Rapid determination of trans-fatty acids in human adipose tissue: Comparison of attenuated total reflection infrared spectroscopy and gas chromatography

A rapid attenuated total reflection (ATR) infrared (IR) spectroscopy procedure was used for quantitating the levels of total trans-fatty acid methyl ester (FAME) derivatives in neat (without solvent) test samples isolated from human adipose tissue. This procedure requires no weighing of the laboratory sample. The single-beam spectrum of the trans-containing FAMEs was ‘ratioed' against that of a reference material having only cis double bonds in order to obtain a symmetric absorption band at 966 cm⁻¹ on a horizontal background. A single-reflection ATR diamond cell that requires only about 1 μl of neat FAMEs was used. The average level of trans-fatty acids in human adipose tissue found by ATR (3.07±0.27%) was generally higher than that obtained by gas chromatography (2.59±0.20%). Reasons for such a difference are discussed.     

Topochemical investigation of early stages of lignin modification within individual cell wall layers of Scots pine (Pinus sylvestris L.) sapwood infected by the brown-rot fungus Antrodia vaillantii (DC.: Fr.) Ryv.

The initiation and progress of wood degradation of Pinus sylvestris sapwood exposed to the brown-rot fungus Antrodia vaillantii was studied on a cellular level by scanning UV microspectrophotometry (UMSP 80, Zeiss, MSP 800 Spectralytics). This improved analytical technique enables direct imaging of lignin modification within individual cell wall layers. The topochemical analyses were supplemented by light and transmission electron microscopy (TEM) studies in order to characterize morphological changes during the first days of degradation. Small wood blocks (1.5 × 1.5 × 5 mm) of Scots pine (P. sylvestris) were exposed to fungal decay by A. vaillantii for 3, 7, 11, 16, and 22 days. No significant weight loss was determined in the initial decay periods within three up to 7 days. After three days of decay the topochemical investigation revealed that the lignin modification starts at the outermost part of the secondary wall layer, especially in the region of the latewood tracheids. During advanced degradation after exposure of 22 days, lignin modification occurs non-homogeneously throughout the tissue. Even among the significantly damaged cells, some apparently unmodified cells still exist. Knowledge about lignin modification at initial stages of wood degradation is of fundamental importance to provide more information on the progress of brown-rot decay.     

Characterisation of the initial degradation stage of Scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) sapwood after attack by brown-rot fungus <Emphasis Type="Italic">Coniophora puteana</Emphasis>

In our study, early period degradation (10 days) of Scots pine (Pinus sylvestris L.) sapwood by the brown-rot fungus Coniophora puteana (Schum.: Fr.) Karst. (BAM Ebw.15) was followed at the wood chemical composition and ultrastructurelevel, and highlighted the generation of reactive oxygen species (ROS). An advanced decay period of 50 days was chosen for comparison of the degradation dynamics. Scanning UV microspectrophotometry (UMSP) analyses of lignin distribution in wood cells revealed that the linkages of lignin and polysaccharides were already disrupted in the early period of fungal attack. An increase in the lignin absorption A₂₈₀ value from 0.24 (control) to 0.44 in decayed wood was attributed to its oxidative modification which has been proposed to be generated by Fenton reaction derived ROS. The wood weight loss in the initial degradation period was 2%, whilst cellulose and lignin content decreased by 6.7% and 1%, respectively. Lignin methoxyl (–OCH₃) content decreased from 15.1% (control) to 14.2% in decayed wood. Diffuse reflectance Fourier-transform infrared (DRIFT) spectroscopy corroborated the moderate loss in the hemicellulose and lignin degradation accompanying degradation. Electron paramagnetic resonance spectra and spin trapping confirmed the generation of ROS, such as hydroxyl radicals (HO^∙), in the early wood degradation period. Our results showed that irreversible changes in wood structure started immediately after wood colonisation by fungal hyphae and the results generated here will assist in the understanding of the biochemical mechanisms of wood biodegradation by brown-rot fungi with the ultimate aim of developing novel wood protection methods.     

