Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]⁺ ions.     

Finite mixture model analysis of microarray expression data on samples of uncertain biological type with application to reproductive efficiency

Common goals of microarray experiments are the detection of genes that are differentially expressed between several biological types and the construction of classifiers that predict biological type of samples. Here we consider a situation where there is no training data. There is considerable interest in comparing expression profiles associated with successful pregnancies (SP) and unsuccessful pregnancies (UP) in model and farm animals. Successful pregnancy rate is known to be much higher in embryos generated by in vitro fertilization (IVF) than in nuclear transfer (NT) embryos, and higher under induced ovulation for large follicles (LF) than for small follicles (SF). The tasks of identifying genes differentially expressed between SP and UP, and predicting SP for future samples are not well accomplished by comparing IVF and NT, or LF and SF. A suitable method is finite mixture model analysis (FMMA), which models each observed class (IVF and NT, or LF and SF) as a mixture of two distributions, one for SP and one for UP, with different known or unknown proportions (here known to be 0.50 SP for IVF and 0.02 SP for NT). The means of the two distributions differ for the differentially expressed genes, which we identify via a likelihood ratio test. We confirm by simulation that FMMA strongly outperforms hierarchical clustering and linear discriminant analysis using the known class labels (NT, IVF). We apply FMMA to a real data set on IVF and NT embryos, and compute their posterior probabilities of SP, which confirm our prior knowledge of the SP proportions for IVF and NT.     

Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4

Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%.     

Recent update on the prevalence of Vibrio species among cultured grouper in Peninsular Malaysia

Vibrio infections are common among marine fish and lead to serious problems in the aquaculture sector. This study reports a recent occurrence of Vibrio species (spp.) isolated from cultured groupers in Peninsular Malaysia using the gyrB and pyrH genes. A total of 147 Vibrio strains were successfully isolated from 77 (64%) groupers using culture method and subjected to gyrB and pyrH sequencing for species identification and confirmation. Results showed that 89% of Vibrio strains were identified and clustered to six groups of Vibrio spp., while 11% were not clustered to any Vibrio spp. using the gyrB sequences. Meanwhile, by analysis of the pyrH sequences all the 147 Vibrio strains (100%) were successfully identified and clustered into 11 groups of Vibrio spp., including the gyrB non‐identified strains. The pyrH gene provides a better resolution for identification of Vibrio spp. compared with the gyrB gene. Thus, the pyrH gene was more suitable for a rapid determination of Vibrio spp. distribution in Peninsular Malaysia. Using the pyrH gene, our study found higher prevalence of Vibrio vulnificus (33%), V. alginolyticus (24%) and V. parahaemolyticus (22%), followed by V. rotiferianus (5%), V. harveyi (3%), V. tubiashii (2%), V. campbellii (2%), V. ponticus (1%), V. diabolicus (1%), V. owensii (1%) and others Vibrio sp. (7%). Thus, the results of this study revealed that the occurrence of pathogenic vibrios among grouper fish is still high in Malaysian aquaculture. In addition, the pyrH gene was proved as a suitable marker for rapid identification of Vibrio species compared with the gyrB gene.     

A first observation of spermatogenesis in mature male scalloped hammerheads (Sphyrna lewini) from Zinkwazi,KwaZulu-Natal,South Africa

Fish Physiology and Biochemistry - Sharks are not only threatened, but also have a low fecundity as they are being overfished. The shark family, Sphyrnidae, consists of nine species of which three...     

Conservation priorities for the different lines of Dutch Red and White Friesian cattle change when relationships with other breeds are taken into account

From a genetic point of view, the selection of breeds and animals within breeds for conservation in a national gene pool can be based on a maximum diversity strategy. This implies that priority is given to conservation of breeds and animals that diverge most and overlap of conserved diversity is minimized. This study investigated the genetic diversity in the Dutch Red and White Friesian (DFR) cattle breed and its contribution to the total genetic diversity in the pool of the Dutch dairy breeds. All Dutch cattle breeds are clearly distinct, except for Dutch Friesian breed (DF) and DFR and have their own specific genetic identity. DFR has a small but unique contribution to the total genetic diversity of Dutch cattle breeds and is closely related to the Dutch Friesian breed. Seven different lines are distinguished within the DFR breed and all contribute to the diversity of the DFR breed. Two lines show the largest contributions to the genetic diversity in DFR. One of these lines comprises unique diversity both within the breed and across all cattle breeds. The other line comprises unique diversity for the DFR but overlaps with the Holstein Friesian breed. There seems to be no necessity to conserve the other five lines separately, because their level of differentiation is very low. This study illustrates that, when taking conservation decisions for a breed, it is worthwhile to take into account the population structure of the breed itself and the relationships with other breeds.     

