Lack Of Reactivity Research Articles

Transformation of chlorpyrifos and chlorpyrifos-methyl in prairie pothole pore waters.

Non-point source pesticide pollution is a concern for wetlands in the prairie pothole region (PPR). Recent studies have demonstrated that reduced sulfur species (e.g., bisulfide and polysulfides) in PPR wetland pore waters directly undergo reactions with chloroacetanilide and dinitroaniline compounds. In this paper, the abiotic transformation of two organophosphate compounds, chlorpyrifos and chlorpyrifos-methyl, was studied in PPR wetland pore waters. Chlorpyrifos-methyl reacted significantly faster (up to 4 times) in pore water with reduced sulfur species relative to hydrolysis. No rate enhancement was observed in the transformation of chlorpyrifos in pore water with reduced sulfur species. The lack of reactivity was most likely caused by steric hindrance from the ethyl groups and partitioning to dissolved organic matter (DOM), thereby shielding chlorpyrifos from nucleophilic attack. Significant decreases in reaction rates were observed for chlorpyrifos in pore water with high concentrations of DOM. Rate enhancement due to other reactive species (e.g., organo-sulfur compounds) in pore water was minor for both compounds relative to the influence of bisulfide and DOM.

Crystal structure and hydrogenation properties of Pd5As

The crystal structure of Pd5As was refined simultaneously from laboratory X-ray, synchrotron and neutron powder diffraction data (space group C2/m, a = 551.82(2) pm, b = 774.50(3) pm, c = 842.13(4), β = 99.037(2)°, Z = 4). This is in contrast to earlier work, which describes the structure in the non-centrosymmetric space group C2. Eight palladium atoms form a bicapped trigonal prism around arsenic. Two such polyhedra are connected by a common edge to double prisms (average distance As–Pd 252 pm). Palladium has got coordination numbers of 11 (average distance 276 pm) or 12 (average distance of 278 pm). The crystal structure of Pd5As represents a strongly distorted cubic closest packing as proven by crystallographic group–subgroup relationships. Pd5As does not take up hydrogen up to 5.0 MPa hydrogen pressure and temperatures up to 723 K. Quantum-mechanical calculations using DFT methods using an ab initio evolutionary algorithm confirm the crystal structure of Pd5As and reveal the stability of a hypothetical hydride Pd5AsH. The lack of reactivity might be due to kinetic hindrance because of an endothermic rearrangement of the metal matrix necessary for hydrogen incorporation. Above 5.8 GPa, however, this phase transition becomes thermodynamically favourable, making hydrogenation of Pd5As likely to occur.

A proof of the direct hole transfer in photocatalysis: The case of melamine

The photoinduced transformation of 2,4,6-triamino-1,3,5-triazine (melamine) was studied by using different advanced oxidation technologies under a variety of experimental conditions. The systems involving homogeneous hydroxyl radicals, as generated by H2O2/hν, Fenton reagent, and sonocatalysis are ineffective. However, melamine is degraded under photocatalytic conditions or by SO4− (S2O82−/hν). The time evolution of long-living intermediates, such as 2,4-diamino-6-hydroxy-1,3,5-triazine (ammeline) and 2-amino-4,6-dihydroxy-1,3,5-triazine (ammelide), has been followed, being 2,4,6-trihydroxy-1,3,5-triazine (cyanuric acid) the final stable product. During both photocatalytic and S2O82−/hν experiments, in the early steps, a fairly stable intermediate evolving to ammelide is observed in a large extent. This intermediate was identified as 2,4-diamino-6-nitro-1,3,5-triazine. This indicates that the primary photocatalytic event is the oxidation of the amino-group to nitro-group through several consecutive fast oxidation steps, and that a hydrolytic step leads to the release of nitrite in solution. To elucidate the nature of the oxidant species hole scavengers such as methanol and bromide ions were added to the irradiated TiO2. They completely stop the degradation, whereas chloride and fluoride ions decrease the degradation rate.The study of the photocatalytic degradation rate of melamine at increasing concentrations using two different commercial titanium dioxides, such as P25 and Merck TiO2, showed an intriguing behavior. A drastic abatement of the melamine transformation rate was observed when coagulation of the P25 slurry occurs due both to the pH change and melamine concentration effect that increase melamine adsorption. In the presence of TiO2 (Merck) the melamine initial degradation rates are significantly lower than those observed in the presence of P25 but are not depressed at larger concentrations. The experimental evidences (e.g., absence of melamine adsorption onto TiO2 surface at low concentrations or at acidic pH or due to the catalyst surface texture, and the lack of reactivity toward OH free and bound) suggest that the effective photocatalytic mechanism is based on an outer sphere direct hole transfer to the melamine. Its formal potential lies in the range 1.9–2.3V vs NHE. Then, the photodegradation of melamine is an efficient tool to evaluate the direct hole transfer ability of a photocatalyst.

