In-Silico-Generated Library for Sensitive Detection of 2-Dimethylaminoethylamine Derivatized FAHFA Lipids Using High-Resolution Tandem Mass Spectrometry.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a family of recently discovered lipids with important physiological functions in mammals and plants. However, low detection sensitivity in negative ionization mode mass spectrometry makes low-abundance FAHFA challenging to analyze. A 2-dimethylaminoethylamine (DMED) based chemical derivatization strategy was recently reported to improve the MS sensitivity of FAHFAs by labeling FAHFAs with a positively ionizable tertiary amine group. To facilitate reliable, high-throughput, and automatic annotation of these compounds, a DMED-FAHFA in silico library containing 4290 high-resolution tandem mass spectra covering 264 different FAHFA classes was developed. The construction of the library was based on the heuristic information from MS/MS fragmentation patterns of DMED-FAHFA authentic standards, and then, the patterns were applied to computer-generated DMED-FAHFAs. The developed DMED-FAHFA in silico library was demonstrated to be compatible with library search software NIST MS Search and the LC-MS/MS data processing tool MS-DIAL to guarantee high-throughput and automatic annotations. Applying the in silico library in Arabidopsis thaliana samples for profiling FAHFAs by high-resolution LC-MS/MS enabled the annotation of 19 DMED-FAHFAs from 16 families, including 3 novel compounds. Using the in silico library largely decreased the false-positive annotation rate in comparison to low-resolution LC-MS/MS. The developed library, MS/MS spectra, and development templates are freely available for commercial and noncommercial use at https://zenodo.org/record/3606905.

Comprehensive Screening and Identification of Fatty Acid Esters of Hydroxy Fatty Acids in Plant Tissues by Chemical Isotope Labeling-Assisted Liquid Chromatography-Mass Spectrometry.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of lipid mediators with promising anti-diabetic and anti-inflammatory properties. Comprehensive screening and identification of FAHFAs in biological samples would be beneficial to the discovery of new FAHFAs and enable greater understanding of their biological functions. Here, we report the comprehensive screening of FAHFAs in rice and  Arabidopsis thaliana by chemical isotope labeling-assisted liquid chromatography-mass spectrometry (CIL-LC-MS). Multiple reaction monitoring (MRM) was used for screening of FAHFAs. With the proposed method, we detected 49 potential FAHFA families, including 262 regioisomers, in tissues of rice and Arabidopsis thaliana, which greatly extends our knowledge of known FAHFAs. In addition, we proposed a strategy to identify FAHFA regioisomers based on their retention on a reversed-phase LC column. Using the proposed identification strategy, we identified 71 regioisomers from 11 FAHFA families based on commercial standards and characteristic chromatographic retention behaviors. The screening technique could allow for the discovery of new FAHFAs in biological samples. The new FAHFAs identified in this work will contribute to the in-depth study of the functions of FAHFAs.

A magnetically enhanced RF discharge source for metastable krypton production.

We describe a high intensity metastable Kr source based on a helical resonator RF discharge. By adding an external B-field to the discharge source, the metastable Kr flux is enhanced. The effect of geometric configuration and magnetic field strength has been studied and optimized experimentally. Compared to the helical resonator discharge source without an external B-field, the new source showed an enhancement factor of 4-5 in producing metastable Kr beams. This improvement has a direct impact on the radio-krypton dating applications as it can increase the atom count rate, resulting in a higher analytical precision.

Classification of contrast-enhanced ultrasonograms in rectal cancer according to tumor inhomogeneity using machine learning-based texture analysis.

Background: Inhomogeneity within tumors can reflect tumor angiogenesis. Existing research into the quantization of angiogenesis mainly focuses on time-intensity curve parameters but has produced inconsistent results. In clinical work, it is difficult to achieve standardization and consistency for manual judgement of the inhomogeneity of contrast-enhanced images, while the artificial intelligence technology may be helpful. The aim of this study was to assess whether computers can assist in the artificial classification of tumor inhomogeneity in contrast-enhanced ultrasound (CEUS) images of rectal cancer. Methods: A total of 500 contrast-enhanced ultrasonograms were retrospectively collected, which was verified of rectal cancer pathologically from 2016 to 2018 as training set. All images are from 18-80 years old patients with rectal cancer in our hospital. These tumors are usually located in the middle and lower segment of the rectum, which can be completely observed on ultrasound. The images were divided into 3 categories according to the inhomogeneous distribution of contrast agents inside the tumors. Computing methods were used to simulate manual classification. Computer processing steps included segmentation, gray level quantization, dimension reduction, and classification. The results of 6 different gray level quantization, 2 dimensionality reduction methods, and 3 classifiers were compared, from which the optimal parameters were selected in each step. The performance of computer classification was evaluated using manual classification results as the reference. Ninety-seven ultrasonograms of contrast-enhanced rectal tumors were collected as validation set from 2018.1 to 2018.6. Results: The optimal gray level was set at 32. Principal component analysis (PCA) was the first choice for dimensionality reduction. The best classifier was support vector machines (SVM). The accuracy of computer classification was 87.80% (439/500). The accuracy of computer classification in the validation cohort was 60.82%. The area under the curve (AUC) of class 1, 2, and 3 were 0.76, 0.41, and 0.48, respectively. Conclusions: Results showed that the computer methods are competent for classifying inhomogeneity of contrast-enhanced rectal cancers inside ultrasonograms.

FAHFA footprint in the visceral fat of mice across their lifespan.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with anti-inflammatory and anti-diabetic effects, having the potential to treat type 2 diabetes (T2D). In view of the important regulatory and therapeutic actions of FAHFAs on age-related diseases such as T2D, we hypothesized that they may also play crucial roles in the growth, development, and aging process. Here, we investigated the FAHFA footprint in the visceral adipose tissue (VAT) of mice across lifespan to attain potential clues for understanding the roles of FAHFAs in growth, development, and aging using chemical isotope labeling assisted liquid chromatography-mass spectrometry. VAT samples were harvested from 80 C57BL/6J male mice of nine different ages (1, 2, 3, 6, 9, 12, 15, 18, and 24 months). The results showed that a total of 51 FAHFA families, including 301 regioisomers, were detected in the VAT of mice of all ages, and the number of FAHFAs (both family and regioisomer) in VAT increased with age, from 35 families (186 ± 0 regioisomers) at 1-month-old mice to 46 families (278 ± 6 regioisomers) in 18-month-old mice. Furthermore, the content of 12 FAHFA families per 100 mg of VAT of mice was highly correlated with age, and was usually low in the middle-age (3-15 months). However, because the VAT mass was 4-5 fold higher in middle-aged mice compared to younger or older mice, the total amount of most of the FAHFA regioisomers in VAT was increased in middle-aged mice. To the best of our knowledge, this is the first study to show that the number of regioisomers from 51 FAHFA families and abundance of 15 FAHFA families are strongly dependent on age, which would be helpful for understanding the mechanisms underlying the effects of FAHFAs on growth, development, and aging.

Highly sensitive determination of fatty acid esters of hydroxyl fatty acids by liquid chromatography-mass spectrometry.

Recently, a new class of endogenous lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), was discovered with anti-diabetic and anti-inflammatory effects in mammals. FAHFAs attracted increasing attention because of their critical physiological function. However, accurate quantitation of FAHFAs is still a challenge due to their high structure similarity and low abundance in biological samples. Herein, we developed a highly sensitive method for the determination of 16 FAHFAs (PAHSAs, OAHSAs, SAHSAs and POHSAs) in biological samples by coupling strong anion exchange solid phase extraction (SAX-SPE) with chemical labeling assisted ultra-high performance liquid chromatography/mass spectrometry (SAX-SPE-CL-UHPLC/MS). In the developed method, SAX-SPE was employed to selectively enrich and purify FAHFAs from biological samples. And then a pair of isotope labeling reagents, 2-dimethylaminoethylamine (DMED) and d4-DMED were used to label the purified samples and standard FAHFAs, respectively. The labeled samples were mixed and further subjected to UHPLC/MS analysis. Our results demonstrated that the detection sensitivities of FAHFAs increased by 7-72 folds upon DMED labeling and the limits of detections (LODs) of labeled FAHFAs ranged from 0.01 to 0.14pg. Moreover, a good separation of FAHFAs isomers was achieved on C18 column in a UHPLC system and all FAHFAs could be analyzed in 20min with sharp peak shape. The established method provided substantial sensitivity, high specificity, and broad linear dynamic range (3 orders of magnitude). Using this method, we successfully measured the contents and distribution of FAHFAs in rat white adipose, lung, kidney, thymus, liver and heart tissues. The results showed that 7 FAHFAs (13-, 12-, 9-, 5-PAHSA, 13-, 12- and 9-SAHSA) were observed in different tissues of rat. In addition, we successfully detected the above 7 FAHFAs in human serum samples; and among the 7 FAHFAs, 13-, 9-PAHSA, 13- and 12-SAHSA were found remarkably decreased in human breast cancer serum. The proposed method could be successfully applied for the detection of FAHFAs in various biological samples, which will facilitate the understanding of the physiological functions of FAHFAs.