eIF5A inhibition influences T cell dynamics in the pancreatic microenvironment of the humanized mouse model of Type 1 Diabetes.

We have developed a transgenic mouse model of Type 1 Diabetes (T1D) in which human GAD65 is expressed in pancreatic ß-cells, and human MHC-II is expressed on antigen presenting cells. Induced GAD65 antigen presentation activates T-cells, which initiates the downstream events leading to diabetes. In our humanized mice, we have shown downregulation of eukaryotic translation initiation factor 5 A (elF5A), expressed only in actively dividing mammalian cells. In-vivo inhibition of elF5A hypusination by deoxyhypusine synthase (DHS) inhibitor "GC7" was studied; DHS inhibitor alters the pathophysiology in our mouse model by catalyzing the crucial hypusination and the rate-limiting step of elF5A activation. In our mouse model, we have shown that inhibition of eIF5A resets the pro-inflammatory bias in the pancreatic microenvironment. There was: (a) reduction of Th1/Th17 response, (b) an increase in Treg numbers, (c) debase in IL17 and IL21 cytokines levels in serum, (d) lowering of anti-GAD65 antibodies, and (e) ablation of the ER stress that improved functionality of the ß-cells, but minimal effect on the cytotoxic CD8 T-cell (CTL) mediated response. Conclusively, immune modulation, in the case of T1D, may help to manipulate inflammatory responses, decreasing disease severity, and may help manage T1D in early stages of disease. Our study also demonstrates that without manipulating the CTLs mediated response extensively, it is difficult to treat T1D.

Single ingestion of di-(2-propylheptyl) phthalate (DPHP) by male volunteers: DPHP in blood and its metabolites in blood and urine.

Di-(2-propylheptyl) phthalate (DPHP) is used as a plasticizer for polyvinyl chloride products. A tolerable daily intake of DPHP of 0.2 mg/kg body weight has been derived from rat data. Because toxicokinetic data of DPHP in humans were not available, it was the aim of the present work to monitor DPHP and selected metabolites in blood and urine of 6 male volunteers over time following ingestion of a single DPHP dose (0.7 mg/kg body weight). Concentration-time courses in blood were obtained up to 24 h for DPHP, mono-(2-propylheptyl) phthalate (MPHP), mono-(2-propyl-6-hydroxyheptyl) phthalate (OH-MPHP), and mono-(2-propyl-6-oxoheptyl) phthalate (oxo-MPHP); amounts excreted in urine were determined up to 46 h for MPHP, OH-MPHP, oxo-MPHP, and mono-(2-propyl-6-carboxyhexyl) phthalate (cx-MPHP). All curves were characterized by an invasion and an elimination phase the kinetic parameters of which were determined together with the areas under the concentration-time curves in blood (AUCs). AUCs were: DPHP > MPHP > oxo-MPHP > OH-MPHP. The amounts excreted in urine were: oxo-MPHP > OH-MPHP> > cx-MPHP > MPHP. The AUCs of MPHP, oxo-MPHP, or OH-MPHP could be estimated well from the cumulative amounts of urinary OH-MPHP or oxo-MPHP excreted within 22 h after DPHP intake. Not considering possible differences in species-sensitivity towards unconjugated DPHP metabolites, it was concluded from a comparison of their AUCs in DPHP-exposed humans with corresponding earlier data in rats that there is no increased risk of adverse effects associated with the internal exposure of unconjugated DPHP metabolites in humans as compared to rats when receiving the same dose of DPHP per kg body weight.

Novel, Selective, and Developable Dopamine D3 Antagonists with a Modified "Amino" Region.

This Minireview describes a presentation made at the XXIV National Meeting in Medicinal Chemistry (NMMC) held in Perugia (Italy), September 11-14, 2016. It relates to the discovery of novel templates of the so-called "amino" region of dopamine D3 receptor antagonists. Moving from the early scaffolds, which were modified in the amine portion, this review discusses the variations that led to the discovery of new systems published in 2016, which allowed the identification of compounds endowed with great selectivity over the dopamine D2 receptor and the human ether-à-go-go-related gene (hERG) ion channel. The main efforts in characterizing these compounds were devoted not only to determining their potency and selectivity relative to closely associated targets (e.g., the dopamine D2 receptor), but to ensure a large therapeutic window versus liability points such as hERG. In particular, we present examples of derivatives with selectivities greater than 2000-fold. Furthermore, much focus is devoted to the overall developability of the scaffolds, ensuring that appropriate physicochemical and pharmacokinetic parameters are present in all compounds progressing through the screening cascade.