What makes children outgrow food allergy?

What makes children outgrow food allergy?

A marine sink for chlorine in natural organic matter

Chloride is abundant in oceans, but is relatively unreactive. Spectroscopic imaging reveals the presence of a chloride sink in organochlorine compounds that can be produced abiotically or by phytoplankton. Chloride—the most abundant ion in sea water1—affects ocean salinity, and thereby seawater density and ocean circulation. Its lack of reactivity gives it an extremely long residence time2. Other halogens are known to be incorporated into marine organic matter3,4,5. However, evidence of similar transformations of seawater chloride is lacking, aside from emissions of volatile organochlorine by marine algae6,7,8. Here we report high organochlorine concentrations from 180 to 700 mg kg−1 in natural particulate organic matter that settled into sediment traps at depths between 800 and 3,200 m in the Arabian Sea, taken between 1994 and 1995. X-ray spectromicroscopic imaging of chlorine bonding reveals that this organochlorine exists primarily in concentrated aliphatic forms consistent with lipid chlorination, along with a more diffuse aromatic fraction. High aliphatic organochlorine in particulate material from cultured phytoplankton suggests that primary production is a source of chlorinated organic matter. We also found that particulate algal detritus can act as an organic substrate for abiotic reactions involving Fe2+, H2O2 or light that incorporate chlorine into organic matter at levels up to several grams per kilogram. We conclude that transformations of marine chloride to non-volatile organochlorine through biological and abiotic pathways represent an oceanic sink for this relatively unreactive element.

P131. Vibrotactile stimulation as a novel method to detect malingered attention or sensory deficits

Introduction To our knowledge there exist no objective methods for the clinical validation of reactions after tactile stimulation although the prevalence of symptom aggravation is high in compensation seeking context (18–40%, Axelrod et al., 2000; Binder, 1993; Mittenberg et al., 2002). In order to develop an electrophysiological method for symptom validation after vibrotactile stimulation we investigated the tactile stimulation of mechanical receptors of single sensory qualities with varied stimulation principles (mechanical pulse, vibration, coating, indentation, passive movement) and established a clinical routine. Methods In the present work LRP (lateralized readiness potentials) and NIRS (near-infrared spectroscopy) were recorded after vibrotactile stimulation of the thumb (60 Hz) in 40 healthy controls and 40 subjects with an episode of depression. Tactile SEP (somatosensory evoked potentials) were conducted to make sure that the stimuli were perceived by the subjects. In condition I the subjects were instructed to react to a vibrational stimulus with least possible delay by pushing a button, in condition II an additional time delay or a lack of reaction was asked to be executed. Stimulus- and response-locked LRP and activation patterns of the NIRS signal (Oxy-Hb and Deoxy-Hb) were compared statistically between both the conditions and the subject groups. Results In condition II the reaction-time was significantly longer and its dispersion was greater ( p p p p Conclusion If our results can be replicated in subjects with substantial brain lesions, a novel objective validation method for both sensory deficits and malingered attention deficits might result. As vibrotactile stimulators have only limited availability yet, EEG and surface myography are used as common techniques. If available, this novel method can be easily implemented to routine procedures.

Hydrazones as Productive Dienophiles in the Inverse Electron Demand Diels–Alder Reactions of 1,3,5‐Triazines

AbstractEnamines, ynamines and amidines are common dienophiles for inverse electron demand Diels–Alder (IEDDA) reactions of 1,3,5‐triazines, however, their wide utilization may be limited due to their poor stability to conventional purification methods such as silica gel chromatography. To address this limitation, oximes and hydrazones are envisioned as potential alternative dienophiles, even though they are primarily used as protecting groups due to their excellent stability or lack of reactivity. Through investigation of substituent effects and optimization of reaction conditions, oximes and hydrazones were developed as productive dienophiles for 1,3,5‐triazine IEDDA reactions, leading to the desired IEDDA products in moderate (for oximes) to excellent (for hydrazones) yields. Hydrazones are air‐stable and can be conveniently purified by conventional methods, therefore the introduction of hydrazones as productive dienophiles further expanded the scope of 1,3,5‐triazine IEDDA reactions.

Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest

Bothrops jararaca is a slender and semi-arboreal medically relevant pit viper species endemic to tropical and subtropical forests in southern Brazil, Paraguay, and northern Argentina (Misiones). Within its geographic range, it is often abundant and is an important cause of snakebite. Although no subspecies are currently recognized, geographic analyses have revealed the existence of two well-supported B. jararaca clades that diverged during the Pliocene ~3.8Mya and currently display a southeastern (SE) and a southern (S) Atlantic rainforest (Mata Atlântica) distribution. The spectrum, geographic variability, and ontogenetic changes of the venom proteomes of snakes from these two B. jararaca phylogroups were investigated applying a combined venom gland transcriptomic and venomic analysis. Comparisons of the venom proteomes and transcriptomes of B. jararaca from the SE and S geographic regions revealed notable interpopulational variability that may be due to the different levels of population-specific transcriptional regulation, including, in the case of the southern population, a marked ontogenetic venom compositional change involving the upregulation of the myotoxic PLA2 homolog, bothropstoxin-I. This population-specific marker can be used to estimate the proportion of venom from the southern population present in the B. jararaca venom pool used for the Brazilian soro antibotrópico (SAB) antivenom production. On the other hand, the southeastern population-specific D49-PLA2 molecules, BinTX-I and BinTX-II, lend support to the notion that the mainland ancestor of Bothrops insularis was originated within the same population that gave rise to the current SE B. jararaca phylogroup, and that this insular species endemic to Queimada Grande Island (Brazil) expresses a pedomorphic venom phenotype. Mirroring their compositional divergence, the two geographic B. jararaca venom pools showed distinct bioactivity profiles. However, the SAB antivenom manufactured in Vital Brazil Institute neutralized the lethal effect of both venoms to a similar extent. In addition, immobilized SAB antivenom immunocaptured most of the venom components of the venoms of both B. jararaca populations, but did not show immunoreactivity against vasoactive peptides. The Costa Rican bothropic–crotalic–lachesic (BCL) antivenom showed the same lack of reactivity against vasoactive peptides but, in addition, was less efficient immunocapturing PI- and PIII-SVMPs from the SE venom, and bothropstoxin-I, a CRISP molecule, and a D49-PLA2 from the venom of the southern B. jararaca phylogroup. The remarkable paraspecificity exhibited by the Brazilian and the Costa Rican antivenoms indicates large immunoreactive epitope conservation across the natural history of Bothrops, a genus that has its roots in the middle Miocene.This article is part of a Special Issue entitled: Omics Evolutionary Ecolog.

Acute brain failure in severe sepsis: a prospective study in the medical intensive care unit utilizing continuous EEG monitoring.

