Design: Sixty-seven high-risk, male volunteers were included in a randomized, crossover consumption trial. After a washout period, all subjects received RW (30 g alcohol/d), the equivalent amount of dealcoholized red wine (DRW), or gin (30 g alcohol/d) for 4 wk. Before and after each intervention period, 7 cellular and 18 serum inflammatory biomarkers were evaluated. Results: Alcohol increased IL-10 and decreased macrophage-derived chemokine concentrations, whereas the phenolic compounds of RW decreased serum concentrations of intercellular adhesion molecule- 1, E-selectin, and IL-6 and inhibited the expression of lymphocyte function-associated antigen 1 in T lymphocytes and macrophage-1 receptor, Sialil-Lewis X, and C-C chemokine receptor type 2 expression in monocytes. Both ethanol and phenolic compounds of RW downregulated serum concentrations of CD40 antigen, CD40 ligand, IL-16, monocyte chemotactic protein-1, and vascular cell adhesion molecule-1. Conclusion: The results suggest that the phenolic content of RW may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of RW may modulate soluble inflammatory mediators in high-risk patients. The trial was registered in the International Standard Randomized Controlled Trial Number Register at http://www. isrctn.org/ as ISRCTN88720134

Headspace needle-trap analysis of priority volatile organic compounds from aqueous samples: application to the analysis of natural and waste waters

Combining headspace (HS) sampling with a needle-trap device (NTD) to determine priority volatile organic compounds (VOCs) in water samples results in improved sensitivity and efficiency when compared to conventional static HS sampling. A 22 gauge stainless steel, 51-mm needle packed with Tenax TA and Carboxen 1000 particles is used as the NTD. Three different HS-NTD sampling methodologies are evaluated and all give limits of detection for the target VOCs in the ng L−1 range. Active (purge-and-trap) HS-NTD sampling is found to give the best sensitivity but requires exhaustive control of the sampling conditions. The use of the NTD to collect the headspace gas sample results in a combined adsorption/desorption mechanism. The testing of different temperatures for the HS thermostating reveals a greater desorption effect when the sample is allowed to diffuse, whether passively or actively, through the sorbent particles. The limits of detection obtained in the simplest sampling methodology, static HS-NTD (5 mL aqueous sample in 20 mL HS vials, thermostating at 50 °C for 30 min with agitation), are sufficiently low as to permit its application to the analysis of 18 priority VOCs in natural and waste waters. In all cases compounds were detected below regulated levels

Randomized, crossover, double-blind, placebo-controlled trial to assess the lipid lowering effect of co-formulated TDF/FTC

Abstract INTRODUCTION: Previous studies have described improvements on lipid parameters when switching from other antiretroviral drugs to tenofovir (TDF) and impairments in lipid profile when discontinuing TDF. [1-3] It is unknown, however, if TDF has an intrinsic lipid-lowering effect or such findings are due to the addition or removal of other offending agents or other reasons. MATERIALS AND METHODS: RESULTS: 46 subjects with a median age of 43 (40-48) years were enrolled in the study: 70% were male, 56% received DRV/r and 44% LPV/r. One subject withdrew the study voluntarily at week 4 and another one interrupted due to diarrhoea at week 24. Treatment with TDF/FTC decreased total, LDL and HDL-cholesterol from 235.9 to 204.9 (p<0.001), 154.7 to 127.6 (p<0.001) and 50.3 to 44.5 mg/dL (p<0.001), respectively. In comparison, total, LDL and HDL-cholesterol levels remained stable during placebo exposure. Week 12 total cholesterol (p<0.001), LDL-cholesterol (p<0.001) and HDL-cholesterol (p=0.011) levels were significantly lower in TDF/FTC versus placebo. Treatment with TDF/FTC reduced the fraction of subjects with abnormal fasting total-cholesterol (≥200 mg/dL) from 86.7% to 56.8% (p=0.001) and LDL-cholesterol (≥130 mg/dL) from 87.8% to 43.9% (p<0.001), which was not observed with placebo. There were no virological failures, and CD4 and triglyceride levels remained stable regardless of exposure. CONCLUSION: Coformulated TDF/FTC has an intrinsic lipid-lowering effect, likely attributable to TDF.