Evaluation of the Biochemical Components and Chromatic Properties of the Juice of <Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Aiton and <Emphasis Type="Italic">Vaccinium oxycoccos</Emphasis> L.

Benzoic acid, total anthocyanins, soluble solids, titratable acidity, and colour properties in juice of the American cranberry Vaccinium macrocarpon and the European cranberry Vaccinium oxycoccos were investigated. Berry juices of V. macrocarpon cultivars were distinguished by their higher total anthocyanin and benzoic acid amounts. These cultivars accumulated on average 43.11 mg/l of benzoic acid and 92.45 mg/l of total anthocyanins. The levels of benzoic acid and total anthocyanins in V. oxycoccos cultivars were 17.52 mg/l and 42.54 mg/l, respectively. The V. macrocarpon cultivars ‘Franklin’, ‘Le Munyon’, ‘Searles’, and ‘Early Richard’ were selected as the best according to the enhanced total anthocyanins and benzoic acid amounts. The separation of anthocyanins by HPLC-UV-VIS revealed the presence of six anthocyanins, with peonidin-3-galactoside being the most prevalent. Galactoside together with glucoside conjugates comprised the largest percentage of total anthocyanins in the juices of V. macrocarpon and V. oxycoccos cultivars.     

Effects of rate of pH fall, time of deboning, aging period, and their interaction on veal quality characteristics

Three groups of veal carcasses were selected on the basis of their pH in the longissimus lumborum muscle at 3 h postmortem (pH3) to study the effects and interaction with time of deboning on quality characteristics of veal. The following groups of 10 calves each were selected: 1) fast pH fall, pH3 < 6.2; 2) intermediate pH fall, 6.2 < pH3 < 6.7; and 3) slow pH fall, pH3 > 6.7. Longissimus thoracis et lumborum (LTL) muscles of sides of the selected carcasses were randomly assigned to be excised at either 24 or 48 h postmortem. Color, water-holding capacity, and shear force measurements were determined after an aging period of 2, 3, 4, 7, and 14 d, respectively. Color of longissimus muscle samples from veal calves with similar preslaughter blood hemoglobin values becomes significantly lighter with a faster pH fall. Muscle color was not affected by time of deboning and overall color of all longissimus samples remained stable during storage and was not affected by rate of pH fall and time of deboning. Cooking losses increased with both a faster rate of pH fall and by deboning at 24 h postmortem. Drip losses during vacuum storage were higher for muscles excised at 24 rather than 48 h postmortem. Both when deboned at 24 and 48 h, postmortem veal carcasses with a lower rate of pH fall had higher shear force (SF) values than did carcasses with a higher rate of pH fall. Deboning at 24 h postmortem resulted in higher SF values than deboning at 48 h postmortem. Differences in SF between 24- and 48-h deboning were larger in slower glycolyzing carcasses. Aging improved tenderness but did not fully reduce the difference in SF values between 24- and 48-h deboning. The results suggest that deboning of veal carcasses before the ultimate pH has been reached may result in muscle contraction, which may exert negative effects on tenderness and water-holding capacity of veal. Effects of time of deboning can, at least partly, be explained by differential effects on shortening of the muscle fibers.     

Mobilization of cesium in organic rich soils: Correlation with production of dissolved organic carbon

A study of the downward movement of ¹³⁷Cs in an undisturbed forest soil is presented. Seasonal variations and depth profiles of ¹³⁷Cs activities were measured in seepage water, which is the transport medium for the downward movement of anthropogenic substances in soils. Furthermore the correlation of ¹³⁷Cs mobilization and production of dissolved organic carbon (DOC) was investigated. Seasonal variations of both ¹³⁷Cs and DOC fluxes in the seepage water in a depth of 5 cm depth were observed, where the maximum fluxes in the summer months were about one order of magnitude higher than the minimum fluxes in the winter months. ¹³⁷Cs fluxes are found to be correlated with DOC fluxes with a correlation coefficient of r = 0.63, and both are highly correlated with soil temperature. This indicates that cesium is bound to soil organic material. The production of DOC is controlled by microbial decomposition of soil organic matter and we assume that this holds true for the ¹³⁷Cs release as well. The actual transport velocity (0.2 ± 0.14 mm/a) of ¹³⁷Cs (calculated by the weighed mean of ¹³⁷Cs concentration in the seepage water and the total ¹³⁷Cs content of the soil) is about one order of magnitude less than the mean transport velocity (1.2 ± 0.3 mm/a) over the past 25 years (calculated from the ¹³⁷Cs depth profile). It is possible that the transport velocity of ¹³⁷Cs in undisturbed soils decreases with time as it binds to aged organic material which is less easily decomposable than fresh organic material.