Investigate the prevalence, risk factors and impact of continuous EEG (cEEG) abnormalities on mortality through the 1-year follow-up period in patients with severe sepsis. Prospective, single-center, observational study of consecutive patients admitted with severesepsis to the Medical ICU at an academic medical center. A total of 98 patients with 100 episodes of severe sepsis were included; 49 patients (50%) were female, median age was 60 (IQR 52-74), the median non-neuro APACHE II score was 23.5 (IQR 18-28) and median non-neuro SOFA score was 8 (IQR 6-11). Twenty-five episodes had periodic discharges (PD), of which 11 had nonconvulsive seizures (NCS). No patient had NCS without PD. Prior neurological history was associated with a higher risk of PD or NCS (45 vs. 17%; CI 1.53-10.43), while the non-neuro APACHE II, non-neuro SOFA, severity of cardiovascular shock and presence of sedation during cEEG were associated with a lower risk of PD or NCS. Clinical seizures before cEEG were associated with a higher risk of nonconvulsive status epilepticus (24 vs. 6%; CI 1.42-19.94) while the non-neuro APACHE II and non-neuro SOFA scores were associated with a lower risk. Lack of EEG reactivity was present in 28% of episodes. In the survival analysis, a lack of EEG reactivity was associated with higher 1-year mortality [mean survival time 3.3 (95% CI 1.8-4.9) vs. 7.5 (6.4-8.7) months; p=0.002] but the presence of PD or NCS was not [mean survival time 3.3 (95% CI 1.8-4.9) vs. 7.5 (6.4-8.7) months; p=0.592]. Lack of reactivity was more frequent in patients on continuous sedation during cEEG. In patients with available 1-year data (34% of the episodes), 82% had good functional outcome (mRS≤3, n=27). There were no significant predictors of functional outcome, late cognition, and no patient with complete follow-up data developed late seizure or new epilepsy. NCS and PD are common in patients with severe sepsis and altered mental status. They were less frequent among the most severely sick patients and were not associated with outcome in this study. Lack of EEG reactivity was more frequent in patients on continuous sedation and was associated with mortality up to 1year after discharge. Larger studies are needed to confirm these findings in a broader population and to further evaluate long-term cognitive outcome, risk of late seizure and epilepsy.

Photoremoval of Protecting Groups: Mechanistic Aspects of 1,3-Dithiane Conversion to a Carbonyl Group

Photodeprotection of 1,3-dithianes in the presence of thiapyrylium was performed to return to the parent carbonyl compound, and the mechanism was studied by steady state photolysis, laser flash photolysis, and theoretical calculations. Electron transfer from dithianes to triplet sensitizers is extremely fast, and the decay of dithiane radical cations was not affected by the presence of water or oxygen as the consequence of a favorable unimolecular fragmentation pathway. Similar behaviors were observed for dithianes bearing electron-releasing or electron-withdrawing substituents on the aryl moiety, evidenced by C-S bond cleavage to form a distonic radical cation species. The lack of reaction under nitrogen atmosphere, requirement of oxygen for good conversion yields, inhibition of the photodeprotection process by the presence of p-benzoquinone, and absence of a labeled carbonyl final product when the reaction is performed in the presence of H2(18)O all suggest that the superoxide anion drives the deprotection reaction. Density functional theory computational studies on the reactions with water, molecular oxygen, and the superoxide radical anion support the experimental findings.

Retrospective Air Quality Simulations of the TexAQS-II: Focused on Emissions Uncertainty

There are several studies on the effects of emissions of highly reactive volatile organic compounds (HRVOC) from the industrial sources in the Houston-Galveston-Brazoria (HGB) area on the high ozone events during the Texas Air Quality Study (TexAQS) in summer of 2000. They showed that the modeled atmosphere lacked reactivity to produce the observed high ozone event and suggested “imputation” of HRVOC emissions from the base inventory. Byun et al. (2007b) showed the imputed inventory leads to too high ethylene concentrations compared to the measurements at the chemical super sites but still too little aloft compared to the NOAA aircraft. The paper suggested that the lack of reactivity in the modeled Houston atmosphere must be corrected by targeted, and sometimes of episodic, increase of HRV OC emissions from the large sources such as flares in the Houston Ship Channel (HSC) distributed into the deeper level of the boundary layer. We performed retrospective meteorological and air quality modeling to achieve better air quality prediction of ozone by comparison with various chemical and meteorological measurements during the Texas Air Quality Study periods in August-September 2006 (TexA QS-Ⅱ). After identifying several shortcomings of the forecast meteorological simulations and emissions inputs, we prepared new retrospective meteorological simulations and updated emissions inputs. We utilized assimilated MM5 inputs to achieve better meteorological simulations (detailed description of MM5 assimilation can be found in F. Ngan et al., 2012) and used them in this study for air quality simulations. Using the better predicted meteorological results, we focused on the emissions uncertainty in order to capture high peak ozone which occasionally happens in the HGB area. We described how the ozone predictions are affected by emissions uncertainty in the air quality simulations utilizing different emission inventories and adjustments.

Gas-phase reactions of CF(+) with molecules of interstellar relevance.