Effect of Confinement on the Properties of Sequestered Mixed Polar Solvents: Enzymatic Catalysis in Nonaqueous 1,4-Bis-2-ethylhexylsulfosuccinate Reverse Micelles.

The influence of different glycerol, N,N-dimethylformamide (DMF) and water mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane reverse micelles (RMs) on the enzymatic hydrolysis of 2-naphthyl acetate by α-chymotrypsin is demonstrated. In the case of the mixtures with DMF and protic solvents it has been previously shown, using absorption, emission and dynamic light-scattering techniques, that solvents are segregated inside the polar core of the RMs. Protic solvents anchor to the AOT, whereas DMF locates to the polar core of the aggregate. Thus, DMF not only helps to solubilize the hydrophobic substrate, increasing its effective concentrations but surprisingly, it does not affect the enzyme activity. The importance of ensuring the presence of RMs, encapsulation of the polar solvents and the corrections by substrate partitioning in order to obtain reliable conclusions is highlighted. Moreover, the effect of a constrained environment on solvent-solvent interactions in homogenous media and its impact on the use of RMs as nanoreactors is stressed.

Volatile organic compounds generated by cultures of bacteria and viruses associated with respiratory infections.

Respiratory infections (RI) can be viral or bacterial in origin. In either case, the invasion of the pathogen results in production and release of various volatile organic compounds (VOCs). The present study examines the VOCs released from cultures of five viruses (influenza A, influenza B, adenovirus, respiratory syncitial virus and parainfluenza 1 virus), three bacteria (Moraxella catarrhalis, Haemophilus influenzae and Legionella pneumophila) and Mycoplasma pneumoniae isolated colonies. Our results demonstrate the involvement of inflammation-induced VOCs. Two significant VOCs were identified as associated with infectious bacterial activity, heptane and methylcyclohexane. These two VOCs have been linked in previous studies to oxidative stress effects. In order to distinguish between bacterial and viral positive cultures, we performed principal component analysis including peak identity (retention time) and VOC concentration (i.e. area under the peak) revealing 1-hexanol and 1-heptadecene to be good predictors.

Whole-cell double oxidation of n-heptane.

Biocascades allow one-pot synthesis of chemical building blocks omitting purification of reaction intermediates and expenses for downstream processing. Here we show the first whole cell double oxidation of n-heptane to produce chiral alcohols and heptanones. The concept of an artificial operon for co-expression of a monooxygenase from Bacillus megaterium (P450 BM3) and an alcohol dehydrogenase (RE-ADH) from Rhodococcus erythropolis is reported and compared to the widely used two-plasmid or Duet-vector expression systems. Both catalysts are co-expressed on a polycistronic constructs (single mRNA) that reduces recombinant DNA content and metabolic burden for the host cell, therefore increasing growth rate and expression level. Using the artificial operon system, the expression of P450 BM3 reached 81mgg(-1) cell dry weight. In addition, in situ cofactor regeneration through the P450 BM3/RE-ADH couple was enhanced by coupling to glucose oxidation by E. coli. Under optimized reaction conditions the artificial operon system displayed a product formation of 656mgL(-1) (5.7mM) of reaction products (heptanols+heptanones), which is 3-fold higher than the previously reported values for an in vitro oxidation cascade. In conjunction with the high product concentrations it was possible to obtain ee values of >99% for (S)-3-heptanol. Coexpression of a third alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) in the same host yielded complete oxidation of all heptanol isomers. Introduction of a second ADH enabled further to utilize both cofactors in the host cell (NADH and NADPH) which illustrates the simplicity and modular character of the whole cell oxidation concept employing an artificial operon system.

Effect of confinement on excited-state proton transfer of firefly's chromophore D-luciferin in AOT reverse micelles.

Excited-state intermolecular proton transfer of D-luciferin in reverse micelles has been investigated using steady-state and time-resolved fluorescence spectroscopy measurement. The different polar cores have been chosen for the study of proton transfer dynamics in aerosol-OT (AOT) reverse micelles. It is shown that aqueous reverse micelle is the suitable environment for the photoprotolytic reaction of D-luciferin. The neutral form of the chromophore is present both in ground and excited state at W0 = 0. The proton transfer in nanometer size water pool of water/AOT/n-heptane begins at W0 = 8 and increases with increasing W0 values. However, the intermolecular excited-state proton transfer (ESPT) of D-luciferin is inhibited in nonaquous reverse micelles with DMF and DMSO as a polar core. Thus, the requirement of ESPT of D-luciferin to take place in reverse micelles consists of polar protic solvent like water as a polar core.

Chemoenzymatic synthesis and evaluation of 3-azabicyclo[3.2.0]heptane derivatives as dopaminergic ligands.