A Headspace needle-trap method for the analysis of volatile organic compounds in whole blood

Needle trap devices (NTDs) are a relatively new and promising tool for headspace (HS) analysis. In this study, a dynamic HS sampling procedure is evaluated for the determination of volatile organic compounds (VOCs) in whole blood samples. A full factorial design was used to evaluate the influence of the number of cycles and incubation time and it is demonstrated that the controlling factor in the process is the number of cycles. A mathematical model can be used to determine the most appropriate number of cycles required to adsorb a prefixed amount of VOCs present in the HS phase whenever quantitative adsorption is reached in each cycle. Matrix effect is of great importance when complex biological samples, such as blood, are analyzed. The evaluation of the salting out effect showed a significant improvement in the volatilization of VOCs to the HS in this type of matrices. Moreover, a 1:4 (blood:water) dilution is required to obtain quantitative recoveries of the target analytes when external calibration is used. The method developed gives detection limits in the 0.020–0.080 μg L−1 range (0.1–0.4 μg L−1 range for undiluted blood samples) with appropriate repeatability values (RSD < 15% at high level and <23% at LOQ level). Figure of merits of the method can be improved by using a smaller phase ratio (i.e., an increase in the blood volume and a decrease in the HS volume), which lead to lower detection limits, better repeatability values and greater sensibility. Twenty-eight blood samples have been evaluated with the proposed method and the results agree with those indicated in other studies. Benzene was the only target compound that gave significant differences between blood levels detected in volunteer non-smokers and smokers

Quantum-to-classical crossover in full counting statistics

The reduction of quantum scattering leads to the suppression of shot noise. In this Letter, we analyze the crossover from the quantum transport regime with universal shot noise to the classical regime where noise vanishes. By making use of the stochastic path integral approach, we find the statistics of transport and the transmission properties of a chaotic cavity as a function of a system parameter controlling the crossover. We identify three different scenarios of the crossover.

Case-Crossover Analysis of Air Pollution Health Effects: A Systematic Review of Methodology and Application

Case-crossover is one of the most used designs for analyzing the health-related effects of air pollution. Nevertheless, no one has reviewed its application and methodology in this context. Objective: We conducted a systematic review of case-crossover (CCO) designs used to study the relationship between air pollution and morbidity and mortality, from the standpoint of methodology and application.Data sources and extraction: A search was made of the MEDLINE and EMBASE databases.Reports were classified as methodologic or applied. From the latter, the following information was extracted: author, study location, year, type of population (general or patients), dependent variable(s), independent variable(s), type of CCO design, and whether effect modification was analyzed for variables at the individual level. Data synthesis: The review covered 105 reports that fulfilled the inclusion criteria. Of these, 24 addressed methodological aspects, and the remainder involved the design’s application. In the methodological reports, the designs that yielded the best results in simulation were symmetric bidirectional CCO and time-stratified CCO. Furthermore, we observed an increase across time in the use of certain CCO designs, mainly symmetric bidirectional and time-stratified CCO. The dependent variables most frequently analyzed were those relating to hospital morbidity; the pollutants most often studied were those linked to particulate matter. Among the CCO-application reports, 13.6% studied effect modification for variables at the individual level.Conclusions: The use of CCO designs has undergone considerable growth; the most widely used designs were those that yielded better results in simulation studies: symmetric bidirectional and time-stratified CCO. However, the advantages of CCO as a method of analysis of variables at the individual level are put to little use

Larock Reaction in the synthesis of heterocyclic compounds

The indole ring is one of the most common features in natural products and small molecules with important bioactivity. Larock reported a new methodology for the synthesis of the indole ring system based on the palladium-catalyzed heteroannulation of 2-iodoaniline and substituted alkyne moieties. This procedure was subsequently extended to the preparation of other nitrogen- and oxygen- containing heterocycles. This is the process of choice for the synthesis of a large number of heterocyclic derivatives, as it provides outstanding regioselectivity and good to excellent yields.

Larock Reaction in the synthesis of heterocyclic compounds

The indole ring is one of the most common features in natural products and small molecules with important bioactivity. Larock reported a new methodology for the synthesis of the indole ring system based on the palladium-catalyzed heteroannulation of 2-iodoaniline and substituted alkyne moieties. This procedure was subsequently extended to the preparation of other nitrogen- and oxygen- containing heterocycles. This is the process of choice for the synthesis of a large number of heterocyclic derivatives, as it provides outstanding regioselectivity and good to excellent yields.