Inhalant deaths in South Australia: a 20-year retrospective autopsy study

A 20-year retrospective study of inhalant deaths in South Australia, autopsied at Forensic Science SA, was undertaken from January 1983 to December 2002. Thirty-nine cases were identified from an autopsy pool of 18,880 cases, with a male to female ratio of 12:1. Sixty-four percent of the victims (N = 25) died during voluntary inhalation of volatile substances and 28% (N = 11) committed suicide utilizing a volatile substance or gas. The remaining 3 cases involved a workplace accident (N = 1) and 2 cases of autoerotic death where inhalants were being used to augment solitary sexual activity. The mean age of the 28 victims of accidental inhalant death of 21 years (range, 13-45 years) was considerably less than that of the 11 suicide victims of 31.5 years (range, 17-48 years). No homicides were found. Approximately one quarter of the victims were Aboriginal (N = 11), 10 of whom had died as a result of gasoline inhalation ("petrol sniffing"). Other common substances of abuse were aliphatic hydrocarbons such as butane. The study has shown that those most at risk for accidental or suicidal inhalant deaths were young males, with 92% of victims overall being male, and with 77% of victims being under 31 years of age. Gasoline inhalation remains a significant problem in Aboriginal communities in South Australia.

Characterization of water-soluble organic compounds in ambient aerosol using ultrahigh-resolution elctrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

Atmospheric aerosol water-soluble organic compounds (WSOC) exist in a complex mixture of thousands of organic compounds which may have a significant influence on the climate-relevant properties of the atmospheric aerosol. To understand the potential influences, the ambient aerosol was collected at a nonurban mountainous site near Steamboat Springs, CO. The WSOC fraction was analyzed using positive and negative electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Approximately 2400 and 4000 molecular formulas were identified from the detected positive and negative ions, respectively. The formulas contained carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sulfur (S) atoms over the mass range of 100-800 Da in both ionization modes. The number range of double bond equivalents (DBE), the mean O:C, H:C, and oxidation state of carbon for the positive ions were 0 – 18, 0.25 ± 0.15, 1.39 ± 0.29, and -0.89 ± 0.23, respectively. Comparatively, the negative ion values were 0 – 14, 0.53 ± 0.20, 1.48 ± 0.30, and -0.41 ± 0.45, respectively. Overall, the positive ion molecular formulas were less oxygenated than negative ions as seen with the lower O:C and OSc values. Molecular formulas of the positive ions classified as aliphatic, olefinic, and aromatic compound classes based on the aromaticity index values. Aliphatic compounds were the CHNO and CHO formulas that had mean DBE values of about 5 and 3, respectively. However, a majority of the CHOS, CHNOS, and CHS formulas were defined as olefinic compounds and had mean DBE values of about 12, 13, and 10, respectively. Overall, more than half of the assigned molecular formulas contained sulfur and were olefinic to aromatic compounds with a DBE range of 7-18. Source of the unsaturated sulfur containing compounds is currently unknown. Several nitrogen containing compounds were in common with the field and laboratory studies of the biomass burning aerosol and aged secondary organic aerosol products of the limonene ozonolysis.

DESIGN AND DEVELOPMENT OF BODIPY-BASED FLUORESCENT PROBES FOR SENSING AND IMAGING OF CYANIDE, Zn (II) IONS, LYSOSOMAL pH AND CANCER CELLS

