DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1 × 10⁶ cells/cm³. This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.     

DNA binding acridine–thiazolidinone agents affecting intracellular glutathione

Three new acridine–thiazolidinone derivatives (2a–2c) have been synthesized and their interactions with calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovarian cancer cell lines A2780) were studied. The compounds 2a–2c possessed high affinity to calf thymus DNA and their binding constants determined by spectrofluorimetry were in the range of 1.37 × 10⁶–5.89 × 10⁶ M^?1. All of the tested derivatives displayed strong cytotoxic activity in vitro, the highest activity in cytotoxic tests was found for 2c with IC₅₀ = 1.3 ± 0.2 μM (HL-60), 3.1 ± 0.4 μM (L1210), and 7.7 ± 0.5 μM (A2780) after 72 h incubation. The cancer cells accumulated acridine derivatives very fast and the changes of the glutathione level were confirmed. The compounds inhibited proliferation of the cells and induced an arrest of the cell cycle and cell death. Their influence upon cells was associated with their reactivity towards thiols and DNA binding activity.     

Curcumin-I Knoevenagel’s condensates and their Schiff’s bases as anticancer agents: Synthesis,pharmacological and simulation studies

Pyrazolealdehydes (4a–d), Knoevenagel’s condensates (5a–d) and Schiff’s bases (6a–d) of curcumin-I were synthesized, purified and characterized. Hemolysis assays, cell line activities, DNA bindings and docking studies were carried out. These compounds were lesser hemolytic than standard drug doxorubicin. Minimum cell viability (MCF-7; wild) observed was 59% (1.0 μg/mL) whereas the DNA binding constants ranged from 1.4 × 10³ to 8.1 × 10⁵ M^?1. The docking energies varied from ?7.30 to ?13.4 kcal/mol. It has been observed that DNA-compound adducts were stabilized by three governing forces (Van der Wall’s, H-bonding and electrostatic attractions). It has also been observed that compounds 4a–d preferred to enter minor groove while 5a–d and 6a–d interacted with major grooves of DNA. The anticancer activities of the reported compounds might be due to their interactions with DNA. These results indicated the bright future of the reported compounds as anticancer agents.     

DNA binding and photocleavage properties and apoptosis-inducing activities of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)2Cl]Cl and its heterometallic derivatives

The interactions of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)₂Cl]Cl (phen = 1,10-phenanthroline, (Py-3′)TPP = 5-(3′-pyridyl-10,15,20-triphenylporphyrin) (1) and its heterometallic derivatives, [Ni(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (2) and [Cu(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (3), with calf thymus DNA have been investigated by spectroscopic and viscosity measurements in this study. The results showed that these synthetic complexes can bind to double strand helix DNA in groove binding mode, and the intrinsic binding constants of complexes 1, 2 and 3, as calculated according to the decay of the Soret absorption, are (1.35 ± 0.5) ×10⁵ M^?1 (s = 4.2), (1.29 ± 0.5) × 10⁵ M^?1 (s = 5.6) and (1.22 ± 0.5) × 10⁵ M^?1 (s = 6.2) (s is the binding-site size), respectively, which are consistent with those obtained from ethidium bromide-quenching experiments. Further investigations on the photocleavage properties of these complexes on plasmid pBR 322 DNA showed that complexes 1, 2 and 3 could cleave single chain DNA and convert DNA molecules from supercoiled form to the nicked form. As determined by MTT assay, the complexes were also identified as potent antiproliferative agents against A375 human melanoma cells, MCF-7 human breast adrenocarcinoma cells, Colo201 human colon adenocarcinoma cells and HepG2 human liver cancer cells. Complex 1 inhibits the growth of A375 cells through induction of apoptotic cell death and G0/G1 cell cycle arrest. Further investigation on intracellular mechanisms indicated that Complex 1 induced depletion of mitochondrial membrane potential (ΔΨ_m) in A375 cells through regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Our results suggest that ruthenium porphyrin complexes could be candidates for further evaluation as chemopreventive and chemotherapeutic agents for human cancers.     

Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes

The quintessential property of developing cardiomyocytes is their ability to beat spontaneously. The mechanisms underlying spontaneous beating in developing cardiomyocytes are thought to resemble those of adult heart, but have not been directly tested. Contributions of sarcoplasmic and mitochondrial Ca²⁺-signaling vs. I_f-channel in initiating spontaneous beating were tested in human induced Pluripotent Stem cell-derived cardiomyocytes (hiPS-CM) and rat Neonatal cardiomyocytes (rN-CM). Whole-cell and perforated-patch voltage-clamping and 2-D confocal imaging showed: (1) both cell types beat spontaneously (60–140/min, at 24 °C); (2) holding potentials between −70 and 0 mV had no significant effects on spontaneous pacing, but suppressed action potential formation; (3) spontaneous pacing at −50 mV activated cytosolic Ca²⁺-transients, accompanied by in-phase inward current oscillations that were suppressed by Na⁺-Ca²⁺-exchanger (NCX)- and ryanodine receptor (RyR2)-blockers, but not by Ca²⁺- and I_f-channels blockers; (4) spreading fluorescence images of cytosolic Ca²⁺-transients emanated repeatedly from preferred central cellular locations during spontaneous beating; (5) mitochondrial un-coupler, FCCP at non-depolarizing concentrations (∼50 nM), reversibly suppressed spontaneous pacing; (6) genetically encoded mitochondrial Ca²⁺-biosensor (mitycam-E31Q) detected regionally diverse, and FCCP-sensitive mitochondrial Ca²⁺-uptake and release signals activating during I_NCX oscillations; (7) I_f-channel was absent in rN-CM, but activated only negative to −80 mV in hiPS-CM; nevertheless blockers of I_f-channel failed to alter spontaneous pacing.     

Evaluation of quantitative recovery of bacterial cells and DNA from different lake sediments by Nycodenz density gradient centrifugation

While purified bacterial cells and DNA – the signature of life – from soil and sediment matrices have been extensively studied in a wide range of environments and in different microbial ecosystems, the paucity of data on DNA extraction from contaminated sediments emphasizes the need for further research on the isolation and quantification of bacterial cells and DNA in sediments. Consequently, the Nycondez gradient centrifugation method was applied to extract bacterial cells from contaminated and uncontaminated sediments. Quantitative estimates of recovered bacterial cells were obtained from direct counts performed using DAPI (4′,6′-diamino-2-phenylindole hypochloride) staining couples with fluorescence microscopy and indirect counts (colony-forming units). The estimation was improved by using an efficient method of comparing sediment types composed of quantifying bacterial densities in three steps: S₁ the initial freshwater sediments; S₂ the first supernatant recovered after mixing the sediments with sodium hexametaphosphate solution followed by centrifugation; and S₃ the extracted cells. Total and extracellular DNA were extracted and quantified in each of the three steps. Additional analysis of faecal indicator bacteria (FIB) including E. coli and Enterococcus (ENT) was also performed in each step. The results display considerable variability in the quantity of bacteria cells depending on sediment type, ranging from 1.2 × 10⁵ to 6.2 × 10⁹ cell g^?1 dry sediments. The treatment with sodium hexametaphosphate solution (2%) leads to the desorption of bacterial populations which were firmly adsorbed on contaminated sediment surfaces resulting in more than 90% of the FIB being recovered. The Nycondez density gradient centrifugation method makes it possible to extract bacterial cells from freshwater sediments without extracellular DNA so it is ideal for metagenomic analysis of bacteria.     

Cytotoxic effect of (1-methyl-1H-imidazol-2-yl)-methanamine and its derivatives in PtII complexes on human carcinoma cell lines: A comparative study with cisplatin