We have studied the gas-phase reactions of CF(+) with 24 neutral species. Reaction rate constants and product branching fractions are measured at 298 K using a flowing afterglow-selected ion flow tube. Experimental work is supported by computational chemistry calculations to provide insight into the reactivity of classes of neutral molecules. Reactions of CF(+) with small triatomic species and oxygen-containing organic molecules produce the stable molecule CO. The product branching fractions are discussed, and the potential energy surfaces for a few representative reactions are examined. CF(+) is highly reactive with complex molecules and will likely be destroyed in dense environments in the interstellar medium. However, the lack of reactivity with small diatomic molecules will likely enable its survival in diffuse regions.

A multipath energy-conserving routing protocol for wireless ad hoc networks lifetime improvement

Ad hoc networks are wireless mobile networks that can operate without infrastructure and without centralized network management. Traditional techniques of routing are not well adapted. Indeed, their lack of reactivity with respect to the variability of network changes makes them difficult to use. Moreover, conserving energy is a critical concern in the design of routing protocols for ad hoc networks, because most mobile nodes operate with limited battery capacity, and the energy depletion of a node affects not only the node itself but also the overall network lifetime. In all proposed single-path routing schemes a new path-discovery process is required once a path failure is detected, and this process causes delay and wastage of node resources. A multipath routing scheme is an alternative to maximize the network lifetime. In this paper, we propose an energy-efficient multipath routing protocol, called AOMR-LM (Ad hoc On-demand Multipath Routing with Lifetime Maximization), which preserves the residual energy of nodes and balances the consumed energy to increase the network lifetime. To achieve this goal, we used the residual energy of nodes for calculating the node energy level. The multipath selection mechanism uses this energy level to classify the paths. Two parameters are analyzed: the energy threshold beta and the coefficient alpha. These parameters are required to classify the nodes and to ensure the preservation of node energy. Our protocol improves the performance of mobile ad hoc networks by prolonging the lifetime of the network. This novel protocol has been compared with other protocols: AOMDV and ZD-AOMDV. The protocol performance has been evaluated in terms of network lifetime, energy consumption, and end-to-end delay.

Effects of field metal-contaminated soils submitted to phytostabilisation and fly ash-aided phytostabilisation on the avoidance behaviour of the earthworm Eisenia fetida

The earthworm Eisenia fetida avoidance behaviour test was used to assess the quality recovery of metal-contaminated soils from lands submitted for 10 years to remediation. Soils were from plots located in the surroundings of a former lead smelter plant of Northern France. Metal concentrations in the soils ranged from 93 to 1231, 56 to 1424, 0.3 to 20 and 15 to 45.5mg metal/kg dry soil for Pb, Zn, Cd and Cu, respectively. Several former agricultural plots were treated either by a single phytostabilisation process involving the plantation of a tree mix or by fly ash aided-phytostabilisation. Silico-aluminous or sulfo-calcic ashes used were ploughed up to a 25- to 30-cm soil depth at a rate of 23.3kg/m2 (i.e., 6 percent W/W). E. fetida was shown to avoid significantly the 10 years ash-treated soils whose habitat function has to be considered as limited. This avoidance would relate to a change of the texture of soils induced by the addition of ashes and consisting in an increased level of fine silts together with a decreased level of clays. By contrast, afforested metal-contaminated soils appeared for E. fetida as more attractive than unplanted ones. Regarding the influence of the metal contamination of the soils on E. fetida, none of the soils tested even the highest contaminated one was significantly avoided by worms. This lack of reaction would result from the low bioavailability of the metals in the soils tested. At the lights of our results and those previously published on both these ashes and these ash-treated soils, the usefulness of these soil treatments is discussed.

Energy Conservation for Ad Hoc On-Demand Distance Vector Multipath Routing Protocol

Routing in ad hoc mobile networks is a problem which has not yet been satisfactorily solved. Traditional routing techniques are not well adapted to new networks. Indeed, their lack of reactivity with respect to the traffic and network changes means traditional routing techniques cannot easily be used except at the price of over-dimensioning of the network resources (network bandwidth, node memory utilization, node CPU load, etc.). In recent years the research community has been interested in the improvement of ad hoc routing, and among the solutions suggested multipath routing has been considered. Multiple paths are exploited in order to ensure reliability and a quick reaction to changes in topology with a low overhead generated by the control messages. In this article we present an extension of the well-known routing protocol AODVM (Ad hoc On-demand Distance Vector Multipath). We propose to improve the multipath routing strategy with a path classification to allow the paths with the best energy level to be chosen.