BODIPY (4,4-Difluoro-3a,4a-diaza-s-indacene) dyes have gained lots of attention in application of fluorescence sensing and imaging in recent years because they possess many distinctive and desirable properties such as high extinction coefficient, narrow absorption and emission bands, high quantum yield and low photobleaching effect. However, most of BODIPY-based fluorescent probes have very poor solubilities in aqueous solution, emit less than 650 nm fluorescence that can cause cell and tissue photodamages compared with bio-desirable near infrared (650-900 nm) light. These undesirable properties extremely limit the applications of BODIPY-based fluorescent probes in sensing and imaging applications. In order to overcome these drawbacks, we have developed a very effective strategy to prepare a series of neutral highly water- soluble BODIPY dyes by enhancing the water solubilities of BODIPY dyes via incorporation of tri(ethylene glycol)methyl ether (TEG) and branched oligo(ethylene glycol)methyl ether (BEG) residues onto BODIPY dyes at 1,7-, 2,6-, 3,5-, 4- and meso- positions. We also have effectively tuned absorptions and emissions of BOIDPY dyes to red, deep red and near infrared regions via significant extension of π-conjugation of BODIPY dyes by condensation reactions of aromatic aldehydes with 2,6-diformyl BODIPY dyes at 1,3,5,7-positions. Based on the foundation that we built for enhancing water solubility and tuning wavelength, we have designed and developed a series of water-soluble, BODIPY-based fluorescent probes for sensitive and selective sensing and imaging of cyanide, Zn (II) ions, lysosomal pH and cancer cells. We have developed three BODIPY-based fluorescent probes for sensing of cyanide ions by incorporating indolium moieties onto the 6-position of TEG- or BEG-modified BOIDPY dyes. Two of them are highly water-soluble. These fluorescent probes showed selective and fast ratiometric fluorescent responses to cyanide ions with a dramatic fluorescence color change from red to green accompanying a significant increase in fluorescent intensity. The detection limit was measured as 0.5 mM of cyanide ions. We also have prepared three highly water-soluble fluorescent probes for sensing of Zn (II) ions by introducing dipicoylamine (DPA, Zn ion chelator) onto 2- and/or 6-positions of BEG-modified BODIPY dyes. These probes showed selective and sensitive responses to Zn (II) ion in the range from 0.5 mM to 24 mM in aqueous solution at pH 7.0. Particularly, one of the probes displayed ratiometric responses to Zn (II) ions with fluorescence quenching at 661 nm and fluorescence enhancement at 521 nm. This probe has been successfully applied to the detection of intracellular Zn (II) ions inside the living cells. Then, we have further developed three acidotropic, near infrared emissive BODIPY- based fluorescent probes for detection of lysosomal pH by incorporating piperazine moiety at 3,5-positions of TEG- or BEG-modified BODIPY dyes as parts of conjugation. The probes have low auto-fluorescence at physiological neutral condition while their fluorescence intensities will significant increase at 715 nm when pH shift to acidic condition. These three probes have been successfully applied to the in vitro imaging of lysosomes inside two types of living cells. At the end, we have synthesized one water- soluble, near infrared emissive cancer cell targetable BODIPY-based fluorescent polymer bearing cancer homing peptide (cRGD) residues for cancer cell imaging applications. This polymer exhibited excellent water-solubility, near infrared emission (712 nm), good biocompatibility. It also showed low nonspecific interactions to normal endothelial cells and can effectively detect breast tumor cells.