The synthesis and pharmacological characterisation of (1-methyl-1H-imidazol-2-yl)-methanamine and its derivatives in Pt^II complexes are described. Six out of eleven new Pt^II complexes showed a significant cytotoxic effect on NCI-H460 lung cancer cell line with EC₅₀ values between 1.1 and 0.115 mM, determined by MTT assay. Compound Pt-4a showed a particularly more potent cytotoxic effect than the previously described Pt^II complex with 2,2′-bipyridine, [Pt(bpy)Cl₂], with an EC₅₀ value equal to 172.7 μM versus 726.5 μM respectively, and similar potency of cisplatin (EC₅₀ = 78.3 μM) in NCI-H460 cell line. The determination of the intracellular and DNA-bound concentrations of ¹⁹⁵Pt, as marker of the presence of the complexes, showed that the cytotoxic compound Pt-4a readily diffused into the cells to a similar extent of cisplatin and directly interacted with the nuclear DNA. Pt-4a induced both p53 and p21^Waf expression in NCI-H460 cells similar to cisplatin. A direct comparison of the cytotoxic effect between compound Pt-4a and cisplatin on 12 different cancer cell lines demonstrated that compound Pt-4a was in general less potent than cisplatin, but it had a comparable cytotoxic effect on non-small-cell lung cancer NCI-H460 cells, and the colorectal cancer cells HCT-15 and HCT-116. Altogether, these results suggested that the Pt^II complex with 1-methyl-1H-imidazol-2-yl)-methanamine (compound Pt-4a), displayed a significant cytotoxic activity in cancer cells. Similarly to cisplatin this compound interacts with nuclear DNA and induces both p53 and p21^waf, and thus it represents an interesting starting point for future optimisation of new Pt^II complexes forming DNA adducts.     

Synthesis and DNA cleavage activity of 2-hydrazinyl-1,4,5,6-tetrahydropyrimidine containing hydroxy group

2-Hydrazinyl-1,4,5,6-tetrahydropyrimidin-5-ol dihydrochloride 2, as well as 2-hydrazinyl-4,5-dihydro-1H-imidazole dihydrochloride 1, was synthesized as metal-free DNA cleaving agent. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of 1 and 2. DNA cleavage efficiency of 2 exhibits remarkable increases compared with its corresponding non-hydroxy compound 1. Kinetic data of DNA cleavage promoted by 2 fit to the Michaelis–Menten-type equation with k_max of 0.0378 ± 0.0013 h^?1 giving 10⁶-fold rate acceleration over uncatalyzed DNA. The acceleration is driven by the spatial proximity of the nucleophilic hydroxy group and the electrophilic activation for the phosphodiester by the ammonium and/or guanidinium groups. In vitro cytotoxic activities toward Hela cells and human leukemia HL-60 cells were also examined, and 2 exhibits stronger cytotoxic activities than 1.     

Cytotoxicity and DNA binding property of phenanthrene imidazole with polyglycol side chains

A series of phenanthrene imidazole with polyglycol side chain (2a–2c and 3a–3c) were synthesized and characterized by IR, NMR and MS. The cytotoxicity of 2a–2c and 3a–3c against cancer cell lines (HL-60, BGC-823, Bel-7402 and KB) in vitro were measured using MTT method. The DNA binding properties of 3a–3c were investigated by UV, fluorescence, CD spectroscopies and thermal denaturation. The results indicate that 2a exhibits higher cytotoxicity than cisplatin against BGC-823 and Bel-7402 cell lines, 3b and 3c exhibit higher cytotoxicity than 2b and 2c against BGC-823, Bel-7402 and KB cell lines. The cytotoxic effect of 2a–2c decrease with the increase of side chains length, the cytotoxic effect of 3a–3c increased with the increasing length of side chains against BGC-823, Bel-7402 and KB cell lines. Compounds 3a–3c intercalated DNA with a vertical orientation in the intercalation pocket. The binding constants of 3a–3c with Ct-DNA are 1.68 × 10⁶, 1.51 × 10⁶ and 0.709 × 10⁶ M^?1, respectively. The binding affinity of 3a–3c with Ct-DNA trended to decrease with the increasing length of polyglycol side chains.     

