Low Steady-state Concentrations Research Articles

Sustainable Hydroponics Using Zero-discharge Nutrient Management and Automated pH Control

Here we review the 400-year history of hydroponic culture and describe a unique management approach that does not require leaching or discarding solution between harvests. Nutrients are maintained at a low and steady concentration by daily additions of a dilute solution that replaces the transpired water along with the nutrients that were removed in growth each day. A stable pH and a low steady-state concentration of ammonium are maintained through automated additions of a solution of nitric acid and ammonium nitrate. Ample solution volume (at least 20 cm deep) stabilizes nutrient concentrations, reduces root density, and improves uniformity. Gentle aeration at ≈100 mL·min−1·L−1 maintains dissolved oxygen near saturation and increases uniformity throughout the rhizosphere. These practices facilitate a uniform, closed, root zone with rigorous pH control that provides the micromolar nutrient concentrations of N and P that are representative of field soils.

Production of Peroxymonocarbonate by Steady-State Micromolar H2O2 and Activated Macrophages in the Presence of CO2/HCO3- Evidenced by Boronate Probes.

Peroxymonocarbonate (HCO4-/HOOCO2-) is produced by the reversible reaction of CO2/HCO3- with H2O2 (K = 0.33 M-1, pH 7.0). Although produced in low yields at physiological pHs and H2O2 and CO2/HCO3- concentrations, HCO4- oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of H2O2. Boronate probes are known examples because HCO4- reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of H2O2 and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO4- formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state H2O2 concentrations (2-18 μM) in Pi buffer at pH 7.4 and 37 °C. Then, CBE (100 μM) was added and fluorescence increase was monitored with time. The results showed that each steady-state H2O2 concentration reacted more rapidly (∼30%) in the presence of CO2/HCO3- (25 mM) than in its absence, and the data permitted the calculation of consistent rate constants. Also, RAW 264.7 macrophages were activated with phorbol 12-myristate 13-acetate (PMA) (1 μg/mL) at pH 7.4 and 37 °C to produce a time-dependent H2O2 concentration (8.0 ± 2.5 μM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO3- equilibrated with 0, 5, or 10% CO2, respectively. In the presence of CBE or Fl-B (30 μM), a time-dependent increase in the fluorescence of the bulk solution was observed, which was higher in the presence of CO2/HCO3- in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO4- and boronate probes can evidence and distinguish it from H2O2 under biologically relevant concentrations of H2O2 and CO2/HCO3-.

Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP.

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription. Materials and methods: In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.

A comparative study of free chlorine and peroxymonosulfate activated by Fe(II) in the degradation of iopamidol: Mechanisms, density functional theory (DFT) calculatitons and formation of iodinated disinfection by-products

Both Fe(II)/free chlorine (Fe(II)/FC) and Fe(II)/peroxymonosulfate (Fe(II)/PMS) have a faster reactive species formation rate than the classic Fenton process, so their efficiencies of removing pollutants are better than that of the Fenton process. In this study, we compared the differences in the mechanisms and kinetics of iopamidol (IPM) degradation by Fe(II)/FC and Fe(II)/PMS. Although the production rate of reactive species of the two processes is at the same level (104 M−1 s−1), the Fe(II)/PMS process performed better regarding IPM removal. Reactive chlorine species (RCS) were the major contributor to the degradation of IPM (73.8%) by Fe(II)/FC, and hydroxyl radicals (HO•) and sulfate radicals (SO4•–) contributed 50.7% and 49.3% to the degradation of IPM by Fe(II)/PMS, respectively. Fe(IV) showed low contributions (<0.1%) to the degradation of IPM during the Fe(II)/FC and Fe(II)/PMS processes because of the low values of kFe(IV), IPM ((25 ± 5) M−1 s−1) and the low steady-state concentration of Fe(IV) in the Fe(II)/FC ((5.1 ± 0.7) × 10−8 M) and Fe(II)/PMS processes ((2.2 ± 0.09) × 10−8 M) at pH 3. Based on density functional theory (DFT) calculations and the MS results, the transformation pathway of IPM has been confirmed to mainly occur on chains B and B’ and the iodine group in between. The formation of disinfection by-products (DBPs) indicates that the Fe(II)/PMS process has lower environmental hazards. In general, we should pay more attention to the type of active species to evaluate the Fenton-like process. This study provides a basis for choosing a suitable alternative to the Fenton process and understanding the process in the Fenton-like procedure.