Contribution of ATP-Sensitive K+ Channels to Contraction of Rat Tail Artery is Significantly Reduced in Males but Not Females Rats in Streptozotocin-Induced Diabetes: Pharmacological Analysis

The aim of this article is to find out possible gender differences of participation of ATP-sensitive potassium channels in regulation of contractility in vascular smooth muscles of rats with diabetes. We used isolated tail artery of both genders rats with streptazotocin-induced diabetes (SID). After 3 weeks, we compared concentration-effects curves obtained with phenylephrine (0.1 to 300 μM) in the presence of 100 μM of diazoxid in control and SID rats of both genders. Diazoxide at 100 μM concentration significantly antagonizes concentration-dependent contraction of tail artery induced by phenylephrine in both male and female control rats. However, this antagonism disappeared in male but not female rats treated by streptozotocin. Our results indicate that SID leads to downregulation or functional malformation (lack of reaction to the opener) of ATP-sensitive potassium channels in males, but not in females rats. This observation has to be confirmed by immunochemistry analysis and taking into account in humans.

Convenient and Practical Alkynylation of Heteronucleophiles with Copper Acetylides

Copper acetylides, readily available reagents which are characterized by their lack of reactivity, can be simply activated by oxidation with oxygen in the presence of simple nitrogen ligands such as TMEDA or imidazole derivatives. Upon activation, these nucleophilic species undergo a formal umpolung and can transfer their alkyne subunit to a wide range of heteronucleophiles, including amides, oxazolidinones, imines, and dialkyl phosphites. This alkynylation, which provides one of the most practical entry to useful building blocks such as ynamides, ynimines, and alkynylphosphonates, proceeds under especially mild conditions and can be easily performed on a multigram scale.

Radical Reactivity of the Fe(III)/(II) Tetramesitylporphyrin Couple: Hydrogen Atom Transfer, Oxyl Radical Dissociation, and Catalytic Disproportionation of a Hydroxylamine.

The chemistry of low-valent iron porphyrin complexes with oxyl radical reagents has been explored. (Meso-tetramesityl porphyrinato) iron(III) hydroxide, (TMP)FeIII(OH) reacts with the hydroxylamine TEMPO-H (1-hydroxy-2,2,6,6-tetramethylpiperdine) to yield the ferrous porphyrin, (TMP)FeII, together with H2O and TEMPO. This reaction has a second order rate constant k1 = 76 ± 5 M-1 1 s-1 and likely occurs by concerted e-/H+ transfer. Hydrazines PhNHNHPh and PhNHNH2 similarly yield (TMP)FeII. A subsequent reaction between TEMPO (2,2,6,6-tetramethylpiperdinyl radical) and (TMP)FeII is observed to reversibly form the TEMPO-ligated ferric porphyrin, (TMP)FeIII(TEMPO). A combination of 1H NMR and optical spectroscopies were used to determine the thermodynamic parameters for TEMPO binding: K4 (25°C) = 535 ± 20 M-1, ΔH°4 = -7.0 ± 1.5 kcal mol-1, ΔS°4= -11 ± 5 cal mol-1 K-1, ΔG‡4(235K) = 21.3 ± 0.5 kcal mol-1, ΔG‡-4(235K) = 16.9 ± 0.5 kcal mol-1. The Fe-O bond is remarkably weak. The stable phenoxyl radical 2,4,6- t Bu3C6H2O• (ArO•) forms a stronger bond to (TMP)FeII to irreversibly make a similar FeIII(OR) complex. Both (TMP)FeII and (TMP)FeIII(OH) are catalysts for the disproportionation of excess TEMPO-H to TEMPO and TEMP-H (2,2,6,6-tetramethylpiperdine). The lack of reactivity between (TMP)FeII and the alkylated TEMPO-H analogue, TEMPO-CH3, suggests that the disproportionation involves a hydrogen atom transfer step. These results highlight the importance and versatility of the heme FeIII/II couple that is often overshadowed by its higher-valent counterparts.

Turning unreactive copper acetylides into remarkably powerful and mild alkyne transfer reagents by oxidative umpolung.