Phosphorus sorption by sediments in a subtropical constructed wetland receiving stormwater runoff

This study investigated the potential of using a mixture of fishpond bund material, completely decomposed granite and river sand as substrate in a constructed wetland for phosphorus removal. Core samples were collected from the newly constructed Hong Kong Wetland Park (HKWP) receiving influent stormwater from a nearby new town, and batch incubation experiments were conducted to determine the P sorption characteristics of sediments. The HKWP sediments adsorbed the majority of available P in the initial 20 min of incubation, with a first-order rate constant of 1.01–2.11 h^?1. Sediments in the reedbeds and freshwater marshes possessed a great capacity for P adsorption with the high Langmuir sorption maxima (478–858 mg kg^?1) and Freundlich adsorption constants (417–672 L kg^?1) obtained, attributable to the high amorphous iron and aluminium concentrations compared to other constructed wetlands. Moreover, sediment equilibrium P concentrations were generally low (4.6–23.6 μg L^?1), facilitating a net P adsorption by sediments under moderate P loadings. Yet, the amount of P adsorbed by the HKWP sediments was limited by the low ambient porewater P concentrations and there was even a risk of P desorption when sediments in the freshwater marshes were resuspended into the water column. While substrates in the HKWP demonstrated a great potential for P adsorption, consideration should also be given to P loadings in influent water to fully utilize the P sorption capacity of sediments and enhance the P removal efficiency of constructed wetlands.     

Comparisons of direct extraction methods of microbial DNA from different paddy soils

Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18–20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical–enzymatic–mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical–enzymatic and chemical–mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals.     

Whole-body cryostimulation increases parasympathetic outflow and decreases core body temperature

The cardiovascular, autonomic and thermal response to whole-body cryostimulation exposure are not completely known. Thus the aim of this study was to evaluate objectively and noninvasively autonomic and thermal reactions observed after short exposure to very low temperatures. We examined 25 healthy men with mean age 30.1±3.7 years and comparable anthropomorphical characteristic. Each subject was exposed to cryotherapeutic temperatures in a cryogenic chamber for 3 min (approx. −120 °C). The cardiovascular and autonomic parameters were measured noninvasively with Task Force^® Monitor. The changes in core body temperature were determined with the Vital Sense^® telemetric measurement system. Results show that 3 min to cryotherapeutic temperatures causes significant changes in autonomic balance which are induced by peripheral and central blood volume changes. Cryostimulation also induced changes in core body temperature, maximum drop of core temperature was observed 50–60 min after the stimulation. Autonomic and thermal reactions to cryostimulation were observed up to 6 h after the exposure and were not harmful for examined subjects.     

Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells

This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm × 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25–125 ng/ml of cell extract by a 1/x² weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of ±15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra^® (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin^® (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus^® (500 mg twice a day). The patients treated by Kaletra^® showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin^® showed mean CC of 2388.11 ng/ml while both patients under Aptivus^® showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.     

Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand

To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (10⁶ M^?1 order) with diminished binding to dsDNA (10⁴ M^?1 order) in 100 mM AcOH-AcOK buffer (pH 5.5) containing 100 mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K = 3.4 × 10⁶ M^?1 and a 2:1 stoichiometry) than dsDNA (K = 7.5 × 10⁴ M^?1) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 0.4 μM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.     

Radioiodinated sunitinib as a potential radiotracer for imaging angiogenesis-radiosynthesis and first radiopharmacological evaluation of 5-[125I]Iodo-sunitinib

Sunitinib® (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR). Radiolabeled inhibitors of RTKs might be useful tools for monitoring RTKs levels in tumour tissue giving valuable information for anti-angiogenic therapy. We report here the synthesis of a ¹²⁵I-labeled derivative of sunitinib® and its first radiopharmaceutical characterization.The non-radioactive reference compound 5-iodo-sunitinib 4 was prepared by Knoevenagel condensation of 5-iodo-oxindole with the corresponding substituted 5-formyl-1H-pyrrole. In a competition binding assay against VEGFR-2 a binding constant (K_d) of 16 nM for 4 was found. The ability of 4 to inhibit tyrosine kinase activity was demonstrated on RTK expressing cells suggesting this radiotracer as a useful tool for monitoring VEGFR expression. 5-[¹²⁵I]lodo-sunitinib, [¹²⁵I]-4 was obtained via destannylation of the corresponding tributylstannyl precursor with [¹²⁵I]NaI in the presence of H₂O₂ in high radiochemical yield (>95%) and radiochemical purity (<98%) after HPLC purification. Determination of human plasma protein binding at time intervals of 0; 1; 2; 4 and 24 h suggested a low non-specific binding of 5-10%. Preliminary biodistribution studies of [¹²⁵I]-4 in healthy CD-1 mice showed a relatively high uptake in VEGFR-2 rich tissues like kidney and lung followed by rapid washout (9.6 and 9.7; 4.5 and 3.8% ID/g of kidney and lung at 1 and 4 h, respectively).     

