Mass Spectrometry Imaging of Bio-oligomer Polydispersity in Plant Tissues by Laser Desorption Ionization from Silicon Nanopost Arrays.

Mass spectrometry imaging (MSI) enables simultaneous spatial mapping for diverse molecules in biological tissues. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has been a mainstream MSI method for a wide range of biomolecules. However, MALDI-MSI of biological homopolymers used for energy storage and molecular feedstock is limited by, e.g., preferential ionization for certain molecular classes. Matrix-free nanophotonic ionization from silicon nanopost arrays (NAPAs) is an emerging laser desorption ionization (LDI) platform with ultra-trace sensitivity and molecular imaging capabilities. Here, we show complementary analysis and MSI of polyhydroxybutyric acid (PHB), polyglutamic acid (PGA), and polysaccharide oligomers in soybean root nodule sections by NAPA-LDI and MALDI. For PHB, number and weight average molar mass, polydispersity, and oligomer size distributions across the tissue section and in regions of interest were characterized by NAPA-LDI-MSI.

Determination of gamma-hydroxybutyric acid in biofluids using a one-step procedure with "in-vial" derivatization and headspace-trap gas chromatography-mass spectrometry.

A headspace-trap gas chromatography-mass spectrometry (HS-trap GC-MS) method was developed to determine GHB, a low molecular weight compound and drug of abuse, in various biological fluids. Combining this relatively novel and fully automated headspace technique with "in-vial" methylation of GHB allowed for a straightforward approach. One single method could be used for all biofluids (urine, plasma, serum, whole blood or lyzed blood), requiring only 100µl of sample. Moreover, our approach involves mere addition of all reagents and sample into one vial. Following optimization of headspace conditions and trap settings, validation was performed. Although sample preparation only consists of the addition of salt and derivatization reagents directly to a 100µl-sample in a HS-vial, adequate method sensitivity and selectivity was obtained. Calibration curves ranged from 5 to 150µg/ml GHB for urine, from 2 to 150µg/ml for plasma, and from 3.5 to 200µg/ml for whole blood. Acceptable precision and accuracy (<13% bias and imprecision) were seen for all quality controls (QC's) (LLOQ-level, low, medium, high), including for the supplementary serum- and lyzed blood-based QC's, using calibration curves prepared in plasma or whole blood, respectively. Incurred sample reanalysis demonstrated assay reproducibility, while cross-validation with another GC-MS method demonstrated that our method is a valuable alternative for GHB determination in toxicological samples, with the advantage of requiring only 100µl and minimal hands-on time, as sample preparation is easy and injection automated.

Determination of GHB and its precursors (GBL and 1,4-BD) in dietary supplements through the synthesis of their isotopologues and analysis by GC-MS method.

Gamma-hydroxybutyric acid (GHB) and its "pro-drugs", gamma-butyrolactone (GBL) and 1,4 butanediol (1,4-BD), are drugs of abuse with depressant effects on the central nervous system. Many analytical methods have been proposed for the quantitative determination of these compounds mainly in biological matrices but only few have been addressed to dietary supplements and foods. Facile synthesis of the GBL and 1,4-BD isotopologues are available by "one pot" Ru-catalyzed homogeneous deuteration of dicarboxylic acids. In this work we propose a new method for determination of GHB, GBL and 1,4-BD in commercially available dietary supplements, based on isotope dilution mass spectrometry (ID-MS). The procedure involves a simple extraction of sample with acidic acetonitrile and direct analysis by GC-ID-MS method without any purification or derivatization. Indeed, the proposed method takes advantage of the complete conversion of GHB (free acid or its salts) to GBL, allowing the quantification of GHB and its pro-drugs. Five levels for each calibration curve have been prepared by diluting working solutions of the analytes to obtain concentrations ranging from 1 to 20mg/mL. The validation procedures have shown an accuracy between 88% and 99% and a precision between 7.3% and 2.9% of each analyte in the sample matrix. Positive ions chemical ionization (PICI) have been employed to preserve the information on molecular ions and to improve specificity and sensitivity of quantitative determination.

Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1.

AIMS: Utilization of cheap and readily available agricultural residues as cheap carbon sources for poly(3-hydroxybutyrate) (PHB) production by Halomonas boliviensis. METHODS AND RESULTS: Wheat bran was hydrolysed by a crude enzyme preparation from Aspergillus oryzae NM1 to provide a mixture of reducing sugars composed mainly of glucose, mannose, xylose and arabinose. Growth of H. boliviensis using a mixture of glucose (0.75% w/v) and xylose (0.25% w/v) in the medium led to a PHB content and concentration of 45 wt% and 1 g l(-1), respectively, after 30 h. A similar PHB concentration was attained when H. boliviensis was grown on wheat bran hydrolysate but with a lower PHB content, 34 wt%. In a batch cultivation mode in a fermentor, using 1.8% (w/v) reducing sugars, the maximum PHB accumulation by H. boliviensis was attained in 20 h, but was reduced to about 30 wt%. By adding butyric acid (0.8% v/v), sodium acetate (0.8% w/v) and decreasing the reducing sugars concentration to 1 x 0% w/v in the medium, PHB accumulation and concentration were increased to 50 wt% and 4 g l(-1), respectively, after 20 h. Butyric acid and sodium acetate for PHB production could also be provided by anaerobic digestion of solid potato waste. CONCLUSIONS: Cheap and readily available agricultural residues can be used as substrates to produce PHB. The production of PHB by H. boliviensis using wheat bran hydrolysate as source of carbon is expected to reduce the production cost and motivates further studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Large-scale commercial utilization of PHB is mainly hampered by its high production cost. Carbon source for PHB production accounts up to 50% of the total production costs. Thus, the use of waste agricultural residues can substantially reduce the substrate cost (and in turn even provide value to the waste), and can downsize the production costs. This improves the market competitiveness. Studies on PHB production by moderate halophiles were recently initiated with H. boliviensis and findings show that it has potential for commercial exploitation. PHB production by H. boliviensis using wheat bran and potato waste is hence interesting.

Simultaneous nitrification, denitrification, and phosphorus removal in a lab-scale sequencing batch reactor.

Simultaneous nitrification and denitrification (SND) via the nitrite pathway and anaerobic-anoxic-enhanced biological phosphorus removal (EBPR) are two processes that can significantly reduce the energy and COD demand for nitrogen and phosphorus removal. The combination of these two processes has the potential of achieving simultaneous nitrogen and phosphorus removal with a minimal requirement for COD. A lab-scale sequencing batch reactor (SBR) was operated in alternating anaerobic-aerobic mode with a low dissolved oxygen (DO) concentration (0.5 mg/L) during the aerobic period, and was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under anaerobic conditions, COD was taken up and converted to polyhydroxyalkanoates (PHAs), accompanied by phosphorus release. In the subsequent aerobic stage, PHA was oxidized and phosphorus was taken up to <0.5 mg/L by the end of the cycle. Ammonia was also oxidized during the aerobic period, but without accumulation of nitrite or nitrate in the system, indicating the occurrence of simultaneous nitrification and denitrification. However, off-gas analysis showed that the final denitrification product was mainly nitrous oxide (N(2)O), not N(2). Further experimental results demonstrated that nitrogen removal was via nitrite, not nitrate. These experiments also showed that denitrifying glycogen-accumulating organisms (DGAOs), rather than denitrifying polyphosphate-accumulating organisms (DPAOs), were responsible for the denitrification activity.

Polyhydroxyalkanoate form and polyphosphate regulation: keys to biological phosphorus and glycogen transformations?

