A Quantitative Analysis of Electrochemical CO2 Reduction on Copper in Organic Amide and Nitrile-Based Electrolytes.

Aqueous electrolytes used in CO2 electroreduction typically have a CO2 solubility of around 34 mM under ambient conditions, contributing to mass transfer limitations in the system. Non-aqueous electrolytes exhibit higher CO2 solubility (by 5-8-fold) and also provide possibilities to suppress the undesired hydrogen evolution reaction (HER). On the other hand, a proton donor is needed to produce many of the products commonly obtained with aqueous electrolytes. This work investigates the electrochemical CO2 reduction performance of copper in non-aqueous electrolytes based on dimethylformamide (DMF), n-methyl-2-pyrrolidone (NMP), and acetonitrile (ACN). The main objective is to analyze whether non-aqueous electrolytes are a viable alternative to aqueous electrolytes for hydrocarbon production. Additionally, the effects of aqueous/non-aqueous anolytes, membrane, and the selection of a potential window on the electrochemical CO2 reduction performance are addressed in this study. Experiments with pure DMF and NMP mainly produced oxalate with a faradaic efficiency (FE) reaching >80%; however, pure ACN mainly produced hydrogen and formate due to the presence of more residual water in the system. Addition of 5% (v/v) water to the non-aqueous electrolytes resulted in increased HER and formate production with negligible hydrocarbon production. Hence, we conclude that aqueous electrolytes remain a better choice for the production of hydrocarbons and alcohols on a copper electrode, while organic electrolytes based on DMF and NMP can be used to obtain a high selectivity toward oxalate and formate.

Anion-exchange membranes with internal microchannels for water control in CO2 electrolysis.

Electrochemical reduction of carbon dioxide (CO2R) poses substantial promise to convert abundant feedstocks (water and CO2) to value-added chemicals and fuels using solely renewable energy. However, recent membrane-electrode assembly (MEA) devices that have been demonstrated to achieve high rates of CO2R are limited by water management within the cell, due to both consumption of water by the CO2R reaction and electro-osmotic fluxes that transport water from the cathode to the anode. Additionally, crossover of potassium (K+) ions poses concern at high current densities where saturation and precipitation of the salt ions can degrade cell performance. Herein, a device architecture incorporating an anion-exchange membrane (AEM) with internal water channels to mitigate MEA dehydration is proposed and demonstrated. A macroscale, two-dimensional continuum model is used to assess water fluxes and local water content within the modified MEA, as well as to determine the optimal channel geometry and composition. The modified AEMs are then fabricated and tested experimentally, demonstrating that the internal channels can both reduce K+ cation crossover as well as improve AEM conductivity and therefore overall cell performance. This work demonstrates the promise of these materials, and operando water-management strategies in general, in handling some of the major hurdles in the development of MEA devices for CO2R.

Mechanism and ultrasensitivity in Hedgehog signaling revealed by Patched1 disease mutations.

Hedgehog signaling is fundamental in animal embryogenesis, and its dysregulation causes cancer and birth defects. The pathway is triggered when the Hedgehog ligand inhibits the Patched1 membrane receptor, relieving repression that Patched1 exerts on the GPCR-like protein Smoothened. While it is clear how loss-of-function Patched1 mutations cause hyperactive Hedgehog signaling and cancer, how other Patched1 mutations inhibit signaling remains unknown. Here, we develop quantitative single-cell functional assays for Patched1, which, together with mathematical modeling, indicate that Patched1 inhibits Smoothened enzymatically, operating in an ultrasensitive regime. Based on this analysis, we propose that Patched1 functions in cilia, catalyzing Smoothened deactivation by removing cholesterol bound to its extracellular, cysteine-rich domain. Patched1 mutants associated with holoprosencephaly dampen signaling by three mechanisms: reduced affinity for Hedgehog ligand, elevated catalytic activity, or elevated affinity for the Smoothened substrate. Our results clarify the enigmatic mechanism of Patched1 and explain how Patched1 mutations lead to birth defects.

Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand.

Hedgehog signaling governs critical processes in embryogenesis, adult stem cell maintenance, and tumorigenesis. The activating ligand, Sonic hedgehog (SHH), is highly hydrophobic because of dual palmitate and cholesterol modification, and thus, its release from cells requires the secreted SCUBE proteins. We demonstrate that the soluble SCUBE-SHH complex, although highly potent in cellular assays, cannot directly signal through the SHH receptor, Patched1 (PTCH1). Rather, signaling by SCUBE-SHH requires a molecular relay mediated by the coreceptors CDON/BOC and GAS1, which relieves SHH inhibition by SCUBE. CDON/BOC bind both SCUBE and SHH, recruiting the complex to the cell surface. SHH is then handed off, in a dual lipid-dependent manner, to GAS1, and from GAS1 to PTCH1, initiating signaling. These results define an essential step in Hedgehog signaling, whereby coreceptors activate SHH by chaperoning it from a latent extracellular complex to its cell-surface receptor, and point to a broader paradigm of coreceptor function.

Distinct Cation Gradients Power Cholesterol Transport at Different Key Points in the Hedgehog Signaling Pathway.