TERPENE AND TERPENOID EMISSIONS AND SECONDARY ORGANIC AEROSOL PRODUCTION

Approximately 90% of fine aerosol in the Midwestern United States has a regional component with a sizable fraction attributed to secondary production of organic aerosol (SOA). The Ozark Forest is an important source of biogenic SOA precursors like isoprene (> 150 mg m-2 d-1), monoterpenes (10-40 mg m-2 d-1), and sesquiterpenes (10-40 mg m-2d-1). Anthropogenic sources include secondary sulfate and nitrate and biomass burning (51-60%), vehicle emissions (17-26%), and industrial emissions (16-18%). Vehicle emissions are an important source of volatile and vapor-phase, semivolatile aliphatic and aromatic hydrocarbons that are important anthropogenic sources of SOA precursors. The short lifetime of SOA precursors and the complex mixture of functionalized oxidation products make rapid sampling, quantitative processing methods, and comprehensive organic molecular analysis essential elements of a comprehensive strategy to advance understanding of SOA formation pathways. Uncertainties in forecasting SOA production on regional scales are large and related to uncertainties in biogenic emission inventories and measurement of SOA yields under ambient conditions. This work presents a bottom-up approach to develop a conifer emission inventory based on foliar and cortical oleoresin composition, development of a model to estimate terpene and terpenoid signatures of foliar and bole emissions from conifers, development of processing and analytic techniques for comprehensive organic molecular characterization of SOA precursors and oxidation products, implementation of the high-volume sampling technique to measure OA and vapor-phase organic matter, and results from a 5 day field experiment conducted to evaluate temporal and diurnal trends in SOA precursors and oxidation products. A total of 98, 115, and 87 terpene and terpenoid species were identified and quantified in commercially available essential oils of Pinus sylvestris, Picea mariana, and Thuja occidentalis, respectively, by comprehensive, two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC × GC-ToF-MS). Analysis of the literature showed that cortical oleoresin composition was similar to foliar composition of the oldest branches. Our proposed conceptual model for estimation of signatures of terpene and terpenoid emissions from foliar and cortical oleoresin showed that emission potentials of the foliar and bole release pathways are dissimilar and should be considered for conifer species that develop resin blisters or are infested with herbivores or pathogens. Average derivatization efficiencies for Methods 1 and 2 were 87.9 and 114%, respectively. Despite the lower average derivatization efficiency of Method 1, distinct advantages included a greater certainty of derivatization yield for the entire suite of multi- and poly-functional species and fewer processing steps for sequential derivatization. Detection limits for Method 1 using GC × GC- ToF-MS were 0.09-1.89 ng μL-1. A theoretical retention index diagram was developed for a hypothetical GC × 2GC analysis of the complex mixture of SOA precursors and derivatized oxidation products. In general, species eluted (relative to the alkyl diester reference compounds) from the primary column (DB-210) in bands according to n and from the secondary columns (BPX90, SolGel-WAX) according to functionality, essentially making the GC × 2GC retention diagram a Carbon number-functionality grid. The species clustered into 35 groups by functionality and species within each group exhibited good separation by n. Average recoveries of n-alkanes and polyaromatic hydrocarbons (PAHs) by Soxhlet extraction of XAD-2 resin with dichloromethane were 80.1 ± 16.1 and 76.1 ± 17.5%, respectively. Vehicle emissions were the common source for HSVOCs [i.e., resolved alkanes, the unresolved complex mixture (UCM), alkylbenzenes, and 2- and 3-ring PAHs]. An absence of monoterpenes at 0600-1000 and high concentrations of monoterpenoids during the same period was indicative of substantial losses of monoterpenes overnight and the early morning hours. Post-collection, comprehensive organic molecular characterization of SOA precursors and products by GC × GC-ToFMS in ambient air collected with ~2 hr resolution is a promising method for determining biogenic and anthropogenic SOA yields that can be used to evaluate SOA formation models.

Studies on the catalytic hydrogenation of Baylis Hillman derivatives of substituted isoxazolecarbaldehydes

Results of the catalytic hydrogenation of Baylis-Hillman adducts obtained from substituted 3-, 4- and 5-isoxazolecarbox-aldehydes and their corresponding acetates in the presence of Raney-Ni and Pd-C are presented. The hydrogenation of Baylis-Hillman adducts of substituted 5-isoxazolecarbaldehydes and 3-isoxazolecarbaldehydes in the presence of Raney-Ni furnishes diastereoselectively syn enaminones over anti and in the presence of boric acid as an additive further enhancement of diastereoselectivity in favor of syn isomer is observed. The Pd-C-promoted hydrogenation of these substrates is also diastereoselective in favor of syn isomer but occurs without the hydrogenolysis of isoxazole-ring. The presence of boric acid as additive in this hydrogenation exhibits no pronounced effect on diastereoselectivity. The Raney-Ni-mediated hydrogenation of Baylis-Hillman adducts of substituted 4-isoxazolecarbaldehydes yield pyridone derivatives and Pd-C-promoted hydrogenation of the same substrate is diastereoselective to afford the anti isomer of the resulting products. The enaminones derived from Baylis-Hillman adducts of 3- and 5-isoxazolecarbaldehydes serve as versatile precursors for '-hydroxy-1, 3-diketones, which undergo acid-catalyzed ring-closure reaction to afford the furanone derivatives in excellent yields

A facile route to the synthesis of novel 2-amino-1,4,5,6-tetrahydropyrimidines from Baylis-Hillman products of acrylonitrile

A facile route for the synthesis of novel 5-substituted-2-amino-1,4,5,6-tetrahydro pyrimidines from the Baylis-Hillman adducts obtained from reaction of aldehydes and acrylonitrile is described.

