A History of Molecular Level Analysis of Natural Organic Matter by FTICR Mass Spectrometry and The Paradigm Shift in Organic Geochemistry

Natural organic matter (NOM) is a complex mixture of biogenic molecules resulting from the deposition and transformation of plant and animal matter. It has long been recognized that NOM plays an important role in many geological, geochemical, and environmental processes. Of particular concern is the fate of NOM in response to a warming climate in environments that have historically sequestered carbon (e.g., peatlands and swamps) but may transition to net carbon emitters. In this review, we will highlight developments in the application of high-field Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) in identifying the individual components of complex NOM mixtures, focusing primarily on the fraction that is dissolved in natural waters (dissolved organic matter or DOM). We will first provide some historical perspective on developments in FTICR technology that made molecular-level characterizations of DOM possible. A variety of applications of the technique will then be described, followed by our view of the future of high-field FTICR MS in carbon cycling research, including a particularly exciting metabolomic approach.     

Large increases in1H metaboliteT 2's after cerebral hypoxia-ischemia correlate with ATP depletion

In vivo proton (¹H) magnetic resonance spectroscopy (MRS) can measure cerebral metabolite concentrations and nuclear relaxation times. Function of the sodium (Na⁺)/potassium (K⁺) pump in cell membranes depends on adequate adenosine triphosphate (ATP) levels: intracellular Na⁺ is normally extruded in exchange for extracellular K⁺. Low ATP will cause pump dysfunction and loss of K⁺ accompanied by influx of Na⁺and water. Raised intracellular water may increase molecular mobility and this might be detectable as increased apparent transverse relaxation times (T ₂'s).¹H-MRS of the brains of newborn piglets during acute hypoxia-ischemia revealed enigmatic increases in the peak area of creatine + phosphocreatine (Cr) relative to those of choline-containing compounds (Cho) andN-acetylaspartate (NAA). Interleaved¹H and phosphorus (³¹P) MRS showed that theT ₂'s of both Cr and lactate (Lac) increased during acute hypoxia-ischemia and these changes correlated with reductions in nucleotide triphosphate (NTP; largely ATP). Within 50 h of metabolic recovery from the primary insult, as delayed energy failure developed, theT ₂'s of Cho, Cr, NAA, and Lac increased greatly. TheseT ₂ changes also correlated with NTP depletion. These observations demonstrate important relationships betweenT ₂'s and function of the ATP-dependent Na⁺/K⁺ pump.     

Visualization of Bile Homeostasis Using <Superscript>1</Superscript>H-NMR Spectroscopy as a Route for Assessing Liver Cancer

Changes in bile synthesis by the liver or alterations in the enterohepatic circulation due to a variety of etiological conditions may represent a novel source of liver disease-specific biomarkers. Bile from patients with liver diseases exhibited significant changes in the levels of glycine- and taurine-conjugated bile acids, phospholipids, cholesterol and urea relative to non-liver disease controls. Cholangiocarcinoma and non-malignant liver diseases (NMLD) showed the most significant alterations. Further, hepatocellular carcinoma (HCC) could be differentiated from NMLD (p = 0.02), as well as non-liver disease controls (p = 0.02) based on the amounts of bile acids, phospholipids and/or cholesterol. HCC also differed with cholangiocarcinoma although not significantly. Urea increases somewhat in non-malignant liver disease relative to non-liver disease controls, while the bile acids, phospholipids and cholesterol all decrease significantly. The ratio between some major bile metabolites also distinguished NMLD (p = 0.004–0.01) from non-liver disease controls. This snapshot view of bile homeostasis, is obtainable from a simple nuclear magnetic resonance (NMR) approach and demonstrates the enormous opportunity to assess liver status, explore biomarkers for high risk diseases such as cancers and improve the understanding of normal and abnormal cellular functions.     