Evaluation of phosphorus distribution and bioavailability in sediments of a subtropical wetland reserve in southeast China

The phosphorus (P) fractions and bioavailable P in the sediments from the Quanzhou Bay Estuarine Wetland Nature Reserve were investigated using chemical extraction methods for the first time to study the distribution and bioavailability of P in the reserve sediments. A hypothesis was presented suggesting that the bioavailable P in the sediments could be evaluated using the P fractions. The total phosphorus (TP), inorganic phosphorus (IP), organic phosphorus (OP), non-apatite phosphorus (NAIP), and apatite phosphorus (AP) contents in the sediments were in the ranges of 303.87–761.59 mg kg⁻¹, 201.22–577.66 mg kg⁻¹, 75.83–179.16 mg kg⁻¹, 28.86–277.90 mg kg⁻¹, and 127.36–289.94 mg kg⁻¹, respectively. The water soluble phosphorus (WSP), readily desorbable phosphorus (RDP), algal available phosphorus (AAP), and NaHCO₃ extractable phosphorus (Olsen-P) contents in the sediments were in the ranges of 0.58–357.17 mg kg⁻¹, 80.77–586.75 mg kg⁻¹, 1.09–24.12 mg kg⁻¹, and 54.96–676.82 mg kg⁻¹, respectively. The correlation analysis results showed that the NAIP was the major component of the bioavailable P and that the impact of the AP on the bioavailable phosphorus may be minimal. Due to the low TP content in the sediments of the Quanzhou Bay Estuarine Wetland Nature Reserve, the potential pollution risks of P in the sediments may not be very high. The results also show that the bioavailable P concentrations in the sediments of the Quanzhou Bay Estuarine Wetland Nature Reserve could not be evaluated by measuring the P fractions and that the hypothesis was untenable.     

Synthesis,biological evaluation of novel 4,5-dihydro-2H-pyrazole 2-hydroxyphenyl derivatives as BRAF inhibitors

A series of novel 4,5-dihydropyrazole derivatives (3a–3t) containing hydroxyphenyl moiety as potential V600E mutant BRAF kinase (BRAF^V600E) inhibitors were designed and synthesized. Docking simulation was performed to insert compounds 3d (1-(5-(5-chloro-2-hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) and 3m (1-(3-(4-chlorophenyl)-5-(3,5-dibromo-2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) into the crystal structure of BRAF^V600E to determine the probable binding model, respectively. Based on the preliminary results, compound 3d and 3m with potent inhibitory activity in tumor growth may be a potential anticancer agent. Results of the bioassays against BRAF^V600E, MCF-7 human breast cancer cell line and WM266.4 human melanoma cell line all showed several compounds had potent activities IC₅₀ value in low micromolar range, among them, compound 3d and compound 3m showed strong potent anticancer activity, which were proved by that 3d: IC₅₀ = 1.31 μM for MCF-7 and IC₅₀ = 0.45 μM for WM266.5, IC₅₀ = 0.22 μM for BRAF^V600E, 3m: IC₅₀ = 0.97 μM for MCF-7 and IC₅₀ = 0.72 μM for WM266.5, IC₅₀ = 0.46 μM for BRAF^V600E, which were comparable with the positive control Erlotinib.     