Combinatorial synthesis and biological evaluation of isoxazole-based libraries as antithrombotic agents

The 3-substitutedphenyl-5-isoxazolecarboxaldehydes have been identified as activated aldehydes for the generation of isoxazole-based combinatorial libraries on solid phase through automation. Three highly functionalized isoxazole-based libraries comprising of 32, 96 and 45 compounds each have been synthesized in parallel format using Baylis Hillman reaction, Michael addition, reductive amination and alkylation reactions. With an objective of lead generation all the three libraries were evaluated for their antithrombin activity in vivo.

Intermolecular crosslinking of fatty acyl chains in phospholipids: Use of photoactivable carbene precursors

Phospholipids containing photolysable carhene precursors (beta-trifluoro-a-diazopropionoxy and m-diazirinophenoxy groups) in w-positions of sn-2 fatty acyl chains were prepared. Photolysis of their vesicles produced crosslinked products in 40-60 % yields. Crosslinking was mostly intermolecular and occurred bv carbene insertion into the C-H bonds of a second fatty acyl chain. Crosslinking products were characterized by (i) their gel permeation behavior, (ii) analysis of produets formed by base-catalyzed transesterification. (iii) degradation with phosphoiipases A2 and C, (iv) gas chromatography/mass spectrometry, and-(v) use of mixtures of phospholipids carrying thf' carhene precursors and a phospholipid containing radioactively labeled fatty acyl groups. Nitrenes generated from the aliphatic or aromatic azido groups in phospholipids were unsatisfactory for forming crosslinks by insertion in C-H bond

Asymmetric cyclopropanation of olefins catalysed by Cu(I) complexes of chiral pyridine-containing macrocyclic ligands (Pc-L*)

The synthesis and characterisation of copper(I) complexes of chiral pyridine-containing macrocyclic ligands (Pc-L*) and their use as catalysts in asymmetric cyclopropanation reactions are reported. All ligands and metal complexes were fully characterised, including crystal structures of some species determined by X-ray diffraction on single crystals. This allowed characterising the very different conformations of the macrocycles which could be induced by different substituents or by metal complexation. The strategy adopted for the ligand synthesis is very flexible allowing several structural modifications. A small library of macrocyclic ligands possessing the same donor properties but with either C-1 or C-2 symmetry was synthesized. Cyclopropane products with both aromatic and aliphatic olefins were obtained in good yields and enantiomeric excesses up to 99%.

Sulforaphane improves cognitive function administered following traumatic brain injury.

Recent studies have shown that sulforaphane, a naturally occurring compound that is found in cruciferous vegetables, offers cellular protection in several models of brain injury. When administered following traumatic brain injury (TBI), sulforaphane has been demonstrated to attenuate blood-brain barrier permeability and reduce cerebral edema. These beneficial effects of sulforaphane have been shown to involve induction of a group of cytoprotective, Nrf2-driven genes, whose protein products include free radical scavenging and detoxifying enzymes. However, the influence of sulforaphane on post-injury cognitive deficits has not been examined. In this study, we examined if sulforaphane, when administered following cortical impact injury, can improve the performance of rats tested in hippocampal- and prefrontal cortex-dependent tasks. Our results indicate that sulforaphane treatment improves performance in the Morris water maze task (as indicated by decreased latencies during learning and platform localization during a probe trial) and reduces working memory dysfunction (tested using the delayed match-to-place task). These behavioral improvements were only observed when the treatment was initiated 1h, but not 6h, post-injury. These studies support the use of sulforaphane in the treatment of TBI, and extend the previously observed protective effects to include enhanced cognition.