In situ EPR and Raman spectroscopy in the curing of bis-methacrylate-styrene resins.

The curing of bis-methacrylate–styrene resins initiated by the cobalt catalyzed decomposition of cumyl hydroperoxide is monitored at ambient temperatures in situ by EPR and Raman spectroscopy. EPR spectroscopy shows the appearance of organic radicals after ca. 1 h from initiation with an increase in intensity from both polystyrene and methacrylate based radical species over a further ca. 2 h period to reach a maximum spin concentration of ca. 2–3 mM. Alkene conversion to polymer was monitored by Raman spectroscopy in real time in situ with EPR spectroscopy and reveals that the appearance of the radical signals is first observed only as the conversion approaches its maximum extent (70% at room temperature), i.e., the resin reaches a glass-like state. The radicals persist for several months on standing at room temperature. Flash frozen samples (77 K) did not show EPR signals within 1 h of initiation. The nature of the radicals responsible for the EPR spectra observed were explored by DFT methods and isotope labelling experiments (D8–styrene) and correspond to radicals of both methacrylate and polystyrene. Combined temperature dependent EPR and Raman spectroscopy shows that conversion increases rapidly upon heating of a cured sample, reaching full conversion at 80 °C with initially little effect on the EPR spectrum. Over time (i.e. subsequent to reaching full conversion of alkene) there was a small but clear increase in the EPR signal due to the methacrylate based radicals and minor decrease in the signal due to the polystyrene based radicals. The appearance of the radical signals as the reaction reaches completion and their absence in samples flash frozen before polymerization has halted, indicate that the observed radicals are non-propagating. The formation of the radicals due to stress within the samples is excluded. Hence, the observed radicals are a representative of the steady state concentration of radicals present in the resin over the entire timespan of the polymerization. The data indicate that the lack of EPR signals is most likely due to experimental aspects, in particular spin saturation, rather than low steady state concentrations of propagating radicals during polymerization.

Mass-Transfer-Limited Biodegradation at Low Concentrations-Evidence from Reactive Transport Modeling of Isotope Profiles in a Bench-Scale Aquifer.

Organic contaminant degradation by suspended bacteria in chemostats has shown that isotope fractionation decreases dramatically when pollutant concentrations fall below the (half-saturation) Monod constant. This masked isotope fractionation implies that membrane transfer is slow relative to the enzyme turnover at μg L–1 substrate levels. Analogous evidence of mass transfer as a bottleneck for biodegradation in aquifer settings, where microbes are attached to the sediment, is lacking. A quasi-two-dimensional flow-through sediment microcosm/tank system enabled us to study the aerobic degradation of 2,6-dichlorobenzamide (BAM), while collecting sufficient samples at the outlet for compound-specific isotope analysis. By feeding an anoxic BAM solution through the center inlet port and dissolved oxygen (DO) above and below, strong transverse concentration cross-gradients of BAM and DO yielded zones of low (μg L–1) steady-state concentrations. We were able to simulate the profiles of concentrations and isotope ratios of the contaminant plume using a reactive transport model that accounted for a mass-transfer limitation into bacterial cells, where apparent isotope enrichment factors *ε decreased strongly below concentrations around 600 μg/L BAM. For the biodegradation of organic micropollutants, mass transfer into the cell emerges as a bottleneck, specifically at low (μg L–1) concentrations. Neglecting this effect when interpreting isotope ratios at field sites may lead to a significant underestimation of biodegradation.