Discrimination of Prey, But Not Plant, Chemicals by Actively Foraging, Insectivorous Lizards, the Lacertid Takydromus sexlineatus and the Teiid Cnemidophorus gularis

Sampling environmental chemicals to reveal prey and predators and to provide information about conspecifics is highly developed in lizards. Actively foraging lizards can discriminate between prey chemicals and control stimuli, but ambush foragers do not exhibit prey chemical discrimination. Recent experiments on a few species of herbivorous lizards have also demonstrated an ability to identify plant food chemicals. We studied chemosensory responses to chemicals from prey and palatable plants in two species of actively foraging, insectivorous lizards. Both the lacertid Takydromus sexlineatus and the teiid Cnemidophorus gularis exhibited strong responses to prey chemicals, but not to plant chemicals. These findings increase confidence in the relationship between prey chemical discrimination and foraging mode, which is based on data for very few species per family. They also provide data showing that actively foraging insectivores in two families do not respond strongly to plant cues. Such information is essential for eventual comparative studies of the relationship between plant diet and responses to food chemicals. The traditional method of presenting stimuli by using hand-held cotton swabs worked well for T. sexlineatus but could not be used for C. gularis due to repeated escape attempts. When stimuli were presented to C. gularis on ceramic tiles and no experimenter was visible, the lizards responded readily. Presentation of stimuli on tiles in the absence of a visible experimenter may be a valuable approach to study of food chemical discrimination by active foragers in which antipredatory behavior interferes with responses to swabs.     

Interfacial viscosity and the stability of emulsions

A viscous traction interfacial viscometer was constructed to study a mineral oil-water-emulsifier system. The effect on interfacial viscosity of the synthetic emulsifiers Span 80 and Pluronic L42 were investigated, along with the bioemulsifier produced by the yeast Torulopsis petrophilum. In the case of the synthetic emulsifiers, it was found that conditions which maximized interfacial viscosity also maximized emulsion stability. The same relation held for T. petrophilum emulsifier once all free fatty acids had been removed from the broth. Emulsion stability was measured by calculating mean diameters, as well as emulsion indices.     

Selection and Synthesis of Energetic Heterocyclic Compounds Suitable for Use in Insensitive Explosive and Propellant Compositions

The rationale behind developing insensitive energetic compounds (IECs) for incorporation into insensitive munition (IM) formulations (rather than the alternative approach of desensitizing higher energy but sensitive compounds) is discussed. With the aim of selecting a maximum of 2–3 IECs suitable for use in insensitive explosive and propellant compositions, a survey of the literature on IECs published in the last 20 years was carried out. From around 50 candidates, a selection was made of eight prime candidates, all heterocyclic compounds (principally monocyclic or fused‐ring bicyclic compounds of the di‐ or triazine, triazole or oxadiazole classes), which displayed explosive performance significantly better than that of the ubiquitous IEC, TATB. The criteria for inclusion of compounds in these listings are described. Screening of the eight candidate compounds against further performance criteria reduced the list to five compounds which were evaluated in detail – these were: CL‐14 (5,7‐diamino‐4,6‐dinitrobenzofuroxan), ANPZ‐i (2,5‐diamino‐3,6‐dinitropyrazine), NNHT (2‐nitrimino‐5‐nitro‐hexahydro‐1,3,5‐triazine), NTAPDO (5‐nitro‐2,4,6‐triaminopyrimidine‐1,3‐dioxide), and PANT [4‐(picrylamino)‐5‐nitro‐1,2,3‐triazole]. A detailed analysis of scale‐up issues associated with each compound was then made, including cost and availability of precursors, hazards (chemical and explosive), effluent streams, and other scale‐up issues (e.g. materials of plant construction). A further downselection using these criteria gave the present short‐list comprising three compounds (the first three listed above) and further evaluation is in progress. The results of this study, funded by UK MOD, comprise the UK contribution to a nine‐nation European research collaboration in the EUCLID Common European Priority Area 14 “Energetic Materials”, as part of a five‐year project which commenced in October 2003.     

