Electrochemical Arsine Generators for Arsenic Determination

Arsine generation is the gateway for several sensitive and selective methods of As determination. An electrochemical arsine generator (EAG) is especially green: we report here the use of two electrode materials, aluminum and highly oriented (ordered) pyrolytic graphite (HOPG) never before used for this purpose. The first is operated on a novel constant voltage mode: current flows only when the sample, deliberately made highly conductive with acid, is injected. As a result, the cathode, despite being a highly active metal that will self-corrode in acid, lasts a long time. This EAG can be made to respond to As­(III) and As­(V) in an equivalent fashion and is fabricated with two readily available chromatographic T-fittings. It permits the use of a wire roll as the cathode, permitting rapid renewal of the electrode. The HOPG-based EAG is easily constructed from ion chromatography suppressor shells and can convert As­(III) to AsH₃ quantitatively but has significantly lower response to As­(V); this difference can be exploited for speciation. The success of Al, an active metal, also dispels the maxim that metals with high hydrogen overpotential are best for electrochemical hydride generation. We report construction, operation, and performance details of these EAGs. Using gas phase chemiluminescence (GPCL) with ozone as a complementary green analytical technique, we demonstrate attractive limits of detection (LODs) (S/N = 3) of 1.9 and 1.0 μg/L As­(V) and As­(III) for the HOPG-based EAG and 1.4 μg/L As­(V) or As­(III) for the Al-based EAG, respectively. Precision at the ∼20 μg/L As­(V) level was 2.4% and 2.1% relative standard deviation (RSD) for HOPG- and Al-based EAGs, respectively. Both HOPG- and Al-based EAGs permitted a sample throughput of 12/h. For groundwater samples from West Texas and West Bengal, India, very comparable results were obtained with parallel measurements by induction coupled plasma-mass spectrometry

Structural–temporal embedding of large-scale dynamic networks with parallel implementation

Due to the widespread network data in the real world, network analysis has attracted increasing attention in recent years. In complex systems such as social networks, entities and their mutual relations can be respectively represented by nodes and edges composing a network. Because occurrences of entities and relations in these systems are often dynamic over time, their networks are called temporal networks describing the process of dynamic connection of nodes in the networks. Dynamic network embedding aims to embed nodes in a temporal network into a low-dimensional semantic space, such that the network structures and evolution patterns can be preserved as much as possible in the latent space. Most existing methods capture structural similarities (relations) of strongly-connected nodes based on their historical neighborhood information, they ignore the structural similarities of weakly-connected nodes that may also represent relations and include no explicit temporal information in node embeddings for capturing periodic dependency of events. To address these issues, we propose a novel temporal network embedding model by extending the structure similarity to cover both strong connections and weak connections among nodes, and including the temporal information in node embeddings. To improve the training efficiency of our model, we present a parallel training strategy to quickly acquire node embeddings. Extensive experiments on several real-world temporal networks demonstrate that our model significantly outperforms the state-of-the-arts in traditional tasks, including link prediction and node classification

Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells

<div><p>Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much <i>P53</i> and <i>MDM2</i> mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher <i>P53</i> gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).</p></div

Transient Nutlin-3 treatment of diploid HCT116 clones induces the appearance of cells with>4N DNA content.

<p><b>A</b>) Diploid HCT116 clones D3 and D8 were untreated (NT) or exposed to Nutlin (NUT 10 µM) for 24 hrs, followed by Nutlin removal. The cells were harvested at the indicated times after Nutlin removal. Fixed cells were stained with propidium iodide (25 µg/ml) and subjected to flow cytometry analysis. <b>B</b>) Cells were untreated (NT) or exposed to Nutlin (NUT 10 µM) for 24 hrs, followed by Nutlin removal. Cell lysates were collected at the indicated time points and analyzed by immunoblotting with the indicated antibodies. Actin was as a loading control. <i>P-Cdc2</i>, phosphor-Cdc2 (Tyr-15).</p

Tetraploid clones show resistance to cisplatin (CP) and ionizing radiation (IR)-induced apoptosis.