A mitochondria-targeted ratiometric fluorescent nanoprobe for imaging of peroxynitrite in living cells

Peroxynitrite (ONOO−) is a series of basic biological oxidants involved in physiological and pathological processes. The detection of ONOO− in biological systems has been challenging due to its extremely short half-life and low steady-state concentration. In this work, a ratiometric fluorescent nanoprobe for ONOO− was constructed by coupling covalently of graphene quantum dots (GQDs) with cyanine 5.5 (Cy5.5). This nanoprobe (GQD-Cy5.5) could selectively accumulate in mitochondrial, appears two strong fluorescence emission peaks at 520 and 694 nm. In the presence of ONOO−, the intensity of fluorescence emission peak at 520 nm increased and the intensity of fluorescence emission peak at 694 nm decreased. The ratio (F520 nm/F694 nm) of fluorescence intensity at two emission peaks had a good linear relationship with the concentration of ONOO− in the range of 0–6.0 μM, and the detection limit was 0.03 μM. The excellent properties of the nanoprobe enable its applications in the ratiometric fluorescence imaging of endogenous ONOO− in cell mitochondria.

Ammonium Removal in Aquaponics Indicates Participation of Comammox Nitrospira

Aquaponic systems are sustainable solutions for food production combining fish growth (aquaculture) and production of vegetables (hydroponic) in one recirculating system. In aquaponics, nitrogen-enriched wastewater from fish in the aquaculture serves as fertilizer for the plants in the hydroponics, while the nitrogen-depleted and detoxified water flows back to the aquaculture. To investigate bacterial nitrogen-cycling in such an aquaponic system, measurements of nitrogen species were coupled with time-resolved 16S rRNA gene profiling and the functional capacity of organisms was studied using metagenomics. The aquaponic system was consistently removing ammonia and nitrite below 23 µM and 19 µM, and nitrate to steady-state concentrations of about 0.5 mM. 16S rRNA gene amplicon sequencing of sediments exposed in the pump sump revealed that typical signatures of canonical ammonia-oxidising microorganisms were below detection limit. However, one of the most abundant operational taxonomic units (OTU) was classified as a member of the genus Nitrospira with a relative abundance of 3.8%. For this genus, also genome scaffolds were recovered encoding the only ammonia monooxygenase genes identified in the metagenome. This study indicates that even in highly efficient aquaponic systems, comammox Nitrospira were found to participate in ammonium removal at low steady-state ammonia concentrations.

Cerebrovascular Accidents in Sickle Cell Disease: Rates and Risk Factors

AbstractCerebrovascular accident (CVA) is a major complication of sickle cell disease. The incidence and mortality of and risk factors for CVA in sickle cell disease patients in the United States have been reported only in small patient samples. The Cooperative Study of Sickle Cell Disease collected clinical data on 4,082 sickle cell disease patients enrolled from 1978 to 1988. Patients were followed for an average of 5.2 ± 2.0 years. Age-specific prevalence and incidence rates of CVA in patients with the common genotypes of sickle cell disease were determined, and the effects of hematologic and clinical events on the risk of CVA were analyzed. The highest rates of prevalence of CVA (4.01%) and incidence (0.61 per 100 patient-years) were in sickle cell anemia (SS) patients, but CVA occurred in all common genotypes. The incidence of infarctive CVA was lowest in SS patients 20 to 29 years of age and higher in children and older patients. Conversely, the incidence of hemorrhagic stroke in SS patients was highest among patients aged 20 to 29 years. Across all ages the mortality rate was 26% in the 2 weeks after hemorrhagic stroke. No deaths occurred after infarctive stroke. Risk factors for infarctive stroke included prior transient ischemic attack, low steady-state hemoglobin concentration and rate of and recent episode of acute chest syndrome, and elevated systolic blood pressure. Hemorrhagic stroke was associated with low steady-state hemoglobin and high leukocyte count.