New 3-azabicyclo[3.2.0]heptane derivatives were synthesized using a multicomponent reaction. Racemic compounds were efficiently resolved by kinetic resolution with immobilized lipase B of Candida antarctica (Novozym 435). The obtained compounds demonstrated greater binding affinity at D(2L) and D(3) dopamine receptors compared to D(1) binding sites, and individual enantiomers of the same compound possessed distinct affinities.

PET Imaging of nicotinic acetylcholine receptors in baboons with 18F-AZAN, a radioligand with improved brain kinetics.

UNLABELLED: There are only 2 currently available radioligands, 2-(18)F-FA and 6-(18)F-FA, for quantitative PET of the main cerebral subtype of nicotinic acetylcholine receptors (α4ß2-nAChRs) in humans. Both exhibit slow distribution kinetics in the brain and require several hours for PET imaging. This makes PET of nAChRs with these radioligands logistically difficult and a serious burden for human subjects. The main purpose of this study was to preclinically evaluate (-)-2-(6-(18)F-fluoro-2,3'-bipyridin-5'-yl)-7-methyl-7-azabicyclo[2.2.1]heptane ((18)F-AZAN), our new radiolabeled antagonist of α4ß2-nAChRs, that has high binding potential and rapid brain kinetics in baboons. METHODS: (18)F-AZAN was synthesized using a modified (18)F-FDG synthesis module. The regional distribution of (18)F-AZAN in the brain was evaluated in baseline and cytisine-blocking studies of 4 male Papio anubis baboons. PET modeling procedures were used for calculation of regional distribution volume (V(T)), nondisplaceable binding potential (BP(ND)), and receptor occupancy. RESULTS: (18)F-AZAN rapidly entered the baboon brain, reached a steady state within 90 min after injection, and specifically labeled cerebral nAChRs. The peak radioactivity in the thalamus was 540 (percentage standardized uptake value) at 18 ± 7 min (n = 4) after bolus injection. Mathematical data analysis demonstrated that scanning for only 90 min is sufficient for determination of PET outcome variables (BP(ND), 3.2 [unitless] and V(T), 32-35 mL/mL in thalamus). The dose-dependent blocking experiments with cytisine demonstrated that (18)F-AZAN binds specifically with ß2-containing (predominantly α4ß2) nAChRs. CONCLUSION: (18)F-AZAN specifically labels nAChRs in baboon brains with a high value of BP(ND) and it requires only 90 min of PET scanning to produce estimates of V(T) and BP(ND) in the various brain regions. The blocking of nAChRs with cytisine is dose-dependent and it showed that (18)F-AZAN is suitable for application in nicotinic drug evaluation. In summary, (18)F-AZAN is superior to 2-(18)F-FA and 6-(18)F-FA for imaging cerebral ß2-containing nAChRs in baboons. Further evaluations of (18)F-AZAN in the human brain are under way.

Application of headspace solid phase dynamic extraction gas chromatography/mass spectrometry (HS-SPDE-GC/MS) for biomonitoring of n-heptane and its metabolites in blood.

Solid phase dynamic extraction (SPDE) is an innovative sample preparation and enrichment technique in connection with gas chromatography (GC). Using SPDE, we developed a method for simultaneous determination of n-heptane and its mono-oxygenated metabolites heptane-4-one, 3-one, 2-one, 4-ol, 3-ol, 2-ol, and 1-ol in blood. After adjustment of various extraction and desorption parameters, method validation resulted in limits of detection (LOD) between 0.006 (heptane-4-one) and 0.021mg/L (heptane-1-ol). Intra-assay coefficients of variation ranged between 4.8% and 20.8% while relative recovery ranged between 100% and 117% (spiked concentration 0.128mg/L, n=8). The method was applied to blood samples, which have been collected from 20 volunteers after controlled inhalative exposure to 167, 333, and 500ppm n-heptane. After 3h of exposure, n-heptane and heptane-2-one were detectable in all samples in concentrations ranging up to 2.903 and 0.495mg/L, while the concentrations of the remaining analytes were closer to the respective LOD or even below. A significant linear relationship with ambient exposure (R(2)=0.701, p<0.001, n=55) was found for n-heptane in blood, which could be helpful for evaluation of biological exposure limits in future. Due to its high abundance in blood, 2-heptanone could be an interesting candidate as a biomarker also in alternative matrices such as urine or saliva.

Removal of saturated aliphatic hydrocarbons (gasoline components) from air via bacterial biofiltration.

