Association of breathing sound spectra with glottal dimensions in exercise-induced vocal cord dysfunction.

The objective of this study was to evaluate associations between the breathing sound spectra and glottal dimensions in exercise-induced vocal cord dysfunction (EIVCD) during a bicycle ergometry test. Nineteen subjects (mean age 21.8 years and range 13-39 years) with suspected EIVCD were studied. Vocal folds were continuously imaged with videolaryngoscopy and breathing sounds were recorded during the bicycle exercise test. Twelve subjects showed paradoxical movement of the vocal folds during inspiration by the end of the exercise. In seven subjects, no abnormal reactions in vocal folds were found; they served as control subjects. The glottal quotient (interarytenoid distance divided by the anteroposterior glottal distance) was calculated. From the same time period, the tracheal-vocal tract resonance peaks of the breathing sound spectra were analyzed, and stridor sounds were detected and measured. Subjects with EIVCD showed significantly higher resonance peaks during the inspiratory phase compared to the expiratory phase (p < 0.014). The glottal quotient decreased significantly in the EIVCD group (p < 0.001), but not in the control group. 8 out of 12 EIVCD patients (67%) showed stridor sounds, while none of the controls did. There was a significant inverse correlation between the frequencies of the breathing sound resonance peaks and the glottal quotient. The findings indicate that the typical EIVCD reaction of a paradoxical approximation of the vocal folds during inspiration, measured here as a decrease in the glottal quotient, is significantly associated with an increase in inspiratory resonance peaks. The findings are applicable in the documentation of EIVCD findings using videolaryngoscopy, in addition to giving clinicians tools for EIVCD recognition. However, the study is limited by the small number of subjects.

Determination of pharmaceuticals in sewage sludge and biochar from hydrothermal carbonization using different quantification approaches and matrix effect studies.

Producing valuable biochar from waste materials using thermal processes like hydrothermal carbonization (HTC) has gained attention in recent years. However, the fate of micropollutants present in these waste sources have been neglected, although they might entail the risk of environmental pollution. Thus, an HPLC-MS/MS method was developed for 12 pharmaceuticals to determine the micropollutant load of biochar, which was made from sewage sludge via HTC within 4 h at 210 °C. Pressurized liquid extraction was applied to extract the compounds. Because of the high load of co-extracted matter, matrix effects in HPLC-MS/MS were investigated using matrix effect profiles. Interfering compounds suppressed 50% of the phenazone signal in sewage sludge and 70% in biochar, for example. The quantification approaches external calibration, internal standard analysis, and standard addition were compared considering recovery rates, standard deviations, and measurement uncertainties. The external analysis resulted in decreased or enhanced recovery rates. Spiking before LC-MS/MS compensated instrumental matrix effects. Still, recovery rates remained below 70% for most compounds because this approach neglects sample losses during the extraction. Internal standards compensated for the matrix effects sufficiently for up to five compounds. The standard addition over the whole procedure proved to compensate for the matrix effects for 11 compounds and achieved recovery rates between 85 and 125%. Additionally, results showed good reproducibility and validity. Only sulfamethoxazole recovery rate remained below 70% in sewage sludge. Real sample analysis showed that three pharmaceuticals were detected in the biochar, while the corresponding sewage sludge source contained 8 of the investigated compounds.

Pharmaceutical load in sewage sludge and biochar produced by hydrothermal carbonization.

We investigated the removal of twelve pharmaceuticals in sewage sludge by hydrothermal carbonization (HTC), which has emerged as a technology for improving the quality of organic waste materials producing a valuable biochar material. In this study, the HTC converted sewage sludge samples to a biochar product within 4h at a temperature of 210 °C and a resulting pressure of about 15 bar. Initial pharmaceutical load of the sewage sludge was investigated as well as the residual concentrations in biochar produced from spiked and eight native sewage sludge samples from three waste water treatment plants. Additionally, the solid contents of source material and product were compared, which showed a considerable increase of the solid content after filtration by HTC. All pharmaceuticals except sulfamethoxazole, which remained below the limit of quantification, frequently occurred in the investigated sewage sludges in the µg/kg dry matter (DM) range. Diclofenac, carbamazepine, metoprolol and propranolol were detected in all sludge samples with a maximum concentration of 800 µg/kgDM for metoprolol. HTC was investigated regarding its contaminant removal efficiency using spiked sewage sludge. Pharmaceutical concentrations were reduced for seven compounds by 39% (metoprolol) to≥97% (carbamazepine). In native biochar samples the four compounds phenazone, carbamazepine, metoprolol and propranolol were detected, which confirmed that the HTC process can reduce the load of micropollutants. In contrast to the other investigated compounds phenazone concentration increased, which was further addressed in thermal behaviour studies including three structurally similar potential precursors.