This is not breaking news: copper acetylides, readily available polymeric rock-stable solids, have been known for more than a century to be unreactive species and piteous nucleophiles. This lack of reactivity actually makes them ideal alkyne transfer reagents that can be easily activated under mild oxidizing conditions. When treated with molecular oxygen in the presence of simple chelating nitrogen ligands such as TMEDA, phenanthroline or imidazole derivatives, they are smoothly oxidized to highly electrophilic species that formally behave like acetylenic carbocations and can therefore be used for the mild and practical alkynylation of a wide range of nitrogen, phosphorus and carbon nucleophiles.

Sensitive and Specific Antibody Probes Directed Against The Erythropoietin Receptor – From Basic Studies To Clinical Implementation

Recombinant erythropoietin (Epo) is an effective anti-anemic agent in most cancer patients and improves their quality of life. Yet, concerns of disease progression and reduced survival in recombinant human Epo (rhuEpo)-treated patients have been raised by Phase III clinical trial data showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Epo has pleiotropic actions and its receptor, EpoR, is expressed by many different cell types outside the erythroid compartment. It was thus proposed that a major possible mechanism for this potentially harmful effect of Epo in cancer patients is the activation of EpoRs on cancer cells. The original clinical studies have been criticized because they deployed polyclonal antibodies later shown to lack specificity for EpoR.Furthermore, multiple isoforms of EpoR caused by differential splicing have been reported, but only at the RNA level, in different cancer cell lines. Investigations into whether these spliced versions actually result in abnormal EpoR forms at the protein level, alter Epo responsiveness and have an impact on tumor progression in vivo, have been hampered by a lack of well characterized monoclonal antibodies.The ‘EpoCan' Consortium, funded by the EU, is directed to promote improved pathological testing of EpoR in patient samples, leading to safer clinical use of rHuEpo and other Erythropoietic Stimulating Agents (ESAs). To date, 25 murine and rat monoclonal antibodies have been produced against the EpoR, using novel genetic and traditional peptide immunization protocols. Of these antibodies, several were found to specifically recognize the receptor in various assays including Western blot (WB), immunoprecipitation (IP), immunofluorescence (IF), flow cytometry (FACS) and immunohistochemistry (IHC). Table 1 lists the antibodies that were selected for further analysis, and their experimental applications. The antibodies were tested on EpoR transfected cells (HEK 293 cells and COS cells) and Epo-dependent UT7 cells which endogenously express EpoR. In addition the antibodies were tested on non-erythroid cells viz. the non-small cell lung carcinoma A549, and breast cancer MDA-MB 231 cell lines. Specificity of the antibodies towards the EpoR in these two latter cell lines was ensured by the lack of reactivity with the corresponding EpoR silenced cells.Table 1Anti EpoR Antibodies - immunizing antigens and applicationsImmunogenSubclone nameSystematic nameIsotypeEpitope locationApplicationsPeptideBCO-3H2-D3GM1201rIgG2bhEpoR cytoplasmic domainWB, IP, IF, IHCPeptideBCO-4B5-C9GM1202rIgG2ah/mEpoR cytoplasmic domainWB, IF, IHCDNAVP-2E8-B6GM1204mIgG1hEpoR extracellular domainIP, IF, FACSDNAVP-4D8-C4GM1205mIgG1hEpoR extracellular domainIP, IF, FACSDNABBQ-9C4-D8GM1206rIgG2ah/mEpoR extracellular domainFACSDNABBQ-10E10-F2GM1207rIgG2ah/mEpoR extracellular domainWBImmunohistochemical analysis was performed on a panel of human non-small cell lung carcinoma sections. Two rat monoclonal antibodies, BCO-3H2-D3 which recognizes full length EpoR and BCO-4B5-C9 which recognizes full length and truncated isoforms, have proved valuable in comparative immunohistochemical and Western blot studies in a range of human tumors and cell lines. These well characterized monoclonal antibodies will enable a careful dissection of EpoR function in cancer cells and the detection of EpoR isoforms in tumor tissue.In the longer term such studies should allow clinicians to balance the benefits and risks of ESA treatment in cancer.THIS WORK WAS SUPPORTED BY THE FP7-HEALTH EUROPEAN COMMISSION EPOCAN GRANT (282551). Disclosures:Grunert:Aldevron Freiburg GmBH: Employment. Thompson:Aldevron Freiburg GmbH: Employment.