Structure and Lateral Organization of Phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor but ubiquitous component of the inner leaflet of the plasma membrane of eukaryotic cells. However, due to its particular complex biophysical properties, it stands out from its neighboring lipids as one of the most important regulators of membrane-associated signaling events. Despite its very low steady-state concentration, PI(4,5)P2 is able to engage in a multitude of simultaneous cellular functions that are temporally and spatially regulated through the presence of localized transient pools of PI(4,5)P2 in the membrane. These pools are crucial for the recruitment, activation, and organization of signaling proteins and consequent regulation of downstream signaling. The present review showcases some of the most important PI(4,5)P2 molecular and biophysical properties as well as their impact on its membrane dynamics, lateral organization, and interactions with other biochemical partners.

Photochemical Properties of Re(CO)3 Complexes with and without a Local Proton Source and Implications for CO2 Reduction Catalysis

Herein, we present a detailed study on the photophysical properties and the excited state reactivity of two mononuclear Re(CO)3 complexes with imdazol-pyridine ligands equipped with and without a local proton source, [Re(CO)3LCl], where for 1: L = 2,4-ditert-butyl-6-(6-(1-methyl-1H-imidazol-2-yl)pyridin-2-yl)phenol and 2: L = 2-(3,5-ditert-butyl-2-methoxyphenyl)-6-(1-methyl-1H-imidazol-2-yl)pyridine. Time-resolved IR and UV/vis spectroscopy revealed that excitation of 1 and 2 is followed by population of the triplet excited state within <100 fs, where structural and vibrational relaxation to the T1 equilibrium structure is observed on the picosecond time scale. The T1 state can be viewed as a MLCT state as all ν(CO) features in the transient infrared (TRIR) spectra are shifted to higher wavenumbers upon excitation, which is indicative for a decreasing Re → CO π-backdonation. The T1 states have considerably long lifetimes at room temperature of 160 ns for 1 and 430 ns for 2 in dmf and they can be successfully quenched by the sacrificial electron donors triethanolamine (TEOA) and 1,3-dimethyl-2-phenyl-2,3-dihydro-1H-benzo[d]imidazole (BIH). The quenching rates are 2 orders of magnitude larger for BIH than for TEOA, as the latter reaction is endergonic. However, both species are not active in the photochemical CO2-to-CO conversion. We rationalize this for 2 by the low steady-state concentration of the initial reduction product, [Re(CO)3LCl]−, which ejects chloride rather fast. Thus, the second, homogeneous electron transfer process between [Re(CO)3LCl]− and [Re(CO)3L(solvent)] forming the active species [Re(CO)3L]−, has a very low probability and decomposition pathways come to the fore. 1 decomposes under irradiation in the presence of BIH or TEOA forming the initial photoproduct 3. We tentatively assume that the ligand in 3 is deprotonated and switches from a N,N- to a N,O–-coordination mode. This indicates that in the excited state the Re–N bond is cleaved quite easily, as this decomposition pathway has not been observed under electrochemical conditions.

Influence of the Ether Functional Group on Ketohydroperoxide Formation in Cyclic Hydrocarbons: Tetrahydropyran and Cyclohexane.