Two-stage biofilters (using perlite and granular activated carbon, GAC, as packing materials) were used for the removal of several linear, branched, and cyclic C(5)-C(8)saturated aliphatic hydrocarbons from air, both as individual chemicals and in mixtures. The acclimation of biofilters from styrene to n-heptane was complete in 14-18 days. The substrate switch resulted in significant changes in pH and microbial composition of biofilters. Subsequent experiments were conducted under steady state conditions at a constant EBRT of 123 s and near-neutral pH, assuring the predominantly bacterial (as opposed to fungal) biofilter population. n-Heptane was removed with consistently high, 87-100%, removal efficiencies (RE) for up to 16 g x m(-3) x h(-1) critical substrate loads in the perlite biofilter, while n-hexane and n-pentane exhibited significantly lower RE under similar conditions. The REs for iso-octane and cyclohexane were less than 10% under similar loads; n-heptane biodegradation was consistently ca. 10% lower in the presence of iso-octane than in its absence. The GAC biofilter showed a significantly lower efficiency than the perlite biofilter (the critical load, yielding RE > 90%, was only 5 g x m(-3) x h(-1) for n-heptane). Evidence obtained indicates that the rate limiting step for mixed culture biofiltration of aliphatic hydrocarbon mixtures is biodegradation rather than mass transfer.

Lipase-catalyzed synthesis of isoamyl acetate in an ionic liquid/n-heptane two-phase system at the microreactor scale.

A continuously operated psi-shaped microreactor was used for lipase-catalyzed synthesis of isoamyl acetate in the 1-butyl-3-methylpyridinium dicyanamide/n-heptane two-phase system. The chosen solvent system with dissolved Candida antarctica lipase B, which was attached to the ionic liquid/n-heptane interfacial area due to its amphiphilic properties, was shown to be highly efficient and enabled simultaneous esterification and product removal. At preliminarily selected conditions regarding the type of acyl donor, its molar ratio to alcohol and enzyme concentration, 48.4 g m(-3) s(-1) of isoamyl acetate was produced, which was almost three-fold better as compared to the intensely mixed batch process. This was mainly a consequence of efficient reaction-diffusion dynamics in the microchannel system, where the developed flow pattern comprising of intense emulsification provided a large interfacial area for the reaction and simultaneous product extraction.

Multiplicity of n-heptane oxidation pathways catalyzed by cytochrome P450.

Modeling, mutagenesis, and kinetic studies have demonstrated that the substrate-binding site of cytochrome P450 is composed of multiple interactive regions that are capable of simultaneously binding two or more xenobiotics. Substrate molecules can interact with each other after docking. Thus, substrates can compete for the activated oxygen-ferrous complex or alter the spatial orientation of other molecules. Cytochrome P450 is a unique enzyme that produces n-heptane metabolites of different oxidation states. Metabolism of n-heptane was investigated with rat liver microsomes and a reconstituted rat liver system. Ethanol, n-propanol, and n-butanol molecules interacted with the n-heptane molecule and resulted in cytochrome P450 spectral changes as well as alterations in the n-heptane metabolic profile. The observed modifications in the biotransformation of n-heptane indicated that there are three distinct pathways for oxidation of n-heptane to heptanols, heptanones, and one-side oxygen-oriented heptanediones.

Enzymatic synthesis of isopropyl myristate using immobilized lipase from Bacillus cereus MTCC 8372.

A purified alkaline thermo-tolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a Poly (MAc-co-DMA-cl-MBAm) hydrogel. The hydrogel showed approximately 94% binding capacity for lipase. The immobilized lipase (2.36 IU) was used to achieve esterification ofmyristic acid and isopropanol in n-heptane at 65 degrees C under continuous shaking. The myristic acid and isopropanol when used at a concentration of 100 mM each in n-heptane resulted in formation of isopropyl myristate (66.0 +/- 0.3 mM) in 15 h. The reaction temperature below or higher than 65 degrees C markedly reduced the formation of isopropyl myristate. Addition of a molecular sieve (3 A x 1.5 mm) to the reaction mixture drastically reduced the ester formation. The hydrogel bound lipase when repetitively used to perform esterification under optimized conditions resulted in 38.0 +/- 0.2 mM isopropyl myristate after the 3rd cycle of esterification.

Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and l-alpha-threofuranosyl ring systems: interactions with P2Y receptors.

The ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y(1) receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y(2) and P2Y(4) receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) l-alpha-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y(1) receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a K(i) value of 22.5 nM in binding to the human P2Y(1) receptor, and antagonized the stimulation of PLC by the potent P2Y(1) receptor agonist 2-methylthio-ADP (30 nM) with an IC(50) of 650 nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A(3)). Thus, this ring system afforded some P2Y receptor selectivity. A l-alpha-threofuranosyl bisphosphate derivative 9 displayed an IC(50) of 15.3 microM for inhibition of 2-methylthio-ADP-stimulated PLC activity. l-alpha-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y(2) (EC(50)=9.9 microM) versus P2Y(4) receptors. The P2Y(1) receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking.