<p><b>A</b>) Five diploid clones isolated from Nutlin treated D3 cells (D3 Diploid) and D8 cells (D8 Diploid), and five tetraploid clones isolated from Nutlin treated D3 cells (D3 Tetraploid) and D8 cells (D8 Tetraploid) were untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 48 hrs. The percentage of cells with sub-G1 DNA was determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). * significance value (P<0.05). <b>B</b>) Tetraploid clones (T3, TD6) and their diploid counterparts (D3, D81B) were untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 72 hrs. The percentage of cells with sub-G1 DNA (propidium iodide staining) was determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). * significance value (P<0.05). <b>C</b>) The indicated diploid and tetraploid clones were untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 24 hrs. p53, p21, and MDM2 protein levels were determined by immunoblotting and quantified. Numbers indicate the relative level of each protein. Actin was used as a loading control. <b>D</b>) The indicated diploid and tetraploid clones untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 48 hrs. Cleaved PARP and Caspase-3 protein levels were determined by immunoblotting and quantified relative to the untreated. <b>E</b>) qRT-PCR was used to determine mRNA levels for the indicated genes in diploid (D3, D81B) and tetraploid (T3, TD6) clones that were either untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 24 hrs. The level of each mRNA transcript in untreated diploid clones (D3 NT, D81B NT) was considered “1.0”, and all other values are plotted relative to it.</p

Effect of the Poly(ethylene glycol) (PEG) Density on the Access and Uptake of Particles by Antigen-Presenting Cells (APCs) after Subcutaneous Administration

Lymphatic trafficking of particles to the secondary lymphoid organs, such as lymph nodes, and the cell types that particles access are critical factors that control the quality and quantity of immune responses. In this study, we evaluated the effect of PEGylation on the lymphatic trafficking and accumulation of particles in draining lymph nodes (dLNs) as well as the cell types that internalized particles. As a model system, 200 nm polystyrene (PS) particles were modified with different densities of poly­(ethylene glycol) (PEG) and administered subcutaneously to mice. PEGylation enhanced the efficiency of particle drainage away from the injection site as well as the access of particles to dendritic cells (DCs). The accumulation of particles in dLNs was dependent on the PEG density. PEGylation also enhanced uptake by DCs while reducing internalization by B cells at the single cell level. Our results indicate that PEGylation facilitated the trafficking of particles to dLNs either through enhanced trafficking in lymphatic vessels or by enhanced internalization by migratory DCs. This study provides insight into utilizing PEGylated particles for the development of synthetic vaccines

Stable tetraploid clones were isolated from diploid D3 and D8 clones after endoreduplication.

<p><b>A</b>) Shown is the procedure to isolate diploid and tetraploid clones from cells transiently exposed to Nutlin. D3 and D8 were untreated (NT) or treated with 10 µM Nutlin (NUT) for 24 hrs, followed by Nutlin removal for an additional 16 hrs. The cells were then live-stained with Hoechst 33342 (5 µg/ml). Cell sorting was performed on a MoFlo cytometer equipped with a UV excitation wavelength laser. Sorted 2N and 8N cells were plated at low density in normal medium (minus Nutlin) and individual clones isolated. <b>B</b>) <i>Top</i>, the comparison of the DNA profiles between D3 and a representative tetraploid clone isolated from D3. <i>Bottom</i>, comparison of the DNA profiles between a representative diploid clone (D81B) isolated from D8 cells, and a representative tetraploid clone (TD6) isolated from D8 cells. <b>C</b>) Representative metaphase spread from diploid D3 cells and tetraploid T3 cells. The number in the bottom right indicates the number of chromosomes counted. <b>D</b>) FISH analysis with chromosome 17 (Chr 17) and p53-specific probes shows tetraploid (T3) cells contain 4 copies of p53 and Chr 17, while diploid (D3) cells contain 2 copies of p53 and Chr 17.</p

Tetraploid clones are more susceptible to Nutlin-induced cell cycle arrest and apoptosis.