Photolytically initiated oxidation experiments were conducted on cyclohexane and tetrahydropyran using multiplexed photoionization mass spectrometry to assess the impact of the ether functional group in the latter species on reaction mechanisms relevant to autoignition. Pseudo-first-order conditions, with [O2]0:[R•]0 > 2000, were used to ensure that R• + O2 → products were the dominant reactions. Quasi-continuous, tunable vacuum ultraviolet light from a synchrotron was employed over the range 8.0-11.0 eV to measure photoionization spectra of the products at two pressures (10 and 1520 Torr) and three temperatures (500, 600, and 700 K). Photoionization spectra of ketohydroperoxides were measured in both species and were qualitatively identical, within the limit of experimental noise, to those of analogous species formed in n-butane oxidation. However, differences were noted in the temperature dependence of ketohydroperoxide formation between the two species. Whereas the yield from cyclohexane is evident up to 700 K, ketohydroperoxides in tetrahydropyran were not detected above 650 K. The difference indicates that reaction mechanisms change due to the ether group, likely affecting the requisite •QOOH + O2 addition step. Branching fractions of nine species from tetrahydropyran were quantified with the objective of determining the role of ring-opening reactions in diminishing ketohydroperoxide. The results indicate that products formed from unimolecular decomposition of R• and •QOOH radicals via concerted C-C and C-O β-scission are pronounced in tetrahydropyran and are insignificant in cyclohexane oxidation. The main conclusion drawn is that, under the conditions herein, ring-opening pathways reduce the already low steady-state concentration of •QOOH, which in the case of tetrahydropyran prevents •QOOH + O2 reactions necessary for ketohydroperoxide formation. Carbon balance calculations reveal that products from ring opening of both R• and •QOOH, at 700 K, account for >70% at 10 Torr and >55% at 1520 Torr. Three pathways are confirmed to contribute to the depletion of •QOOH in tetrahydropyran including (i) γ-•QOOH → pentanedial + •OH, (ii) γ-•QOOH → vinyl formate + ethene + •OH, and (iii) γ-•QOOH → 3-butenal + formaldehyde + •OH. Analogous mechanisms in cyclohexane oxidation leading to similar intermediates are compared and, on the basis of mass spectral results, confirm that no such ring-opening reactions occur. The implication from the comparison to cyclohexane is that the ether group in tetrahydropyran increases the propensity for ring-opening reactions and inhibits the formation of ketohydroperoxide isomers that precede chain-branching. On the contrary, the absence of such reactions in cyclohexane enables ketohydroperoxide formation up to 700 K and perhaps higher temperature.

Pharmacokinetics, excretion, distribution, and metabolism of 60-kDa polyethylene glycol used in BAY 94-9027 in rats and its value for human prediction

The covalent binding of proteins with polyethylene glycol (PEG) molecules is a valuable tool to extend the half-life of many biotherapeutics, including factor VIII (FVIII) products to treat patients with haemophilia A. Although PEG has low toxicity, accumulation of large PEG molecules (>20–30 kDa) with long-term exposure is a potential concern. Thus, it is important to determine whether sufficient excretion processes exist for PEG molecules used in biotherapeutics. BAY 94-9027 is an extended–half-life FVIII product modified through addition of a 60-kDa (branched: dual 30-kDa) PEG molecule. BAY 1025662 is the 60-kDa PEG moiety used for PEGylation of BAY 94-9027. This study investigated the pharmacokinetic (PK) properties, distribution, and excretion of BAY 1025662 in rats in order to predict estimated 60-kDa PEG PK properties in patients. Plasma concentrations in male rats after a single 11-mg/kg intravenous dose of BAY 1025662 (approximating the cumulative PEG-60 exposure in patients during 30 years of BAY 94-9027 treatment) decreased with an initial half-life of 119 h (5 days) in the interval of 114–336 h post administration. Single-dose mass balance studies using radiolabeled BAY 1025662 ([prop-14C]BAY 1025662) showed that 30.4% of radioactivity was excreted within 1 week and 79.1% by Day 168 (primarily in urine). The terminal half-life of radioactivity elimination was approximately 24 days in blood and plasma and was 31–68 days in the majority of other organs up to Day 168. Elimination was nearly complete at the end of the experiment on Day 168; only ~4% of residual radioactivity was present in the animal body. There was no irreversible binding of radioactivity to any tissues and no penetration of the blood-brain barrier. Based on these results, very low steady-state concentrations of 60-kDa PEG were predicted in patients treated with BAY 94-9027, and the validity of these predictions was supported by clinical studies in which almost all 179 patients receiving BAY 94-9027 for prophylaxis had undetectable PEG in plasma for up to >5 years; those with detectable PEG levels demonstrated concentrations within the predicted range. These combined preclinical and clinical observations suggest that excretion processes are in place for high-molecular-weight PEGs such as the PEG-60 moiety used in BAY 94-9027.