Cholesterol plays two critical roles in Hedgehog signaling, a fundamental pathway in animal development and cancer: it covalently modifies the Sonic hedgehog (SHH) ligand, restricting its release from producing cells, and directly activates Smoothened in responding cells. In both contexts, a membrane protein related to bacterial RND transporters regulates cholesterol: Dispatched1 controls release of cholesterylated SHH, and Patched1 antagonizes Smoothened activation by cholesterol. The mechanism and driving force for eukaryotic RND proteins, including Dispatched1 and Patched1, are unknown. Here, we show that Dispatched1 acts enzymatically to catalyze SHH release. Dispatched1 uses the energy of the plasma membrane Na+ gradient, thus functioning as an SHH/Na+ antiporter. In contrast, Patched1 repression of Smoothened requires the opposing K+ gradient. Our results clarify the transporter activity of essential eukaryotic RND proteins and demonstrate that the two main cation gradients of animal cells differentially power cholesterol transport at two crucial steps in the Hedgehog pathway.

Modified cation-exchange membrane for phosphate recovery in an electrochemically assisted adsorption-desorption process.

A novel ion separation methodology using a cation-exchange membrane modified with iron oxide nanoparticles (Fe3O4 NPs) coated with polyhexamethylene guanidine (PHMG) is proposed. The separation is performed in an electrodialysis cell, where firstly phosphate is electro-adsorbed to the PHMG@Fe3O4 NP coating, followed by a desorption step by applying an electric current.

Sending and Receiving Hedgehog Signals.

Communication between cells pervades the development and physiology of metazoans. In animals, this process is carried out by a relatively small number of signaling pathways, each consisting of a chain of biochemical events through which extracellular stimuli control the behavior of target cells. One such signaling system is the Hedgehog pathway, which is crucial in embryogenesis and is implicated in many birth defects and cancers. Although Hedgehog pathway components were identified by genetic analysis more than a decade ago, our understanding of the molecular mechanisms of signaling is far from complete. In this review, we focus on the biochemistry and cell biology of the Hedgehog pathway. We examine the unique biosynthesis of the Hedgehog ligand, its specialized release from cells into extracellular space, and the poorly understood mechanisms involved in ligand reception and pathway activation at the surface of target cells. We highlight several critical questions that remain open.

Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog.

The Hedgehog cell-cell signaling pathway is crucial for animal development, and its misregulation is implicated in numerous birth defects and cancers. In unstimulated cells, pathway activity is inhibited by the tumor suppressor membrane protein, Patched. Hedgehog signaling is triggered by the secreted Hedgehog ligand, which binds and inhibits Patched, thus setting in motion the downstream events in signal transduction. Despite its critical importance, the mechanism by which Hedgehog antagonizes Patched has remained unknown. Here, we show that vertebrate Patched1 inhibition is caused by direct, palmitate-dependent interaction with the Sonic Hedgehog ligand. We find that a short palmitoylated N-terminal fragment of Sonic Hedgehog binds Patched1 and, strikingly, is sufficient to inhibit it and to activate signaling. The rest of Sonic Hedgehog confers high-affinity Patched1 binding and internalization through a distinct binding site, but, surprisingly, it is not absolutely required for signaling. The palmitate-dependent interaction with Patched1 is specifically impaired in a Sonic Hedgehog mutant causing human holoprosencephaly, the most frequent congenital brain malformation, explaining its drastically reduced potency. The palmitate-dependent interaction is also abolished in constitutively inhibited Patched1 point mutants causing the Gorlin cancer syndrome, suggesting that they might adopt a conformation distinct from the wild type. Our data demonstrate that Sonic Hedgehog signals via the palmitate-dependent arm of a two-pronged contact with Patched1. Furthermore, our results suggest that, during Hedgehog signaling, ligand binding inhibits Patched by trapping it in an inactive conformation, a mechanism that explains the dramatically reduced activity of oncogenic Patched1 mutants.

The Tlo proteins are stoichiometric components of Candida albicans mediator anchored via the Med3 subunit.

The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis.

The Paf1 complex represses SER3 transcription in Saccharomyces cerevisiae by facilitating intergenic transcription-dependent nucleosome occupancy of the SER3 promoter.

Previous studies have shown that repression of the Saccharomyces cerevisiae SER3 gene is dependent on transcription of SRG1 from noncoding DNA initiating within the intergenic region 5' of SER3 and extending across the SER3 promoter region. By a mechanism dependent on the activities of the Swi/Snf chromatin remodeling factor, the HMG-like factor Spt2, and the Spt6 and Spt16 histone chaperones, SRG1 transcription deposits nucleosomes over the SER3 promoter to prevent transcription factors from binding and activating SER3. In this study, we uncover a role for the Paf1 transcription elongation complex in SER3 repression. We find that SER3 repression is primarily dependent on the Paf1 and Ctr9 subunits of this complex, with minor contributions by the Rtf1, Cdc73, and Leo1 subunits. We show that the Paf1 complex localizes to the SRG1 transcribed region under conditions that repress SER3, consistent with it having a direct role in mediating SRG1 transcription-dependent SER3 repression. Importantly, we show that the defect in SER3 repression in strains lacking Paf1 subunits is not a result of reduced SRG1 transcription or reduced levels of known Paf1 complex-dependent histone modifications. Rather, we find that strains lacking subunits of the Paf1 complex exhibit reduced nucleosome occupancy and reduced recruitment of Spt16 and, to a lesser extent, Spt6 at the SER3 promoter. Taken together, our results suggest that Paf1 and Ctr9 repress SER3 by maintaining SRG1 transcription-dependent nucleosome occupancy.

SERPINB5 and AKAP12 - expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma.

BACKGROUND: Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. METHODS: Metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. RESULTS: In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05). CONCLUSIONS: AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.