Oxovanadium(IV) complexes with Knoevenagel Schiff base condensate as impending chemotherapeutic agents: Synthesis,characterization, biological screening and anti-proliferative assay

Two novel oxovanadium(IV) complexes [VOL1]SO₄ (1) and [VOL2]SO₄ (2) containing Knoevenagel condensate Schiff base ligand (L1/L2) have been synthesized and characterized by physical, spectral and analytical methods. These complexes are reported as ionic in nature on the basis of elemental composition and molar conductance, and possess square pyramidal geometry around the central metal ions. The binding interactions of (1) and (2) with calf thymus DNA (CT DNA) were explored by absorption spectrophotometric titration, cyclic voltammetry data and viscosity measurements. The calculated intrinsic binding constant values (K_b) for (1) and (2) obtained from UV–Vis absorption studies are 0.4 × 10⁵ and 5.6 × 10⁵ (M⁻¹) respectively. These experimental results indicate that (1) and (2) are intercalative binders and avid binder to CT DNA with different affinities. These complexes exhibit significant oxidative cleavage of supercoiled plasmid (pUC18) DNA in the presence of activators. In particular, the in vitro antimicrobial efficacy of oxovanadium(IV) complexes reveal that they are more active than free ligands. Besides, the in vitro cytotoxic effect of the titled complexes were examined on a bundle of human tumor cell lines such as MCF-7 and HeLa cancerous cell lines by the MTT method. Interestingly, complex (2) exhibits more potent cytotoxic activity than the other complex and standard drug (cisplatin). The mode of cell death was assessed by Hoechst 33258 staining morphological studies.     

Development of a new epidermal growth factor receptor positron emission tomography imaging agent based on the 3-cyanoquinoline core: Synthesis and biological evaluation

The epidermal growth factor receptor (EGFR/c-ErbB1/HER1) is overexpressed in many cancers including breast, ovarian, endometrial, and non-small cell lung cancer. An EGFR specific imaging agent could facilitate clinical evaluation of primary tumors and/or metastases. To achieve this goal we designed and synthesized a small array of fluorine containing compounds based on a 3-cyanoquinoline core. A lead compound, 16, incorporating 2′-fluoroethyl-1,2,3-triazole was selected for evaluation as a radioligand based on its high affinity for EGFR kinase (IC₅₀ = 1.81 ± 0.18 nM), good cellular potency (IC₅₀ = 21.97 ± 9.06 nM), low lipophilicity and good metabolic stability. ‘Click’ labeling afforded [¹⁸F]16 in 37.0 ± 3.6% decay corrected radiochemical yield based on azide [¹⁸F]14 and 7% end of synthesis (EOS) yield from aqueous fluoride. Compound [¹⁸F]16 was obtained with >99% radiochemical purity in a total synthesis time of 3 h. The compound showed good stability in vivo and a fourfold higher uptake in high EGFR expressing A431 tumor xenografts compared to low EGFR expressing HCT116 tumor xenografts. Furthermore, the radiotracer could be visualized in A431 tumor bearing mice by small animal PET imaging. Compound [¹⁸F]16 therefore constitutes a promising radiotracer for further evaluation for imaging of EGFR status.     

Evaluation of new thiadiazoles and benzothiazoles as potential radioprotectors: Free radical scavenging activity in vitro and theoretical studies (QSAR,DFT)

Thiol and aminothiol compounds are among the most efficient chemical radioprotectors. To increase their efficiency, we synthesized two new classes of thiol and aminothiol compounds derived from benzothiazole (T1, T2, AM1, AM2) and thiadiazole (T3, T4, AM3) structures. We examined them for their ability to scavenge free radicals (DPPH^·, ABTS^·+, ^·OH). Thiol derivatives with a thiadiazole structure are the most active compounds scavenging DPPH^· and ABTS^·+ free radicals, with an IC₅₀ of 0.053 ± 0.006 and 0.023 ± 0.002 mM, respectively, for the derivative T3. Moreover, compounds T1, T2, and T3 at 60 μM gave 83% protection against 2-deoxyribose degradation by ^·OH. The ability of these compounds to protect DNA against ^·OH produced by a Fenton reaction and γ-irradiation (15 Gy)-induced strand breaks was also evaluated on pBR322 plasmid DNA. In both tests thiol derivatives were the most efficient compounds. Derivatives T2 and T3 totally inhibit DNA strand breaks at the concentration of 50 μM. The protection afforded by these derivatives was comparatively higher than that of the radioprotectors WR-2721 and WR-1065. Our data indicate that these two compounds are free radical scavengers and potential antioxidant agents. Finally, DFT and QSAR studies were performed to support the experimental observations.