Modulation of Catalytic Promiscuity during Hydrogen Sulfide Oxidation.

The mitochondrial sulfide oxidation pathway prevents the toxic accumulation of hydrogen sulfide (H2S), a signaling molecule that is maintained at low steady-state concentrations. Sulfide quinone oxidoreductase (SQR), an inner mitochondrial membrane-anchored protein, catalyzes the first and committing step in this pathway, oxidizing H2S to persulfide. The catalytic cycle comprises sulfide addition to the active site cysteine disulfide in SQR followed by sulfur transfer to a small molecule acceptor, while a pair of electrons moves from sulfide, to FAD, to coenzyme Q. While its ability to oxidize H2S is well characterized, SQR exhibits a remarkable degree of substrate promiscuity in vitro that could undermine its canonical enzyme activity. To assess how its promiscuity might be contained in vivo, we have used spectroscopic and kinetic analyses to characterize the reactivity of alternate substrates with SQR embedded in nanodiscs ( ndSQR) versus detergent-solubilized enzyme ( sSQR). We find that the membrane environment of ndSQR suppresses the unwanted addition of GSH but enhances sulfite addition, which might become significant under pathological conditions characterized by elevated sulfite levels. We demonstrate that methanethiol, a toxic sulfur compound produced in significant quantities by colonic and oral microbiota, can add to the SQR cysteine disulfide and also serve as a sulfur acceptor, potentially interfering with sulfide oxidation when its concentrations are elevated. These studies demonstrate that the membrane environment and substrate availability combine to minimize promiscuous reactions that would otherwise disrupt sulfide homeostasis.

Oxidative degradation of phenols in sono-Fenton-like systems upon high-frequency ultrasound irradiation

The kinetics of oxidative degradation of phenol and chlorophenols upon acoustic cavitation in the megahertz range (1.7 MHz) is studied experimentally in model systems, and the involvement of in situ generated reactive oxygen species (ROSs) is demonstrated. The phenols subjected to high frequency ultrasound (HFUS) are ranked in terms of their rate of conversion: 2,4,6-trichlorophenol > 2,4-dichlorophenol ~ 2-chlorophenol > 4-chlorophenol ~ phenol. Oxidative degradation upon HFUS irradiation is most efficient at low concentrations of pollutants, due to the low steady-state concentrations of the in situ generated ROSs. A dramatic increase is observed in the efficiency of oxidation in several sonochemical oxidative systems (HFUS in combination with other chemical oxidative factors). The system with added Fe2+ (a sono-Fenton system) derives its efficiency from hydrogen peroxide generated in situ as a result of the recombination of OH radicals. The S2O82-/Fe2+/HFUS system has a synergetic effect on substrate oxidation that is attributed to a radical chain mechanism. In terms of the oxidation rates, degrees of conversion, and specific energy efficiencies of 4-chlorophenol oxidation based on the amount of oxidized substance per unit of expended energy the considered sonochemical oxidative systems form the series HFUS < S2O82-/HFUS < S2O82-/Fe2+/HFUS.

Abstract 5099: Development of novel antimetabolite, DFP-11207, with self-toxicity protection and its promising preclinical and clinical profiles