Bioconcentration of n-dodecane and its highly branched isomer 2,2,4,6,6-pentamethylheptane in fathead minnows.

Petroleum products are complex mixtures of hydrocarbons. They are important as constituents of fuels and lubricants, and as key raw materials for the chemicals industry. Since there is a potential for accidental releases to the aquatic environment, bioaccumulation of higher hydrocarbons is of concern. Here, the bioconcentration behaviour of two representative hydrocarbons, the dodecane isomers n-dodecane and 2,2,4,6,6-pentamethylheptane (PMH), was investigated in fathead minnows at concentrations in water below their maximum aqueous solubility. The concentration of n-dodecane in fish did not exceed our method limit of detection of 60 microg/kg. In contrast, PMH could be quantified in fish. No significant increase in the ratio of PMH concentrations in fish to water could be detected indicating that an exposure time of 4-10 days is sufficient to approach steady-state. For n-dodecane the upper limit of the bioconcentration factor (BCF) is estimated by dividing the method limit of detection by the exposure concentration and a value of 240 l/kg is derived. For PMH the bioconcentration factor, estimated as the average fish/water concentration ratio during the steady-state part of the experiment, ranges between 880 and 3,500 l/kg. The BCFs of both compounds are small compared to their hydrophobicity. Given that both linear and branched hydrocarbons are known to be biotransformed by fish, it appears that efficient metabolism of the test compounds in fathead minnows prevents bioaccumulation.

In vitro permeability and binding of hydrocarbons in pig ear and human abdominal skin.

Human skin has continual exposure to chemicals due to various occupational activities. Chemicals that get on skin have the potential to be absorbed. Hence, the potential human health hazards of a chemical must include an estimate for percutaneous absorption. An inexpensive, easy, and adequate model for the quantitative measurement of skin penetration of chemicals from JP-8 is absent. Cutaneous penetration studies in vitro through human skin are severely limited due to the lack of availability of the human skin. In this study, we have shown that pig ear skin can be used as a model for risk assessment from the percutaneous absorption of chemicals. We determined flux and permeability coefficient (Kp) of three chemicals--heptane, hexadecane, and xylene--from their permeation profile through porcine and human skin. Binding of these chemicals to porcine stratum corneum (SC) and human SC were also determined. Factors of difference (FOD) in the permeability of pig and human skin were 1.71, 1.28, and 1.16, respectively, for heptane, hexadecane, and xylene. FOD in binding of heptane, hexadecane, and xylene to pig and human SC were found to be 1.04, 0.76, and 1.31, respectively. Since, FOD for permeability and binding parameters were less than 2, hence, we conclude that pig ear skin can be used as model for humans for risk assessment from percutaneous absorption of chemicals.

Bioconversion of hydrophobic compounds in a continuous closed-gas-loop bioreactor: feasibility assessment and epoxide production.

Microorganisms can be used as catalysts to produce organic compounds in a highly chemo-, regio- and enantioselective manner, and whole cells do not require the costly addition of cofactors for redox reactions. However, bioconversions are slow compared to alternative chemical reactions, and the biocatalyst works at its best in an aqueous medium, while the transformations of interest frequently involve compounds with a low-aqueous solubility and that are toxic to microorganisms. This results in low-volumetric productivity in classical bioreactors. The Continuous Closed-Gas-Loop Bioreactor is described here-a reactor system with high productivity, but without the problems associated with two-phase systems, such as an emulsified product stream and phase toxicity. Its working principle is to recirculate a gas phase through a bioreaction compartment and a saturator/absorber module where the product accumulates as a clear organic solution. A wide range of bioconversions should be possible in this set-up, and proof of concept was established for the epoxidation of 1,7-octadiene to (R)-1,2-epoxyoct-7-ene by a native strain of Pseudomonas oleovorans. This reaction represents a group of terminal alkene epoxidations where the bioconversion substrate does not support growth of the microorganism. Practical results at a 5l-scale are presented for this bioconversion for both batch and continuous operation with respect to the aqueous phase, showing continuous stable epoxidation at productivities >14 micromol min(-1) L(-1) (U L(-1)). The results confirm that the metabolism does not allow a simple optimization strategy, because growth and biotransformation substrates compete for the same enzyme sites, and conversely growth on a substrate using this very enzyme system is necessary for longterm bioconversion. Integrated removal of the CO(2) formed via the liquid overflow was estimated from theory and verified in experimental work.