Improvement of decay resistance of wood via combination treatment on wood cell wall: Swell-bonding with maleic anhydride and graft copolymerization with glycidyl methacrylate and methyl methacrylate

Poplar wood (Populus ussuriensis Kom) was modified by a novel combined two-step treatment to improve its decay resistance. Maleic Anhydride (MAN) was first employed to swell and bond to wood cell wall, and then mixed monomers of glycidyl methacrylate/methyl methacrylate (GMA/MMA) were used to graft copolymerization within wood cell lumen. The swelling and bonding of cell wall by MAN, interfacial compatibility between resultant polymer from GMA/MMA monomers and wood cell wall, and decay resistance of all composites were tested and analyzed by Scanning electron microscopy–Energy dispersive X-ray (SEM–EDX), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) apparatus. The results indicate that the volume of poplar wood treated by MAN swells about 9% with about 15% of weight percent gain, and MAN chemically bonds to the cell wall through substitution reaction with hydroxyl group, and the grafting adduct mainly remains as an amorphous form. The resultant Poplar-MAN shows improved decay resistance of 69.79% against brown fungus (Gloeophyllum trabeum (Pers. ex Fr.) Murr.) and 81.42% against white fungus (Phanerochaete chrysosporium Burdsall.) over those of untreated Poplar, respectively. After the combined two-step treatment, GMA and MMA are copolymerized within wood cell lumen, and the resultant polymer is also grafted onto wood cell wall, resulting in the improvement of interfacial compatibility between polymer and wood substance without obvious gaps. The decay resistance of the resultant composite from the combined two-step treatment against the brown decay fungus and the white decay fungus is improved by 97.64% and 99.17%, respectively, compared with those of untreated poplar wood; and also more excellent than those of MMA treated wood, GMA/MMA monomers treated wood, organic 3-Iodo-2-Propynyl Butyl Carbamate (IPBC) treated wood and inorganic boron compounds treated wood, respectively.     

Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

ASSESSMENT OF Cryptococcus albidus FOR BIOPULPING OF EUCALYPTUS

Cryptococcus albidus shows delignification activity in nature. It was used for the biopulping of eucalyptus wood (Eucalyptus grandis) to access its potential for industrial application in the pulp and paper industry. Enzyme analysis on days 15, 30, and 60 showed the presence of laccase and xylanase as key enzymes. The production of endo-glucanase (CMCase) and exo-glucanase (FPase) was very low. Scanning electron microscopy (SEM) showed the surface colonization of wood and loosening of wood fibers in C. albidus-treated samples. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of eucalyptus wood. Denaturing gradient gel electrophoresis (DGGE) analysis on days 15, 30, and 60 confirmed the presence of C. albidus throughout the experiments. Cryptococcu albidus was able to suppress the growth of a native population. Further, after 60 days both the control and treated eucalyptus wood chips were given kraft pulping treatment. The kappa number of pulp of control wood was 21 and for treated wood was 17. Kappa number is considered a measure of lignin content in wood; hence the treatment of eucalyptus by C. albidus (biopulping) was effective in reducing its lignin content and can be used for biopulping in the pulp and paper industry.     

A technique for monitoring mammalian cell growth and inhibition in situ via Fourier transform infrared spectroscopy

A single culture of Chinese hamster ovary cells was grown on germanium attenuated total reflectance (ATR) crystals and continuously monitored in situ via ATR/Fourier transform infrared (FT-IR) spectroscopy for approximately 60 h. The cells were seeded into a specially designed flow cell which controlled physiological conditions, flow rate, and addition of growth medium or metabolic inhibitors. Infrared spectra were taken at 20-min intervals until a confluent monolayer was formed. Several strong bands are evident in the spectra which can be generally ascribed to molecular features of cellular components. Cell growth kinetics were measured as a function of infrared band intensity over time and exhibited the normal lag phase, logarithmic growth, and stationary phase on reaching confluence. Spectra of growing cells, normalized to the area under the spectral region 1800-1000 cm-1, were subtracted from reference spectra of confluent cells at 60 h. Difference spectra showed that the largest differences were observed between confluent cells and cells in early growth stages. Differences may reflect cell morphological changes, biochemical activity, and degree of ATR crystal exposure to the bulk medium. ATR/FT-IR spectroscopy of living Chinese hamster ovary cells was also used in a toxicological study to monitor the effects of hydroxyurea, an inhibitor of DNA synthesis. Delayed growth was observed in the cell growth curve of the hydroxyurea-treated cells during the course of treatment as compared to the control culture.     