Experimental studies with both synthetic and real domestic wastewater showed that poly-3-hydroxy-butyrate (3HB) and poly-3-hydroxy-valerate (3HV) formed in direct proportion to the acetate/propionate (Ace/Pro) ratio of the influent wastewater during Enhanced Biological Phosphorus Removal (EBPR). Acetic acid resulted in higher anaerobic phosphorus (P) release, polyhydroxyalkanoate (PHA) yield, 3HB content, and glycogen (CH) degradation. Linear regression showed that anaerobic P release (Prel) and CH degradation (CHdeg) were both a function of Ace-->3HB, but not of Pro-->3HV. Aerobic P uptake (Pup) correlated best with preceding Prel, rather than PHA (but note Prel correlated with Ace-->3HB). Aerobic CH formation (CHform) correlated best with CHdeg and 3HB. The results imply the acetate/propionate content of influent has a major influence on PHA, CH, and P transformations. Short-term increases in acetic or propionic acid increased Prel, but were always offset by corresponding changes in Pup to yield the same net P removal as the control reactor. Thus net P removal, and EBPR process performance, was probably a function of the population selected (i.e. XPAO fraction) during long-term cultivation.

Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.).

Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.

Gas chromatographic analysis of polyhydroxybutyrate in activated sludge: a round-robin test.

Polyhydroxyalkanoates (PHA) and poly-beta-hydroxybutyrate (PHB) in particular have become compounds which is routinely investigated in wastewater research. The PHB analysis method has only recently been applied to activated sludge samples where PHA contents might be relatively low. This urges the need to investigate the reproducibility of the gas chromatographic method for PHB analysis. This was evaluated in a round-robin test in 5 European laboratories with samples from lab-scale and full-scale enhanced biological phosphorus removal systems. It was shown that the standard deviation of measurements in each lab and the reproducibility between the labs was very good. Experimental results obtained by different laboratories using this analysis method can be compared. Sludge samples with PHB contents varying between 0.3 and 22.5 mg PHB/mg sludge were analysed. The gas chromatographic method allows for PHV, PH2MB and PH2MV analysis as well.

Accumulation of poly-beta-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803.

Accumulation of poly-beta-hydroxybutyrate (PHB) by photoautotrophic microorganisms makes it possible to reduce the production cost of PHB. The Synechocystis sp. PCC6803 cells grown in BG11 medium under balanced, nitrogen-starved or phosphorus-starved conditions were observed by transmission electron microscope. Many electron-transparent granules in the nitrogen-starved cells had a diameter up to 0.8 micron. In contrast, the number of granules in the normally cultured cells decreased obviously and only zero to three much smaller granules were in each cell. These granules were similar to those in bacteria capable of synthesizing PHB. They were proved to be PHB by gas chromatography after subjecting the cells to methanolysis. Effects of glucose as carbon source and light intensity on PHB accumulation in Synechocystis sp. PCC6803 under nitrogen-starved cultivation were further studied. Glucose and illumination promoted cell growth but did not favor PHB synthesis. After 7 days of growth under nitrogen-starved photoautotrophic conditions, the intracellular level of PHB was up to 4.1% of cellular dry weight and the PHB concentration in the culture broth was 27 mg/l.

Effects of excess dietary leucine and leucine catabolites on growth and immune responses in weanling pigs.

Two experiments with weanling pigs were conducted to study the effects on growth and immune responses of excess dietary L-leucine (LEU) and dietary supplementation with the LEU catabolites, alpha-ketoisocaproic acid (KIC) and beta-hydroxymethyl butyrate (HMB). In Exp. 1, 80 pigs were randomly allocated according to initial BW and ancestry to five replications of four dietary treatments (four pigs/pen). The control diet contained wheat, oat groats, menhaden fish meal, and dried whey and provided 1.12% LEU. Treatment diets were the control plus 1.12% LEU, 1.12% KIC, or .4% HMB. The experiment lasted 6 wk. In Exp. 2, 36 pigs were randomly allocated to nine replications of four dietary treatments in a 2 x 2 factorial arrangement. Treatments consisted of two concentrations of dietary LEU and a daily i.m. injection of dexamethasone (DEX) or saline. Pigs were fed a control corn-soybean meal and dried whey diet (1.56% LEU) or the control diet plus 1.56% of crystalline LEU. Pigs were individually penned and the experiment lasted 4 wk. Growth performance, plasma free amino acids, plasma urea nitrogen, and humoral and cellular immune responses were measured. Results indicated that LEU concentrations in practical diets and supplementation with KIC and HMB (Exp. 1) did not detrimentally affect growth and immune response. The high LEU concentration and DEX injection used in Exp. 2, however, were detrimental to both growth and immune response.