A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue

Recently, the pro‐inflammatory effects of linoleic acid (LNA) have been re‐examined. It is now becoming clear that relatively few studies have adequately assessed the effects of LNA, independent of obesity. The purpose of this work was to compare the effects of several fat‐enriched but non‐obesigenic diets on inflammation to provide a more accurate assessment of LNA's ability to induce inflammation. Specifically, 8‐week‐old male C57Bl/6 mice were fed either saturated (SFA), monounsaturated (MUFA), LNA, or alpha‐linolenic acid enriched diets (50 % Kcal from fat, 22 % wt/wt) for 4 weeks. Chow and high‐fat, hyper‐caloric diets were used as negative and positive controls, respectively. Expression of pro‐inflammatory and pro‐coagulant markers from epididymal fat, liver, and plasma were measured along with food intake and body weights. Mice fed the high SFA, MUFA, and high‐fat diets exhibited increased pro‐inflammatory markers in liver and adipose tissue; however, mice fed LNA for four weeks did not display significant changes in pro‐inflammatory or pro‐coagulant markers in epididymal fat, liver, or plasma. The present study demonstrates that LNA alone is insufficient to induce inflammation. Instead, it is more likely that hyper‐caloric diets are responsible for diet‐induced inflammation possibly due to adipose tissue remodeling.     

The Potential of the Combination of CRISPR/Cas9 and Pluripotent Stem Cells to Provide Human Organs from Chimaeric Pigs

Clinical organ allotransplantation is limited by the availability of deceased human donors. However, the transplantation of human organs produced in other species would provide an unlimited number of organs. The pig has been identified as the most suitable source of organs for humans as organs of any size would be available. Genome editing by RNA-guided endonucleases, also known as clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), in combination with induced pluripotent stem cells (iPSC), may have the potential to enable the creation of human organs from genetically-modified chimaeric pigs. These could potentially provide an unlimited supply of organs that would not be rejected by the recipient’s immune system. However, substantial research is needed to prove that this approach will work. Genetic modification of chimaeric pigs could also provide useful models for developing therapies for various human diseases, especially in relation to drug development.     

Characterization of 1,5‐pentanediol dibenzoate as a potential “green” plasticizer for poly(vinyl chloride)

1,5‐Pentanediol dibenzoate (PDDB) was evaluated as a potential “green” plasticizer for poly(vinyl chloride) (PVC) at concentrations ranging between 20 and 80 parts by weight per hundred parts of resin. The results of glass transition temperature (T_g) and tensile tests of PDDB blends with PVC were compared with those for blends of the commercial plasticizers di(2‐ethylhexyl) phthalate (DEHP), di(ethylene glycol) dibenzoate (DEGDB), and di(propylene glycol) dibenzoate (DPGDB) in PVC. The depression in T_g and the tensile properties were comparable for a PDDB/PVC blend at a fixed composition to those of blends with DEHP, DEGDB, and DPGDB. The PDDB was subjected to biodegradation using co‐metabolism by the common soil bacterium Rhodococcus rhodochrous (ATCC 13808). After 16 days of growth, nearly all of the PDDB was degraded, and only small amounts of transient, unidentified metabolites were observed in the growth medium during the experiment. J. VINYL ADDIT. TECHNOL., 2009. © 2009 Society of Plastics Engineers     

Relative rates and mechanisms of biodegradation of diester plasticizers mediated by Rhodococcus rhodochrous

Di‐ester plasticizers undergo slow degradation resulting in the release of recalcitrant, toxic metabolites in the environment. It is demonstrated here that the first and rate‐limiting step in their co‐metabolism by Rhodococcus rhodochrous is the hydrolysis of one ester bond. This mechanism is different than one proposed earlier for direct biodegradation of di‐2‐ethylhexyl phthalate. Also, using butyl butyrate as a reference compound to compare the relative rates of hydrolysis of different di‐ester plasticizers, biodegradation rates were found to be strongly influenced by steric hindrances and solubility. Esterase activity associated with the hydrolysis of these bonds was located in the envelope of the bacterial cell. The esterase activity was not induced by specific substrates and was detected at all stages of growth. These considerations are important for the hydrolysis of recalcitrant plasticizers by a process of co‐metabolism, which leads to a slow but steady overall rate of biodegradation. This suggests that as the growing reservoir of plasticizers in the environment slowly undergoes biotransformation, recalcitrant toxic metabolites will be continuously released.