Abstract Background: For over 50 years, 5-fluorouracil (5-FU) has played a critical role in the systemic chemotherapy of various gastrointestinal cancers including gastric, colorectal and pancreatic cancer which are the leading causes of cancer death globally. Although systemic cytotoxics such as 5-FU can modestly prolong overall survival in the adjuvant and advanced settings, but at the cost of unpleasant toxicities. Therefore better drug formulations are desirable. Methods: To this end, we developed a new conceptual fluoropyrimidine, DFP-11207 that is engineered to reduce toxicity without loss of antitumor activity as well in addition to providing sustained concentrations of the active anticancer moiety. DFP-11207 contains three components in one formulation: 1-ethoxymethyl-5-fluorouracil (EM-FU) as a prodrug of 5-FU, 5-chloro-2,4-dihyroxypyridine (CDHP) as a potent inhibitor of 5-FU degradation and cytrazinic acid (CTA) as a gastrointestinal regulator of 5-FU phosphorylation. Results: In in vitro metabolism studies using cell-free extracts from plasma, liver and tumors or intact tumor cells, DFP-11207 was rapidly hydrolyzed to 3 components and subsequently EM-FU was specifically converted to 5-FU by liver microsomes, and CDHP and CTA strongly inhibited 5-FU degradation and phosphorylation, respectively. Following consecutive oral administration to human tumor-bearing nude rats, DFP-11207 attained favorable antitumor efficacy and long-sustained PK profiles with lack of GI- and myelo-toxicities. Next, in investigational clinical study in patients with solid tumors, 12 patients were treated at 8 unique dose levels of DFP-11207, ranging from 40 to 440mg/m2/day by each 1 pt. MTD and RD of daily and 28-day consecutive DFP-11207 was found to be 440 (n=2) and 330 mg/m2 (n=6), respectively. The main AEs were nausea,, anemia, neutropenia, febrile neutropenia but these events were very mild, and no thrombocytopenia was observed as expected. Furthermore, review of the preliminary PK data, DFP-11207 at 330 mg/m2 resulted in a desirable low but efficacious (~15- 30 ng/ml) steady-state plasma concentration of 5-FU. Interestingly, some of patients heavily treated with therapeutic drugs had a stable-disease for long periods. Conclusion: Our preclinical and early clinical data with DFP-11207 suggest that it’s a promising compound for the treatment of gastrointestinal cancers and can overcome the shortcomings of all other oral fluoropyrimidines. Citation Format: Masakazu Fukushima, Kenzo Iizuka, Kokoro Eshima, Chung Zhang, Cheng Jin, Kiyoshi Eshima, Jaffer Ajani. Development of novel antimetabolite, DFP-11207, with self-toxicity protection and its promising preclinical and clinical profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5099. doi:10.1158/1538-7445.AM2017-5099

The lncRNA VELUCT strongly regulates viability of lung cancer cells despite its extremely low abundance.

Little is known about the function of most non-coding RNAs (ncRNAs). The majority of long ncRNAs (lncRNAs) is expressed at very low levels and it is a matter of intense debate whether these can be of functional relevance. Here, we identified lncRNAs regulating the viability of lung cancer cells in a high-throughput RNA interference screen. Based on our previous expression profiling, we designed an siRNA library targeting 638 lncRNAs upregulated in human cancer. In a functional siRNA screen analyzing the viability of lung cancer cells, the most prominent hit was a novel lncRNA which we called Viability Enhancing LUng Cancer Transcript (VELUCT). In silico analyses confirmed the non-coding properties of the transcript. Surprisingly, VELUCT was below the detection limit in total RNA from NCI-H460 cells by RT-qPCR as well as RNA-Seq, but was robustly detected in the chromatin-associated RNA fraction. It is an extremely low abundant lncRNA with an RNA copy number of less than one copy per cell. Blocking transcription with actinomycin D revealed that VELUCT RNA was highly unstable which may partially explain its low steady-state concentration. Despite its extremely low abundance, loss-of-function of VELUCT with three independent experimental approaches in three different lung cancer cell lines led to a significant reduction of cell viability: Next to four individual siRNAs, also two complex siPOOLs as well as two antisense oligonucleotides confirmed the strong and specific phenotype. In summary, the extremely low abundant lncRNA VELUCT is essential for regulation of cell viability in several lung cancer cell lines. Hence, VELUCT is the first example for a lncRNA that is expressed at a very low level, but has a strong loss-of-function phenotype. Thus, our study proves that at least individual low-abundant lncRNAs can play an important functional role.