<p><b>A</b>) Five diploid clones isolated from Nutlin treated D3 cells (D3 Diploid) and D8 cells (D8 Diploid), and five tetraploid clones isolated from Nutlin treated D3 cells (D3 Tetraploid) and D8 cells (D8 Tetraploid) were untreated or treated with increasing Nutlin (1.0–5.0 µM) for 24 hrs. The percent G1 and S-phase cells was determined by flow cytometry, and the fold change of G1/S ratio is plotted. Untreated diploid clones G1/S ratio was given a value of 1.0. Shown is the average of three separate experiments, +/- SE. <b>B</b>) Tetraploid clones (T3, TD6) and diploid counterparts (D3, D81B) were untreated or treated with Nutlin (1.0–5.0 µM) for 24 hrs. The fold change of G1/S ratio is plotted. Shown is the average of three separate experiments, +/- SE. <b>C</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with increasing Nutlin (1.0–5.0 µM) 24 hrs. P53 and p21 protein levels were quantified using Image-J software. Numbers indicate the relative levels of each protein. P53 and p21 levels in untreated diploid clones was given a value of “1.0”. Actin was used as loading control. <b>D</b>) Five diploid clones isolated from Nutlin treated D3 cells (D3 Diploid) and D8 cells (D8 Diploid), and five tetraploid clones isolated from Nutlin treated D3 cells (D3 Tetraploid) and D8 cells (D8 Tetraploid) were untreated (NT) or treated with Nutlin (20 µM) 72 hrs and apoptosis determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). *(P<0.05). <b>E</b>) Representative diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated (NT) or treated with Nutlin (20 µM) for 72 hrs. The percentage of cells with sub-G1 DNA content (propidium iodide staining) was determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). * significance value (P<0.05). <b>F</b>) The indicated diploid and tetraploid clones were untreated (NT) or exposed to Nutlin (20 µM) for 48 hrs. Cleaved PARP and Caspase-3 protein levels were determined by immunoblotting and quantified relative to the untreated.</p

Tetraploid clones express more p53-MDM2 complexes and more p53 after Nutlin treatment than diploid counterparts.

<p><b>A</b>) P53 and MDM2 mRNA levels were compared in untreated tetraploid clones (T3, TD6) and their diploid counterparts (D3, D81B). <b>B</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with proteasome inhibitor MG132 (10 µM) for 8 hrs. <i>Upper</i> Levels of MDM2, p53, and actin (loading control) in untreated and MG132 treated cells are shown. <i>Lower</i> To determine levels of p53-MDM2 complexes in diploid and tetraploid cells, protein lysates were immunoprecipitated with anti-p53 antibody, followed by immunoblotting for MDM2. <b>C</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with Nutlin (10 µM) for 24 hrs. p53 and MDM2 protein levels were detected by immunoblotting and quantified using Image-J software. The relative amount of p53 and MDM2 protein in the untreated diploid clones was given a value of “1.0”. Numbers indicate the relative level of each protein. Actin was used as a loading control.</p

Tetraploid clones are more resistant than diploid clones to Cisplatin (CP) and ionizing radiation (IR) and more sensitive to Nutlin in a long term survival assay.

<p><b>A</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with CP (10 µM) for 24 hrs. Cells were rinsed and re-fed with drug free media and stained after 2–3 weeks. The colonies were counted and normalized with plating efficiency of untreated controls. Shown are the mean results from three separate experiments. <b>B</b>) Indicated clones were untreated or exposed to IR (3 Gy). Colonies were counted 2–3 weeks later and normalized with plating efficiency of untreated controls. Shown are the mean results from three separate experiments. <b>C</b>) Diploid and tetraploid clones were grown in normal medium (minus Nutlin) or grown in continuous Nutlin (1 µM) and stained after 2–3 weeks. Colony number was normalized with the plating efficiency of untreated controls. <b>D</b>) Indicated clones were untreated or treated with Nutlin (20 µM) for 72 hrs after which the cells were rinsed and re-fed with normal medium (minus Nutlin) for 2–3 weeks. The colonies were then counted and normalized with the plating efficiency of untreated controls. Shown are the mean results from three separate experiments. <i>bars</i> indicate standard error (SE). * significance value (P<0.05).</p