Analysing Sr isotopes in low-Sr samples such as single insects with inductively coupled plasma tandem mass spectrometry using N2 O as a reaction gas for in-line Rb separation.

RATIONALE: Strontium isotopes are valuable markers of provenance in a range of disciplines. Limited amounts of Sr in low-mass samples such as insects mean that conventional Sr isotope analysis precludes their use for geographic origins in many ecological studies or in applications such as biosecurity. Here we test the viability of using inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) with N2 O as a reaction gas for accurately determining Sr isotopes in insects with Sr < 100 ng. METHODS: Strontium isotopes were determined in solution mode using ICP-MS/MS with 0.14 L/min N2 O as a reaction gas to convert Sr+ into SrO+ for in-line separation of 87 Sr from 87 Rb. The Sr isotope reference standards NIST SRM 987, NIST SRM 1570a and NIST SRM 1547 were used to assess accuracy and reproducibility. Ten insect species collected from the wild as a proof-of-principle application were analysed for Sr concentration and Sr isotopes. RESULTS: Using ICP-MS/MS we show for the first time that internal mass bias correction of 87 Sr16 O/86 Sr16 O based on 88 Sr16 O/86 Sr16 O works to give for NIST SRM 987 a 87 Sr/86 Sr ratio of 0.7101 ± 0.012 (RSD = 0.17%) and for NIST SRM 1570a a 87 Sr/86 Sr ratio of 0.7100 ± 0.009 (RSD = 0.12%), which are within error of the accepted values. The first 87 Sr/86 Sr ratio of NIST SRM 1547 is 0.7596 ± 0.0014. Strontium analyses were run on 0.8 mL of 0.25-0.5 ppb Sr, which equates to 2-4 ng of Sr. Strontium isotope analysis with a precision of >99.8% can be achieved with in-line separation of 87 Sr from 87 Rb at least up to solutions with 25 ppb Rb. CONCLUSIONS: A minimum of 5 mg of insect tissue is required for Sr isotope analysis. This new ICP-MS/MS method enables Sr isotope analysis in single insects, allowing population-scale studies to be feasible and making possible applications with time-critical uses such as biosecurity.

A Turn-on Fluorescence Sensor for Heparin Detection Based on a Release of Taiwan Cobra Cardiotoxin from a DNA Aptamer or Adenosine-Based Molecular Beacon.

This study presents two sensitive fluorescent assays for sensing heparin on the basis of the electrostatic interaction between heparin and Naja naja atra cardiotoxin 3 (CTX3). Owing to CTX3-induced folded structure of an adenosine-based molecular beacon (MB) or a DNA aptamer against CTX3, a reduction in the fluorescent signal of the aptamer or MB 5'-end labeled with carboxyfluorescein (FAM) and 3'-end labeled with 4-([4-(dimethylamino)phenyl]azo)-benzoic acid (DABCYL) was observed upon the addition of CTX3. The presence of heparin and formation of the CTX3-heparin complex caused CTX3 detachment from the MB or aptamer, and restoration of FAM fluorescence of the 5'-FAM-and-3'-DABCYL-labeled MB and aptamer was subsequently noted. Moreover, the detection of heparin with these CTX3-aptamer and CTX3-MB sensors showed high sensitivity and selectivity toward heparin over chondroitin sulfate and hyaluronic acid regardless of the presence of plasma. The limit of detection for heparin in plasma was determined to be 16 ng/mL and 15 ng/mL, respectively, at a signal-to-noise ratio of 3. This study validates the practical utility of the CTX3-aptamer and CTX3-MB systems for determining the concentration of heparin in a biological matrix.

The Association Between p38 MAPK-Mediated TNF-α/TNFR2 up-Regulation and 2-(4-Aminophenyl)-7-Methoxybenzothiazole-Induced Apoptosis in Human Leukemia U937 Cells.

The primary cause of treatment failures in acute myeloid leukemia is usually associated with defects in the apoptotic pathway. Several studies suggest that 2-(4-aminophenyl)-7-methoxybenzothiazole (7-OMe-APBT) may potentially induce apoptosis of cancer cells. Thus, the present study was conducted to explore the cytotoxic effect of 7-OMe-APBT on human leukemia U937 cells. The apoptosis of human leukemia U937 cells induced by 7-OMe-APBT was characterized by an increase in mitochondrial membrane depolarization, procaspase-8 degradation, and tBid production. Down-regulation of FADD blocked 7-OMe-APBT-induced procaspase-8 degradation and rescued the viability of 7-OMe-APBT-treated cells, suggesting the involvement of a death receptor-mediated pathway in 7-OMe-APBT-induced cell death. Increased TNF-α expression, TNFR2 expression, and p38 MAPK phosphorylation were noted in 7-OMe-APBT-treated cells. Pretreatment with a p38 MAPK inhibitor abolished 7-OMe-APBT-induced TNF-α and TNFR2 up-regulation. 7-OMe-APBT stimulated p38 MAPK/c-Jun-mediated transcriptional up-regulation of TNFR2, while the increased TNF-α mRNA stability led to TNF-α up-regulation in 7-OMe-APBT-treated cells. Treatment with 7-OMe-APBT up-regulated protein phosphatase 2A catalytic subunit α (PP2Acα) expression via the p38 MAPK/c-Jun/ATF-2 pathway, which, in turn, promoted tristetraprolin (TTP) degradation. Pretreatment with a protein phosphatase 2A inhibitor or TTP over-expression abrogated TNF-α up-regulation in 7-OMe-APBT-treated cells. Abolishment of TNF-α up-regulation or knock-down of TNFR1/TNFR2 by siRNA restored the viability of 7-OMe-APBT-treated cells. Taken together, our data indicate a connection between p38 MAPK-mediated TNF-α and TNFR2 up-regulation and 7-OMe-APBT-induced TNF-α-mediated death pathway activation in U937 cells. The same pathway also elucidates the mechanism underlying 7-OMe-APBT-induced death of human leukemia HL-60 cells.

Palladium(0)-catalyzed single and double isonitrile insertion: a facile synthesis of benzofurans, indoles, and isatins.

A palladium(0)-catalyzed cascade process consisting of isonitrile insertion and α-Csp(3)-H cross-coupling can be achieved for the synthesis of benzofurans and indoles. The construction of isatins by a Pd-catalyzed cascade reaction incorporating double isonitrile insertion, amination, and hydrolysis has also been achieved. The key features of this work include diverse heterocycle synthesis, phosphine-ligand-free reaction conditions, a one-pot procedure, simple and commercially available starting materials, broad functional-group compatibility, and moderate to good reaction yields.

Apoptosis induced by 2-aryl benzothiazoles-mediated photodynamic therapy in melanomas via mitochondrial dysfunction.

A mild and efficient synthetic development of 2-arylbenzothiazoles 5 mediated by ceric ammonium nitrate (CAN) via intramolecular cyclization of N-phenyl-thiobenzamides 4 was achieved. Further compounds 5 were reduced to corresponding amines 6, and their photodynamic therapy (PDT) effect was evaluated on malignant human melanoma A375 cells. Amine 6l plus ultraviolet A (UVA) induced caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 positive CytoDeath staining, and subsequent apoptotic cell death. Our data disclosed that treatment of A375 cells with 6l plus UVA resulted in a decrease in mitochondrial membrane potential (ΔΨmt), oxidative phosphorylation system (OXPHOS) subunits, and adenosine triphosphate (ATP) but an increase in mitochondrial DNA 4977-bp deletion via reactive oxygen species (ROS) generation. Transmission electron microscopy (TEM) observations also showed major ultrastructural alterations of mitochondria. Additionally, 6l plus UVA was also shown to reduce murine melanoma size in a mouse model. The present study supports the hypothesis that 6l-PDT may serve as a potential ancillary modality for the treatment of melanoma.

Synthesis, DNA-binding abilities and anticancer activities of triazole-pyrrolo[2,1-c][1,4]benzodiazepines hybrid scaffolds.

We synthesized a new series of PBD-hybrid derivatives having tethered triazoles and investigated for their cytotoxicity. The studies indicated that cis-olefin compounds induce higher cytotoxicity with increase in the G1 cell cycle phase compared with the trans-compounds. Quantitative RT-PCR assay indicated that compounds (16a-d) induced G1 phase arrest through down-regulation of cyclin D1 and up-regulation of p21, p27, and p53 mRNA expressions. Compounds 16a-d induced A375 early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide. Moreover, the Western blot analysis showed that A375 treated by compounds (16a-d) resulted in decreased levels of Bcl-2 and Bcl-xL, increased levels of Bax and Bad, and caspase/PARP degradation to identify apoptotic cells.

A convenient method to construct (Z)-oxazines via 6-exo-dig iodocyclization and synthesis of indolin-3-one.

An efficient regio-, stereo- and chemo-specific synthesis of 1,3-benzoxazines via 6-exo-dig cyclization to afford the Z-isomer is reported. The structure and connectivity were confirmed unambiguously on the basis of (1)H NMR, NOESY, and ORTEP. Furthermore, DFT studies revealed that the Z-isomer was more stable than the E-isomer. Iodine substituted 1,3-benzoxazines were very useful precursors for cross coupling reactions. Suzuki reaction was carried out successfully and the resulting product was transformed to 1-(4-nitrobenzoyl)-2,2-diphenylindolin-3-one in the presence of a Lewis acid.

DC-81-enediyne induces apoptosis of human melanoma A375 cells: involvement of the ROS, p38 MAPK, and AP-1 signaling pathways.

Melanoma is one of the most chemoresistant cancers in patient care. The remission rate of current therapy remains low. DC-81, an antitumor antibiotic produced by Streptomyces species, belongs to pyrrolo[2,1-c][1,4]benzodiazepine (PBD), which is a potent inhibitor of nucleic acid synthesis. An enediyne contains either DNA intercalating groups or DNA minor groove binding functions and these are potent DNA-damaging agents due to their ability to generate benzenoid diradicals. We have previously reported an efficient synthesis and antitumor activity of a series of novel PBD hybrids linked with enediyne. The purpose of this study was to examine the mechanism of the antiproliferative effect of DC-81-enediyne agent on human melanoma A375 cells. DC-81-enediyne induced an increase in Ca(2+) level and reactive oxygen species (ROS) generation as detected by flow cytometric assay. Western blot analysis showed that DC-81-enediyne induced the phosphorylation of p38 and activating transcription factor 2 (ATF-2). By using the luciferase reporter assay, activating protein-1 (AP-1) activity was further enhanced after A375 cells were treated with graded concentrations of DC-81-enediyne. DC-81-enediyne treatment-induced A375 cell apoptosis was significantly abrogated by the addition of Ca(2+), ROS, and p38 inhibitors. Collectively, our studies indicate that DC-81-enediyne induces A375 cell apoptosis through an increased Ca(2+) and ROS generation, which involves p38 phosphorylation and enhanced ATF-2/AP-1 expressions, leading to caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 CytoDeath staining, and subsequent apoptotic cell death.

Flexibilins A-C, new cembrane-type diterpenoids from the Formosan soft coral, Sinularia flexibilis.

Three new cembrane-type diterpenoids, flexibilins A-C (1-3), along with a known cembrane, (-)-sandensolide (4), were isolated from the soft coral, Sinularia flexibilis. The structures of cembranes 1-4 were elucidated by spectroscopic methods. The structure of 4, including its absolute stereochemistry, was further confirmed by single-crystal X-ray diffraction analysis. Cembrane 2 displayed a moderate inhibitory effect on the release of elastase by human neutrophils.

A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1α-mediated signaling.

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1α (SDF-1α) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1α-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1α-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1α-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency.

Physico-chemical properties of sediments governing the bioavailability of heavy metals in urban waterways.

Bioavailability is a critical facet of metal toxicity. Although past studies have investigated the individual role of sediment physico-chemical properties in relation to the bioavailability of heavy metals, their collective effects are little-known. Further, limited knowledge exists on the contribution of nutrients to metal bioavailability. In this study, the influence of physico-chemical properties of sediments, including total organic carbon (TOC), total phosphorus (TP), total nitrogen (TN), cation exchange capacity (CEC), specific surface area (SSA), and mineralogical composition to metal bioavailability is reported. The weak-acid extraction method was used to measure Cd, Cr, Cu, Ni, Pb and Zn as the potentially bioavailable fraction in sediments in an urban creek. The results confirmed that Cu has strong selectivity for organic matter (r = 0.814, p < 0.01). Cr bioavailability was influenced by either sediment mineralogy, nutrients, CEC or SSA. Zn, Ni and Pb showed strong affinity to mineral oxides, though their preferred binding positions were with nutrients, particularly organic matter (r = 0.794, 0.809, and 0.753, p < 0.01, respectively). The adsorption of Cd was strongly influenced by the competition with other metals and its bioavailability was weakly influenced by ion exchange (CEC: r = 0.424, p < 0.01). The study results indicate that nitrogen and phosphorus compounds can elevate metal bioavailability due to complexation reactions. Generally, the estuarine area was more favourable for the adsorption of weakly-bound metals. This is concerning as estuaries generate high biogeochemical activity and are economically important.

Monitoring chloramines and bromamines in a humid environment using selected ion flow tube mass spectrometry.

The selectivity and sensitivity of selected ion flow tube mass spectrometry (SIFT-MS) for individual breath analysis of haloamines has been improved by heating the flow tube in a commercial instrument to around 106 degrees C. Data is presented showing the marked reduction in the number density of water clusters of product ions of common breath metabolites that are isobaric with the product ions from monochloramine and monobromamine that are used to monitor the haloamine concentrations. These results have direct relevance to the real-time monitoring of chloramines in drinking water, swimming pools and food processing plants. However, once the isobaric overlaps from water cluster ions are reduced at the higher temperatures, there is no conclusive evidence showing the presence of haloamines on single breath exhalations in the mid parts per trillion range from examination of the breaths of volunteers.

Synthesis, and biological evaluation of 2-(4-aminophenyl)benzothiazole derivatives as photosensitizing agents.

Photodynamic therapy (PDT) employing exogenous photosensitizers is currently being approved for treatment of basal cell carcinoma (BCC). 2-(4-Aminophenyl)benzothiazoles (6) consist of chromophoric structure and absorb light in the UVA (315-400 nm). These results encouraged us to design and synthesize a diversity of 2-phenylbenzothiazoles (6). Studies on the apoptotic mechanism involved in photosensitive effects induced by UVA-activated 6 in BCC cells are carried out in the present article. 6-UVA-treated cells displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, and activation of caspase-3. 6-UVA induced a decrease in mitochondrial membrane potential (Deltapsi(mt)) and ATP via enhanced ROS generation and promoted phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK expression. These results suggest that 6-UVA elicits photosensitive effects in mitochondria processes which involve ERK and p38 activation, and ultimately lead to BCC cell apoptosis.

Lewis Acid Catalyzed Atom-Economic Synthesis of C2-Substituted Indoles from o-Amido Alkynols.

Herein we have disclosed a Zn(OTf)2 catalyzed synthesis of C2-alkyl substituted indole derivatives via unprecedented carbonyl group migration from o-amido alkynols. The key features of this protocol involve N,O-carbonyl group migration, broad substrate scope with varied functionality tolerance, moderate to good yields, and 100% atom economy. The crossover experiments proved that the migration is happening via an intramolecular pathway.

Synthesis and antitumor activity of novel enediyne-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids.

A series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) hybrids linked with enediyne is described. These compounds were prepared by linking C-8 of DC-81 (1) with an enediyne (10-16) through carbon chain linkers to afford PBD hybrid agents 17-23 in good yields. Most of the hybrids on human cancer cell lines exhibited higher cytotoxicity, and an increase in the sub-G1 population than 1. In a previous article, we have demonstrated that DC-81-indole conjugate agents (3-6) are potent inducers of cell apoptosis in melanoma. In the present article, we investigated whether DC-81-enediyne agents possess more cytotoxicity than 6 on human 293T cells. Our data revealed that treatment of 293T cells with DC-81-enediyne resulted in a significant increase of annexin V binding, caspase-3 degradation, and p53 arrest to identify apoptotic cells than 6. These results suggest that the DC-81-enediyne agents are more efficient in inducing apoptosis than DC-81-indole in 293T cells.

Induction of apoptosis by DC-81-indole conjugate agent through NF-kappaB and JNK/AP-1 pathway.

DC-81, an antitumor antibiotic produced by Streptomyces species, belongs to the pyrrolo[2,1- c][1,4]benzodiazepine (PBD) family, which are potent inhibitors of nucleic acid synthesis. We previously reported an efficient synthesis of PBD hybrids linked with indole carboxylates. Recently, we have also shown that a PBD hybrid (IN6CPBD) agent can activate the apoptotic pathway mediated by mitochondria. In this study, we will examine the transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) that functionally regulate cell proliferation, transformation, and apoptosis. To investigate the IN6CPBD-induced alterations in NF-kappaB and AP-1 activity that involve cell cycle regulation, we exposed human melanoma A375 cells to different concentrations of IN6CPBD. Our data revealed that treatment of A375 cells with IN6CPBD resulted in a marked loss of cells from the G2/M phase of the cell cycle and an increase in Ca (2+) and cAMP and promoted phosphorylation of Jun N-terminal kinase (JNK) expression. By using the luciferase reporter assay, the NF-kappaB activities were decreased; however, AP-1 activity was further enhanced after A375 cells were treated with graded concentrations of IN6CPBD. Blockade of NF-kappaB or JNK activity further enhanced caspase-3 substrate PARP cleavage and subsequent apoptotic cell death.

Synthesis and biological evaluation of thiobenzanilides as anticancer agents.

A series of novel thiobenzanilides is described. These compounds have been previously found to show strong biological activity such as antimycotic and antifungal actions. This is the first demonstration on the mechanism of the anticancer effect of thiobenzanilide agents (4a-c) on human melanoma A375 cells. The cytotoxic studies of compounds 4a-c on human melanoma A375 cells indicate thiobenzanilides induced higher cytotoxicity than nitrobenzanilides (3a-c). In addition, DNA flow cytometric analysis shows that 4a-c displays a significant G2/M phase arrest, which progresses to early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide (PI). Because cellular apoptosis is often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in 4a-c-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with 4a-c resulted in the loss of mitochondrial membrane potential (DeltaPsi(mt)), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and activation of caspase-3. Thus, we suggest that 4a-c agents are potent inducers of cell apoptosis in A375 cells.

A C-3 methylated isocembranoid and 10-oxocembranoids from a formosan soft coral, Sinularia grandilobata.

Five new cembranoids, designated grandilobatins A-E (1- 5), were isolated from the soft coral Sinularia grandilobata. Grandilobatin C (3) was found to have a novel skeleton with the C-4 methyl group of the normal cembranoid rearranged to C-3, while the other metabolites were identified as new 10-oxocembranoids. Metabolite 4 has weak cytotoxicity toward MDA-MB-23 human breast tumor cells. Also, 4 significantly inhibited the accumulation of the pro-inflammatory iNOS protein of LPS-stimulated RAW264.7 macrophage cells at 50 microM.

Detection of Naja atra Cardiotoxin Using Adenosine-Based Molecular Beacon.

This study presents an adenosine (A)-based molecular beacon (MB) for selective detection of Naja atra cardiotoxin (CTX) that functions by utilizing the competitive binding between CTX and the poly(A) stem of MB to coralyne. The 5'- and 3'-end of MB were labeled with a reporter fluorophore and a non-fluorescent quencher, respectively. Coralyne induced formation of the stem-loop MB structure through A2-coralyne-A2 coordination, causing fluorescence signal turn-off due to fluorescence resonance energy transfer between the fluorophore and quencher. CTX3 could bind to coralyne. Moreover, CTX3 alone induced the folding of MB structure and quenching of MB fluorescence. Unlike that of snake venom α-neurotoxins, the fluorescence signal of coralyne-MB complexes produced a bell-shaped concentration-dependent curve in the presence of CTX3 and CTX isotoxins; a turn-on fluorescence signal was noted when CTX concentration was ≤80 nM, while a turn-off fluorescence signal was noted with a further increase in toxin concentrations. The fluorescence signal of coralyne-MB complexes yielded a bell-shaped curve in response to varying concentrations of N. atra crude venom but not those of Bungarus multicinctus and Protobothrops mucrosquamatus venoms. Moreover, N. nigricollis venom also functioned as N. atra venom to yield a bell-shaped concentration-dependent curve of MB fluorescence signal, again supporting that the hairpin-shaped MB could detect crude venoms containing CTXs. Taken together, our data validate that a platform composed of coralyne-induced stem-loop MB structure selectively detects CTXs.

Photodynamic Therapy Using Indolines-Fused-Triazoles Induces Mitochondrial Apoptosis in Human Non-Melanoma BCC Cells.

BACKGROUND/AIM: Cancer is one of the most dreadful diseases in humans and among them non-melanoma skin cancer (NMSC) is an increasing problem in the world, that occurs more frequently in people with fair skin. Photodynamic therapy (PDT), a non-invasive treatment is widely used for the prevention and treatment of BCC cells. We previously reported an efficient synthesis of novel indolines-fused-triazoles and studied their photophysical studies. This study delineated the signaling pathways involved in the PDT effect of triazoles on BCC cells under UVA irradiation. MATERIALS AND METHODS: Cell survival was evaluated by the MTT assay. The uptake of 1j in BCC cells was determined by using its fluorescence properties. Intracellular ROS and mitochondrial membrane potential (ΔΨmt) were measured using DCFH-DA probe and DiOC6 dye, respectively. Cytochrome c release was determined using immunofluorescent staining. RESULTS: Our data disclosed that treatment of BCC cells with 1j-UVA resulted in increased ROS generation, loss of mmp (ΔΨmt), decreased levels of Bcl-2 and Bcl-xL, increased levels of Bax and Bad, cytochrome c release, and caspase-3/PARP degradation to identify apoptotic cell death. CONCLUSION: The present study suggest that 1j-PDT may serve as a potential ancillary modality for the treatment of NMSC.