SYNTHESIS AND BIOLOGICAL EVALUATION OF ANTITUMOR ALDOPHOSPHAMIDE ACETALS

Although the major metabolic pathways of cyclophosphamide are well established, the mechanism of antitumor drug selectivity is highly controversial. However, it is widely accepted that aldophosphamide, one of the primary metabolites, plays a crucial role in drug selectivity. In an attempt to gain a better understanding of the mechanism of selectivity of cyclophosphamide, a series of aldophosphamide analogs have been synthesized.^ The new analogs, unlike aldophosphamide, are relatively stable in neutral solution; however, they are converted rapidly to aldehydo intermediates in the presence of carboxylate esterase. Due to structural differences, these analogs may be classified into three different groups, arbitrarily designated as A, B, C, depending upon the facility with which the intermediate aldehydes form 4-hydroxy cyclic tautomers. The half-life of the aldehydo/4-hydroxy cyclic tautomeric mixture is longer for bis(acetoxy)aldophosphamide acetal I (a representative of group A), shorter for the n-ethyl analog III (B), and shortest for the N,N-dimethyl analog IV (C). The ratio of aldophosphamide: 4-hydroxycyclophosphamide at pseudoequilibrium is 1: 4 for compound I, 1: 2 for compound III and 0: 1 for compound IV. The therapeutic efficacy of these compounds are group A $>$ group B $>$ group C. It is apparent that the equilibrium position between the aldehydo and 4-hydroxy cyclic tautomers, which determines their stability, is a crucial determinant of both the cytotoxicity and antitumor selectivity. These findings, taken in conjunction with the aldehyde dehydrogenase selectivity hypothesis, may provide an explanation for the unique antitumor activity of cyclophosphamide. ^

Fine structure of synapses on dendritic spines

Camillo Golgi's "Reazione Nera" led to the discovery of dendritic spines, small appendages originating from dendritic shafts. With the advent of electron microscopy (EM) they were identified as sites of synaptic contact. Later it was found that changes in synaptic strength were associated with changes in the shape of dendritic spines. While live-cell imaging was advantageous in monitoring the time course of such changes in spine structure, EM is still the best method for the simultaneous visualization of all cellular components, including actual synaptic contacts, at high resolution. Immunogold labeling for EM reveals the precise localization of molecules in relation to synaptic structures. Previous EM studies of spines and synapses were performed in tissue subjected to aldehyde fixation and dehydration in ethanol, which is associated with protein denaturation and tissue shrinkage. It has remained an issue to what extent fine structural details are preserved when subjecting the tissue to these procedures. In the present review, we report recent studies on the fine structure of spines and synapses using high-pressure freezing (HPF), which avoids protein denaturation by aldehydes and results in an excellent preservation of ultrastructural detail. In these studies, HPF was used to monitor subtle fine-structural changes in spine shape associated with chemically induced long-term potentiation (cLTP) at identified hippocampal mossy fiber synapses. Changes in spine shape result from reorganization of the actin cytoskeleton. We report that cLTP was associated with decreased immunogold labeling for phosphorylated cofilin (p-cofilin), an actin-depolymerizing protein. Phosphorylation of cofilin renders it unable to depolymerize F-actin, which stabilizes the actin cytoskeleton. Decreased levels of p-cofilin, in turn, suggest increased actin turnover, possibly underlying the changes in spine shape associated with cLTP. The findings reviewed here establish HPF as an appropriate method for studying the fine structure and molecular composition of synapses on dendritic spines.

The role of glucosinolates and the jasmonic acid pathway in resistance of Arabidopsis thaliana against molluscan herbivores

Although slugs and snails play important roles in terrestrial ecosystems and cause considerable damage on a variety of crop plants, knowledge about the mechanisms of plant immunity to molluscs is limited. We found slugs to be natural herbivores of Arabidopsis thaliana and therefore investigated possible resistance mechanisms of this species against several molluscan herbivores. Treating wounded leaves with the mucus residue (‘slime trail’) of the Spanish slug Arion lusitanicus increased wound-induced jasmonate levels, suggesting the presence of defence elicitors in the mucus. Plants deficient in jasmonate biosynthesis and signalling suffered more damage by molluscan herbivores in the laboratory and in the field, demonstrating that JA-mediated defences protect A. thaliana against slugs and snails. Furthermore, experiments using A. thaliana mutants with altered levels of specific glucosinolate classes revealed the importance of aliphatic glucosinolates in defending leaves and reproductive structures against molluscs. The presence in mollusc faeces of known and novel metabolites arising from glutathione conjugation with glucosinolate hydrolysis products suggests that molluscan herbivores actively detoxify glucosinolates. Higher levels of aliphatic glucosinolates were found in plants during the night compared to the day, which correlated well with the nocturnal activity rhythms of slugs and snails. Our data highlight the function of well-known antiherbivore defence pathways in resistance against slugs and snails and suggest an important role for the diurnal regulation of defence metabolites against nocturnal molluscan herbivores.