Oxidation of Crocein Orange G by lignin peroxidase isoenzymes. Kinetics and effect of H2O2.

The ligninolytic enzyme system of Phanerochaete chrysosporium is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium of P. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from the N-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H2O2 concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 microM COG was obtained when the H2O2 concentration was 150 microM. This was also the lowest H2O2 concentration for maximal decolorization of 100 microM COG, regardless of the amount of lignin peroxidase used in the reaction.

Semiautomatic quantification of spiking in patients with continuous spikes and waves in sleep: sensitivity to settings and correspondence to visual assessment.

OBJECTIVE: To define the optimal analysis protocol for semiautomatic quantification of spike index (SI) in continuous spikes and waves in sleep (CSWS). METHODS: Ten overnight EEGs (nine patients) with abundant spiking were used to quantify SI with a previously published semiautomatic quantification based on spike detection with BESA software. We studied (i) dependency of SI on maximal interspike interval (maxISI) defining the continuous discharge, (ii) sensitivity of SI to variations in the spike search protocol, and (iii) stability of SI over time. Finally, the semiautomatic method was compared with the quantification based on visual scoring by two neurophysiologists. RESULTS: MaxISI of 3s appeared to yield the best combination of sensitivity and stability in SI quantification. The SI of the first hour of sleep did not differ significantly from the SI of the whole night. Mean error of the semiautomatic method compared to visual scoring was only seven percentage units. CONCLUSIONS: Semiautomatic quantification of SI functions well with maxISI of 3s, and the first hour of sleep represents the whole night SI with a clinically relevant accuracy. SIGNIFICANCE: This method opens a possibility for objective quantification of near-continuous epileptiform spiking during sleep, and it supports the use of shorter epochs for quantitative assessment of CSWS.

Expression in Pichia pastoris and purification of Aspergillus awamori glucoamylase catalytic domain.

In this paper we report the expression in Pichia pastoris, purification, and characterization of the Aspergillus awamori glucoamylase catalytical domain (GAc). Pichia pastoris produced GAc to the level of 0.4 g per liter medium. This production level is about the same level as that gained for recombinant GA from Aspergillus and about 100-fold more than previously achieved by Saccharomyces cerevisiae. The GAc expressed in Pichia pastoris was purified by two independent chromatographic methods employing ion exchange or affinity chromatography to apparent homogeneity. The purified protein has a molecular weight of about 75,000 and specific activity of 78 units per milligram protein. The propeptide present in the glucoamylase N terminus was found to be removed correctly by P. pastoris. Glucoamylase produced by P. pastoris is N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed in P. pastoris. O-glycosides (studied to our knowledge for the first time in P. pastoris in this report) contribute about half of the total carbohydrate content in GAc. Purified GAc appears as multiple hands on isoelectric focusing with p1 values around 3.5, a value that is little higher than that for GAc produced in S. cerevisiae. GAc could be used as a versatile tool in studying protein expression in P. pastoris: as an affinity handle for other secreted proteins produced in P. pastoris, as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing in P. pastoris.

Human placental alkaline phosphatase: expression in Pichia pastoris, purification and characterization of the enzyme.

The soluble form of human placental alkaline phosphatase (PLAP) was expressed in the methylotrophic yeast Pichia pastoris and the expression product was purified and characterized. Yeast-derived PLAP (yPLAP) was secreted into the medium to the level of 2 mg/liter. yPLAP displayed kinetic properties similar to those reported earlier for the membrane-bound PLAP. Purified yPLAP had specific activity of 774 U/mg and appeared in two subunit sizes, ca. 62 and 65 kDa. This difference was due to heterogenous N-glycosylation. Purified yPLAP appeared as multiple forms in isoelectric focusing in pI range of 4.2 to 5.2. The expression system is discussed in comparison to previously reported expression systems.

Identification of a cyclase gene dictating the C-9 stereochemistry of anthracyclines from Streptomyces nogalater.

Nogalamycin is an anthracycline antibiotic produced by Streptomyces nogalater. Its aglycone has a unique stereochemistry (7S, 9S, 10R) compared to that of most other anthracyclines (7S, 9R, 10R). The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogalamycin, was used to generate nogalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.

The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures.

Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes.