scholarly journals Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

2020 ◽  
Vol 6 (32) ◽  
pp. eabb3521 ◽  
Author(s):  
Xixian Wang ◽  
Yi Xin ◽  
Lihui Ren ◽  
Zheng Sun ◽  
Pengfei Zhu ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Signal Quality ◽  
Label Free ◽  
Theoretical Limit ◽  
Degree Of Unsaturation ◽  
Conflicting Demands ◽  
Low Levels ◽  
Droplet Sorting ◽  
Positive Dielectrophoresis

The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min−1 throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min−1. From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid–rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman–independent phenotypes greatly expands RACS’ application.

scholarly journals Cocktail biosynthesis of triacylglycerol by rational modulation of diacylglycerol acyltransferases in industrial oleaginous Aurantiochytrium

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Chuanzeng Lan ◽  
Sen Wang ◽  
Huidan Zhang ◽  
Zhuojun Wang ◽  
Weijian Wan ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Ex Vivo ◽  
Saturated Fatty Acids ◽  
Degree Of Unsaturation ◽  
Promising Target ◽  
Oleaginous Microorganism ◽  
Key Enzyme ◽  
Functional Distribution

Abstract Background Triacylglycerol (TAG) is an important storage lipid in organisms, depending on the degree of unsaturation of fatty acid molecules attached to glycerol; it is usually used as the feedstock for nutrition or biodiesel. However, the mechanism of assembly of saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) into TAGs remains unclear for industrial oleaginous microorganism. Results Diacylglycerol acyltransferase (DGAT) is a key enzyme for TAG synthesis. Hence, ex vivo (in yeast), and in vivo functions of four DGAT2s (DGAT2A, DGAT2B, DGAT2C, and DGAT2D) in industrial oleaginous thraustochytrid Aurantiochytrium sp. SD116 were analyzed. Results revealed that DGAT2C was mainly responsible for connecting PUFA to the sn-3 position of TAG molecules. However, DGAT2A and DGAT2D target SFA and/or MUFA. Conclusions There are two specific TAG assembly routes in Aurantiochytrium. The “saturated fatty acid (SFA) TAG lane” primarily produces SFA-TAGs mainly mediated by DGAT2D whose function is complemented by DGAT2A. And, the “polyunsaturated fatty acid (PUFA) TAG lane” primarily produces PUFA-TAGs via DGAT2C. In this study, we demonstrated the functional distribution pattern of four DGAT2s in oleaginous thraustochytrid Aurantiochytrium, and provided a promising target to rationally design TAG molecular with the desired characteristics.

scholarly journals Toward Quantitative in vivo Label-Free Tracking of Lipid Distribution in a Zebrafish Cancer Model

2021 ◽  
Vol 9 ◽  
Author(s):  
Marco Andreana ◽  
Caterina Sturtzel ◽  
Clemens P. Spielvogel ◽  
Laszlo Papp ◽  
Rainer Leitgeb ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Cancer Cells ◽  
Lipid Accumulation ◽  
Fatty Acid Uptake ◽  
Alternative Mechanism ◽  
Acid Uptake ◽  
Label Free ◽  
Tumor Onset

Cancer cells often adapt their lipid metabolism to accommodate the increased fatty acid demand for membrane biogenesis and energy production. Upregulation of fatty acid uptake from the environment of cancer cells has also been reported as an alternative mechanism. To investigate the role of lipids in tumor onset and progression and to identify potential diagnostic biomarkers, lipids are ideally imaged directly within the intact tumor tissue in a label-free way. In this study, we investigated lipid accumulation and distribution in living zebrafish larvae developing a tumor by means of coherent anti-Stokes Raman scattering microscopy. Quantitative textural features based on radiomics revealed higher lipid accumulation in oncogene-expressing larvae compared to healthy ones. This high lipid accumulation could reflect an altered lipid metabolism in the hyperproliferating oncogene-expressing cells.

Effect of fatty acid structure on esterification by the small intestine in vitro

1963 ◽  
Vol 204 (5) ◽  
pp. 821-824 ◽  
Author(s):  
Alvin M. Gelb ◽  
Jacques I. Kessler
Keyword(s):  
Fatty Acids ◽  
Small Intestine ◽  
Fatty Acid ◽  
Small Bowel ◽  
Chain Length ◽  
Degree Of Unsaturation ◽  
Fatty Acid Structure ◽  
Acid Structure

The effect of chain length and degree of unsaturation of fatty acids (FA) on in vitro esterification by slices of hamster small intestine was observed in a medium containing C14-labeled FA. After incubation, lipids were extracted and separated and the radioactivity in the esterified lipids was measured. Comparative experiments, in which results were expressed as per cent of substrate esterified per 100 mg tissue, indicate that for saturated FA, maximal esterification occurred with myristic acid, 14 carbons. As chain length was either increased or decreased, percentage esterification decreased. FA with 8 carbons or less were only minimally esterified. Among 18-carbon FA, two unsaturated bonds significantly decreased percentage esterification, although one unsaturated bond did not. These results suggest that, at least in vitro, the small bowel esterifies FA at varying rates depending upon chain length and degree of unsaturation. These differences are in the same direction as differences in absorption and partition of FA in vivo previously reported by others.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

scholarly journals GPR40 Is Necessary but Not Sufficient for Fatty Acid Stimulation of Insulin Secretion In Vivo

Diabetes ◽  
2007 ◽  
Vol 56 (4) ◽  
pp. 1087-1094 ◽  
Author(s):  
M. G. Latour ◽  
T. Alquier ◽  
E. Oseid ◽  
C. Tremblay ◽  
T. L. Jetton ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fatty Acid ◽  
Insulin Secretion In Vivo ◽  
Stimulation Of

scholarly journals Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2543
Author(s):  
Ruidong Ni ◽  
Suzeeta Bhandari ◽  
Perry R. Mitchell ◽  
Gabriela Suarez ◽  
Neel B. Patel ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fatty Acid ◽  
Apis Mellifera ◽  
Chemical Synthesis ◽  
Acid Amide ◽  
Fatty Acid Amides ◽  
Fatty Acid Amide

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

Sign in / Sign up

Export Citation Format

Share Document