Optimization of a DNSH passive sampling method to measure airborne carbonyls

Various airborne aldehydes and ketones (i.e., airborne carbonyls) present in outdoor, indoor, and personal air pose a risk to human health at present environmental concentrations. To date, there is no adequate, simple-to-use sampler for monitoring carbonyls at parts per billion concentrations in personal air. The Passive Aldehydes and Ketones Sampler (PAKS) originally developed for this purpose has been found to be unreliable in a number of relatively recent field studies. The PAKS method uses dansylhydrazine, DNSH, as the derivatization agent to produce aldehyde derivatives that are analyzed by HPLC with fluorescence detection. The reasons for the poor performance of the PAKS are not known but it is hypothesized that the chemical derivatization conditions and reaction kinetics combined with a relatively low sampling rate may play a role. This study evaluated the effect of absorption and emission wavelengths, pH of the DNSH coating solution, extraction solvent, and time post-extraction for the yield and stability of formaldehyde, acetaldehyde, and acrolein DNSH derivatives. The results suggest that the optimum conditions for the analysis of DNSHydrazones are the following. The excitation and emission wavelengths for HPLC analysis should be at 250nm and 500nm, respectively. The optimal pH of the coating solution appears to be pH 2 because it improves the formation of di-derivatized acrolein DNSHydrazones without affecting the response of the derivatives of the formaldehyde and acetaldehyde derivatives. Acetonitrile is the preferable extraction solvent while the optimal time to analyze the aldehyde derivatives is 72 hours post-extraction. ^

Geochemical and nannofossil analyses of Cenomanian/Turonian sediments of DSDP Hole 75-530A

One of the key objectives of Deep Sea Drilling Project (DSDP) Leg 75 was to shed light on the underlying causes of Cretaceous oceanic anoxia in the South Atlantic by addressing two major hypotheses: productivity productivity-driven anoxia vs. enhanced ocean stratification leading to preservation of organic matter and black shale deposition. Here we present a detailed geochemical dataset from sediments deposited during the Cenomanian/Turonian (C/T) transition and the global oceanic anoxic event 2 (OAE 2) at DSDP Site 530A, located off-shore Namibia (southeast Angola Basin, north of Walvis Ridge). To characterise the succession of alternating black and green shales at this site and to reconstruct the evolution of their paleoenvironmental setting, we have combined data derived from investigations on bulk organic matter, biomarkers and the inorganic fraction. The location of the C/T boundary itself is biostratigraphically not well constrained due to the carbonate-poor (but organic matter-rich) facies of these sediments. The bulk d13Corg record and compound-specific d13C data, in combination with published as well as new biostratigraphic data, enabled us to locate more precisely the C/T boundary at DSDP Site 530A. The compound-specific d13C record is the first of this kind reported from C/T black shales in the South Atlantic. It is employed for paleoenvironmental reconstructions and chemostratigraphic correlation to other C/T sections in order to discuss the paleoceanographic aspects and implications of the observations at DSDP Site 530A in a broader context, e.g., with regard to the potential trigger mechanisms of OAE 2, global changes in black shale deposition and climate. On a stratigraphic level, an approximation and monitoring of the syndepositional degree of oxygen depletion within the sediments/bottom waters in comparison to the upper water column is achieved by comparing normalised concentrations of redox-sensitive trace elements with the abundance of highly source specific molecular compounds. These biomarkers are derived from photoautotrophic and simultaneously anoxygenic green sulphur bacteria (Chlorobiacea) and are interpreted as paleoindicators for events of photic zone euxinia. In contrast to a number of other OAE 2 sections that are characterised by continuous black shale sequences, DSDP Site 530A represents a highly dynamic setting where newly deposited black shales were repeatedly exposed to conditions of subtle bottom water re-oxidation, presumably leading to their progressive alteration into green shales. The frequent alternation between both facies and the related anoxic to slight oxygenated conditions can be best explained by variations in vertical extent of an oxygen minimum zone in response to changes in a highly productive western continental margin setting driven by upwelling.