Apparent Quantum Efficiency Spectra for Superoxide Photoproduction and Its Formation of Hydrogen Peroxide in Natural Waters

Superoxide (O2−) is a key intermediate in the cycling of organic matter and trace metals in natural waters but production rates are difficult to determine due to low steady-state concentrations, rapid decay rates, and unstable standards. On the other hand, superoxide’s dismutation product, hydrogen peroxide (H2O2), is relatively stable in filtered water. Thus, if the stoichiometry between O2− and H2O2 is known, one can derive superoxide data from H2O2 measurements. The relationship between O2− and H2O2 remains uncertain in seawater but work by Petasne and Zika (1987) presented a method for examining the relationship between O2− and H2O2 during irradiations of coastal seawater using superoxide dismutase (SOD), which forces a 2:1 stoichiometry between O2− and H2O2. Here we report the first O2− apparent quantum yield (AQY) spectra following their approach; performing irradiations of various fresh and seawater samples and measuring H2O2 accumulation with and without added SOD. For all but a single riverine sample, H2O2 AQY spectra fell in a narrow range, but O2− AQY spectra varied such that O2−:H2O2 ratios were always greater than 2 and were highest for the clear waters of the Gulf Stream (~3.4 O2− per H2O2 generated). Because this approach eliminates the need to measure O2− production rates directly, it represents a simple way to refine the stoichiometric relationships that would potentially allow global estimates of O2− photoproduction rates, O2− steady-state concentrations ([O2−]ss), and related surface ocean redox reactions based on more manageable H2O2 photochemical studies.

Probe Compounds to Assess the Photochemical Activity of Dissolved Organic Matter.

The photochemical properties of dissolved organic matter (DOM) have been of interest to scientists and engineers since the 1970s. Upon light absorption, chromophoric DOM (CDOM) can sensitize the formation of different short-lived reactive intermediates (RIs), including hydroxyl radical (•OH), singlet oxygen (1O2) and superoxide radical anion (O2•-). In addition, a fraction of the excited singlet states in CDOM decays into excited triplet states (3CDOM*), which are also important photochemical transients in environmental systems. These RIs have a significant impact on different processes in sunlit waters, including degradation of organic contaminants and the inactivation of pathogens. Due to their transient nature and low steady-state concentrations, the use of common analytical techniques for the direct measurement of these species is impractical. Therefore, specific probe compounds (PCs) are used. PCs include furfuryl alcohol for 1O2, and terephthalic acid for •OH. In this publication, we present a critical review of the use of PCs for the assessment of the formation of photochemically generated RIs. We first introduce the concept of a PC, including the kinetic treatment and necessary assumptions needed to conduct a specific measurement. Afterward, we present short overviews of the most studied RIs and review relevant issues regarding the use of specific PCs for their measurement. We finalize by offering recommendations regarding the use of PCs in environmental photochemistry.

Acetaldehyde as an Intermediate in the Electroreduction of Carbon Monoxide to Ethanol on Oxide‐Derived Copper

AbstractOxide‐derived copper (OD‐Cu) electrodes exhibit unprecedented CO reduction performance towards liquid fuels, producing ethanol and acetate with &gt;50 % Faradaic efficiency at −0.3 V (vs. RHE). By using static headspace‐gas chromatography for liquid phase analysis, we identify acetaldehyde as a minor product and key intermediate in the electroreduction of CO to ethanol on OD‐Cu electrodes. Acetaldehyde is produced with a Faradaic efficiency of ≈5 % at −0.33 V (vs. RHE). We show that acetaldehyde forms at low steady‐state concentrations, and that free acetaldehyde is difficult to detect in alkaline solutions using NMR spectroscopy, requiring alternative methods for detection and quantification. Our results represent an important step towards understanding the CO reduction mechanism on OD‐Cu electrodes.