Glycolytic changes after local hyperthermia in mouse liver and adenocarcinoma.

Glycolytic changes in an adenocarcinoma and in liver of C57B1 mice were determined for up to twelve hours after local hyperthermia at 43 degrees C for 30 min. The metabolites studied included glucose, glucose-6-phosphate, pyruvate, lactate, acetoacetate and beta-hydroxybutyrate. Both the glucose and glucose-6-phosphate levels decreased significantly in liver and tumour and remained low for up to twelve hours. The lactate levels increased slightly immediately after heating but were decreased at later times. However, the hepatic pyruvate levels decreased for up to two hours after heating but increased later reaching control levels. In both liver and tumour the levels of beta-hydroxybutyrate were significantly enhanced immediately after hyperthermia, whereas those of acetoacetate were lowered.

Electroencephalographic characteristics of lithium hydroxybutyrate.

Lithium hydroxybutyrate influence on excitability, functional mobility and frequency range power of the cortex electrograms, midbrain reticular formation, posterior hypothalamus caudate nucleus, dorsal hippocampus, basolateral amygdala and medial thalamus in rabbits has been investigated. It has been shown that the drug suppresses the non-specific activating systems of the midbrain and posterior hypothalamus, intensifies work of the caudatocortical inhibitory mechanisms and the forebrain limbic formations (the hippocampus and amygdala).

Effect of hyperthermia on the lactic acid and beta-hydroxybutyric acid content in tumour.

The effects of hyperthermia on the content of lactic acid and beta-hydroxybutyric acid in the SCK mammary carcinoma and the leg muscle of A/J mice were studied. The contents of lactic acid in the SCK tumour before heating was 9.32 mumol/g, and the content of beta-hydroxybutyric acid was only 0.013 mumol/g. The lactic acid content in the tumour increased to 17.5 mumol/g at 0 h after heating at 41.5 degrees C for 30 min and then decreased to the control level 3 h later. When heated at 43.5 degrees C for 30 min, the lactic acid content in the tumour increased to 24 mumol/g at the end of heating and remained elevated for 24 h. The content of beta-hydroxybutyric acid increased continuously reaching 0.45 mumol/g at 5 h after heating at 43.5 degrees C for 30 min, and then declined thereafter. The contents of lactic acid and beta-hydroxybutyric acid in the muscle also increased after heating, but these increases were far less than those observed in the tumours. The absolute amount of lactic acid in the heated tumours was far greater than that of beta-hydroxybutyric acid, and thus appeared to play the major role for the increased acidity in the heated tumours.

Chemical and physical characteristics of the deoxycholate-soluble and magnesium-reaggregated membrane nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium.

The inactive components of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase present in the 0.4% deoxycholate-soluble fraction obtained from Bacillus megaterium KM membranes were reaggregated into active NADH oxidase by dilution in the presence of Mg(2+). The reaggregated oxidase was different from the original membrane with respect to sedimentation behavior in a sucrose gradient and morphological appearance. The deoxycholate-insoluble portion of the membrane had membrane-like structure whereas the reaggregated oxidase appeared to be a filamentous aggregate of small particles. The reaggregated oxidase and the deoxycholate-insoluble membrane residue were similar to the original membrane with respect to total protein and total lipid content. The inactive components of the NADH oxidase system exist in deoxycholate as two molecular species which were separable by sucrose density gradient centrifugation or gel filtration in deoxycholate-containing solutions. Both components and dilution in the presence of Mg(2+) were necessary for restoration of oxidase activity. The smaller-molecular-weight component contained all of the NADH-2,6-dichlorophenolindophenol oxidoreductase activity of the original membrane.