Resistance of Scots pine wood to Brown-rot fungi after long-term forest fertilization

The susceptibility of Scots pine (Pinus sylvestris L.) sap- and heartwood against the wood decaying brown-rot fungus (Coniophora puteana) was investigated after long-term forest fertilization at three different sites in central Finland. Different wood properties: wood extractives, wood chemistry, and wood anatomy were used to explain sap- and heartwood decay. Scots pine sapwood was more susceptible to decay than its heartwood. In one site, sapwood seemed to be more resistant to wood decay after forest fertilization whereas the susceptibility of heartwood increased. Significant changes in the sapwood chemistry were found between treatment and sites, however, no relationship between wood chemistry and wood decay was observed in the factor analysis. The results of this study show that there was an inconsistent relationship between decay susceptibility and fertilization and the measured physical and chemical attributes of the wood were not consistently correlated with the decay rate.     

Alkaline phosphatase at the cell wall of the yeast phase ofParacoccidioides brasiliensis

The activity of alkaline phosphatase demonstrated by histochemical techniques was shown at the cell wall of the yeast form ofParacoccidioides brasiliensis at 3, 6, and 9 days of culture. The results showed a very active deposition at the cell wall as early as 9 days of culture of the fungus which made us think an inactive salt precipitate was also present.     

The lethal effects of gamma irradiation on larvae of the Huhu beetle, Prionoplus reticularis: a potential quarantine treatment for New Zealand export pine trees

Gamma irradiation was investigated as a possible method for disinfestation of huhu beetle larvae, Prionoplus reticularis White, in Pinus radiataD. Don. Larvae of four representative size classes were irradiated at six doses, and the lethal dose (LD₉₉) calculated from mortality data 3 days and 10 days post treatment. All larval size classes showed a similar sensitivity to gamma irradiation and required 3677 Gray (Gy) and 2476 Gy for a LD₉₉ 3 and 10 days post-treatment, respectively. The penetration of gamma irradiation into pine wood was found to be lowest in freshly cut logs, and decreased linearly at a rate of 0.698 Gy mm^–1 of wood. The penetration was greatest in wood that had been stored for 2 years, and decreased 0.512 Gy mm^–1 of wood. These results are likely to be correlated with wood moisture content. Gamma irradiation appears to be a potential alternative method to fumigation for quarantine treatment of P. reticularis.     

Inhibitory Effect of <i>N</i>, <i>N</i>-Dimethylhexadecylamine on the Growth of White-Rot Fungus <i>Trametes versicolor</i> (L.) in Wood

Wood is an organic material that is a source of carbon of organisms called Wood-decay fungi, and to preserve the wood, various toxic compounds to man and the environment have been used. To analyze the effect of N,N-Dimethylhexadecylamine (DMHDA) on wood attacked by the rotting fungus Trametes versicolor L. We used an in vitro system to expose the fungus T. versicolor to different concentrations of the DMHDA (50, 150 and 450 μM). We quantified the diameter of mycelial growth and laccase activity, also, under these experimental conditions we studied morphological details of the organisms using different scanning equipment including scanning electron microscopy. The growth of T. versicolor exposed to DMHDA for 60 days, showed a concentration-dependent dose behavior, also, the electron microscopy analysis revealed that the morphology and mycelial density was affected by the DMHDA, showing a formation of atypical morphological and thickener folds. Finally, the pieces of wood treated with DMHDA and exposed to the fungus had a lower mass loss, after a period of 60 days of exposure, the values obtained were 0.7, 1.0 and 0.5 g of mass lost for the control, LoC and LoDMHDA treatments respectively. Wood-rot fungi have represented economic losses worldwide, the strategies used have been supported by toxic compounds for the environment. The DMHDA both in the Petri dish system and as a wood preservative was shown to significantly inhibit the growth of T. versicolor.     

Mid-infrared spectroscopy is a fast screening method for selecting Arabidopsis genotypes with altered leaf cuticular wax

Arabidopsis eceriferum (cer) mutants with unique alterations in their rosette leaf cuticular wax accumulation and composition established by gas chromatography have been investigated using attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy in combination with univariate and multivariate analysis. Objectives of this study were to evaluate the utility of ATR-FTIR for detection of chemical diversity in leaf cuticles, obtain spectral profiles of cer mutants in comparison with the wild type, and identify changes in leaf cuticles caused by drought stress. FTIR spectra revealed both genotype- and treatment-dependent differences in the chemical make-up of Arabidopsis leaf cuticles. Drought stress caused specific changes in the integrated area of the CH₃ peak, asymmetrical and symmetrical CH₂ peaks, ester carbonyl peak and the peak area ratio of ester CO to CH₂ asymmetrical vibration. CH₃ peak positively correlated with the total wax accumulation. Thus, ATR-FTIR spectroscopy is a valuable tool that can advance our understanding of the role of cuticle chemistry in plant response to drought and allow selection of superior drought-tolerant varieties from large genetic resources.     

Infrared monitoring of the adhesion ofCatenaria anguillulae zoospores to solid surfaces

Electron microscopic studies of nematodes infected with the chytridiomycetous fungusCatenaria anguillulae indicated that zoospores of the fungus adhered to the cuticle of nematodes by a layer of extracellular polymers. The chemical composition of the adhesive polymers and their interaction with a solid surface were examined with Fourier transform infrared spectroscopy, using an attenuated total reflectance cell. On-line monitoring of the adhesion of zoospores to a germanium crystal with this technique showed that the adhesive polymers consisted of a protein(s) containing amide I and II bands. The adsorption of these proteins, measured as the increase in the amide II band, had a rapid initial phase of ca. 20 minutes, followed by a slower increase during the course of incubation. Fluorescein isothiocyanate staining of the attached cells at the end of the experiment showed that the adhesion of the zoospores occurred before the formation of the cyst wall.     

A Cytochemical Study of Extracellular Sheaths Associated with Rigidoporus lignosus during Wood Decay

An ultrastructural and cytochemical investigation of the development of Rigidoporus lignosus, a white-rot fungus inoculated into wood blocks, was carried out to gain better insight into the structure and role of the extracellular sheaths produced by this fungus during wood degradation. Fungal sheaths had a dense or loose fibrillar appearance and were differentiated from the fungal cell wall early after wood inoculation. Close association between extracellular fibrils and wood cell walls was observed at both early and advanced stages of wood alteration. Fungal sheaths were often seen deep in host cell walls, sometimes enclosing residual wood fragments. Specific gold probes were used to investigate the chemical nature of R. lignosus sheaths. While labeling of chitin, pectin, β-1,4- and β-1,3-glucans, β-glucosides, galactosamine, mannose, sialic acid, RNA, fucose, and fimbrial proteins over fungal sheaths did not succeed, galactose residues and laccase (a fungal phenoloxidase) were found to be present. The positive reaction of sheaths with the PATAg test indicates that polysaccharides such as β-1,6-glucans are important components. Our data suggest that extracellular sheaths produced by R. lignosus during host cell colonization play an important role in wood degradation. Transportation of lignin-degrading enzymes by extracellular fibrils indicates that alteration of plant polymers may occur within fungal sheaths. It is also proposed that R. lignosus sheaths may be involved in recognition mechanisms in fungal cell-wood surface interactions.     

Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions.     

Investigation on catechin as a beech wood decay biomarker

A high-performance liquid chromatography (HPLC) method based on the evolution of wood extractives was developed to follow the first stages of fungal degradation of beech wood exposed to Coriolus versicolor. The nature and the quantity of the extracts initially present in wood depended on the extraction conditions and also on the wood-drying conditions (time and temperature). The most interesting fraction was soxhlet extracted with acetone at 56 °C for 6 h. The best conditions to avoid extractives degradation consisted of a moderate drying at 55 °C for 48 h allowing identification of catechin as potential tracer. After 2 weeks of wood blocks exposure to C. versicolor, analysis of their acetonic extractives showed that catechin signal initially detected in beech wood, had totally disappeared. Treatment of wood with an appropriate fungicide such as propiconazole before exposure to C. versicolor, prevents the catechin amount from any variation. The comparison of these results with the classical weight loss (WL) measurements obtained after long-time experiments on treated and untreated wood blocks shows that the catechin amount evolution, monitored during 2 weeks, correlates with the wood resistance evaluated during 16 weeks, allowing the use of this flavonoid as a valuable biomarker of wood decay.     

Monitoring a recombinant Pichia pastoris fed batch process using Fourier transform mid-infrared spectroscopy (FT-MIRS)

The use of Fourier transform mid-infrared spectroscopy (FT-MIRS) to predict the concentrations of key analytes in fed-batch cultivations of an industrial strain of Pichia pastoris in a chemical complex medium was investigated. Models for glycerol, methanol (substrates), and product (an heterologous protein) were built, and evaluated. The use of a multi-bounce attenuated total reflectance (HATR) accessory aided spectral acquisition in optically dense samples. Generally, all models were robust and performed well on external validation, using data from processes not present in the original modelling exercise. Substrate models lacked the complexity of some previous IR models, and the models performed adequately even at low analyte concentration (<1 g l^–1). Thus, simultaneous, rapid monitoring of low concentrations of multiple analytes in a complex bioprocess matrix with little or no sample pre-treatment is achievable using ATR FT-MIRS.     

Rapid selection of mutants of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> for carbohydrate and fatty acid metabolism by fourier transform infrared spectroscopy and gas chromatography combined with multivariate analysis

Transgene-tagged mutants of Chlamydomonas reinhardtii were generated by random insertional mutagenesis for screening of mutants of carbohydrate and fatty acid metabolism. Approximately 2,500 insertion mutants tagged with the aph7″ gene were produced from one mutagenesis in three weeks. To establish a rapid screening system for numerous insertional lines, whole cell extracts of 100 insertional lines were subjected to Fourier transform infrared spectroscopy (FT-IR) and gas chromatography (GC) analysis combined with multivariate analysis. Mutant lines 28, 67, and 90 showed dramatic differences in the carbohydrate (1,000∼1,200 cm⁻¹) and amide (1,500∼1,700 cm⁻¹) regions of the FT-IR spectrum compared to wild type strain CC-124. Separate GC analysis also showed that 16:0 iso, palmitic acid (16:0), and oleic acid (18:1) were the major fatty acids in the wild type strain. In mutant 80, the relative content ratio of 16:0 iso in total fatty acids was significantly lower than in wild type, whereas the ratios of palmitic acid and oleic acid to 16:0 iso were higher. In mutant 95, the ratio of 16:0 iso to total fatty acids was increased, whereas ratios of palmitic acid and oleic acid to 16:0 iso were decreased. In particular, mutant 57 showed remarkably different fatty acid patterns with novel peaks of long-chain fatty acids having more than 20 carbon atoms. The results of this study show that FT-IR and GC combined with multivariate analysis enable rapid selection of mutants of carbohydrate and fatty acid metabolism in C. reinhardtii.     

Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora

White zones produced on biodegraded Pinus radiata wood chips were characterized by micro-localized-FTIR (Fourier Transformed Infra Red) spectroscopy and scanning electron microscopy. Both techniques permitted assignment of the white zones to a selective lignin removal process. Although both fungi studied have degraded lignin selectively in these restricted superficial areas, chemical analysis of the wood chips indicated that Ganoderma australe removed 16% of the initial amount of glucan at the 20% weight loss level. Ceriporiopsis subvermispora did not remove glucan at weight loss values below 17%. Prolonged biodegradation resulted in reduction of white zones by G. australe, and increased white zones from C. subvermispora decayed samples. This revised version was published online in July 2006 with corrections to the Cover Date.