Three-year study of pre-existing drug resistance substitutions and efficacy of bictegravir/emtricitabine/tenofovir alafenamide in HIV-1 treatment-naive participants.

OBJECTIVES: Two Phase 3, randomized, double-blind, active-controlled studies of initial HIV-1 treatment demonstrated that bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) was non-inferior to dolutegravir/abacavir/lamivudine (DTG/ABC/3TC; Study 1489) or to DTG+F/TAF (Study 1490) through 144 weeks. In both studies, there was no emergent resistance to study drugs. Here, the 3 year resistance analysis and impact of baseline resistance substitutions on treatment response are described. METHODS: Population sequencing of HIV-1 protease and reverse transcriptase (RT) was performed at screening. Retrospective baseline next generation sequencing of protease, RT and integrase (IN) was analysed at a ≥ 15% cutoff. Resistance analyses were performed on participants with confirmed viral rebound of HIV-1 RNA ≥200 copies/mL through Week 144 or last visit who did not resuppress to <50 copies/mL while on study drug. RESULTS: Transmitted primary drug resistance substitutions were present in the following proportions of participants: integrase strand transfer inhibitor (INSTI) resistance (-R) in 1.3% (17/1270) of participants; NRTI-R in 2.7% (35/1274); NNRTI-R in 14.1% (179/1274); and PI-R in 3.5% (44/1274). These pre-existing resistance substitutions not associated with study drug did not affect treatment outcomes. One participant in the B/F/TAF group had pre-existing bictegravir and dolutegravir resistance substitutions (Q148H+G140S in integrase) at baseline and suppressed and maintained HIV-1 RNA <50 copies/mL through Week 144. In total, 21 participants qualified for resistance testing [1.3% (8/634) B/F/TAF; 1.9% (6/315) DTG/ABC/3TC; 2.2% (7/325) DTG+F/TAF]; none had emergent resistance to study drugs. CONCLUSIONS: Treatment with B/F/TAF, DTG/ABC/3TC, or DTG+F/TAF achieved high, durable rates of virological suppression in HIV-1 treatment-naive participants. The presence of pre-existing resistance substitutions did not affect treatment outcomes, and there was no treatment-emergent resistance.

Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78-100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.

Transcriptome analysis and identification of genes associated with ω-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens.

BACKGROUND: Perilla (Perilla frutescens (L.) var frutescens) produces high levels of α-linolenic acid (ALA), a ω-3 fatty acid important to health and development. To uncover key genes involved in fatty acid (FA) and triacylglycerol (TAG) synthesis in perilla, we conducted deep sequencing of cDNAs from developing seeds and leaves for understanding the mechanism underlying ALA and seed TAG biosynthesis. RESULTS: Perilla cultivar Dayudeulkkae contains 66.0 and 56.2 % ALA in seeds and leaves, respectively. Using Illumina HiSeq 2000, we have generated a total of 392 megabases of raw sequences from four mRNA samples of seeds at different developmental stages and one mature leaf sample of Dayudeulkkae. De novo assembly of these sequences revealed 54,079 unique transcripts, of which 32,237 belong to previously annotated genes. Among the annotated genes, 66.5 % (21,429 out of 32,237) showed highest sequences homology with the genes from Mimulus guttatus, a species placed under the same Lamiales order as perilla. Using Arabidopsis acyl-lipid genes as queries, we searched the transcriptome and identified 540 unique perilla genes involved in all known pathways of acyl-lipid metabolism. We characterized the expression profiles of 43 genes involved in FA and TAG synthesis using quantitative PCR. Key genes were identified through sequence and gene expression analyses. CONCLUSIONS: This work is the first report on building transcriptomes from perilla seeds. The work also provides the first comprehensive expression profiles for genes involved in seed oil biosynthesis. Bioinformatic analysis indicated that our sequence collection represented a major transcriptomic resource for perilla that added valuable genetic information in order Lamiales. Our results provide critical information not only for studies of the mechanisms involved in ALA synthesis, but also for biotechnological production of ALA in other oilseeds.

Expression of Castor LPAT2 Enhances Ricinoleic Acid Content at the sn-2 Position of Triacylglycerols in Lesquerella Seed.

Lesquerella is a potential industrial oilseed crop that makes hydroxy fatty acid (HFA). Unlike castor its seeds are not poisonous but accumulate lesquerolic acid mostly at the sn-1 and sn-3 positions of triacylglycerol (TAG), whereas castor contains ricinoleic acid (18:1OH) at all three positions. To investigate whether lesquerella can be engineered to accumulate HFAs in the sn-2 position, multiple transgenic lines were made that express castor lysophosphatidic acid acyltransferase 2 (RcLPAT2) in the seed. RcLPAT2 increased 18:1OH at the sn-2 position of TAGs from 2% to 14%-17%, which resulted in an increase of tri-HFA-TAGs from 5% to 13%-14%. Our result is the first example of using a LPAT to increase ricinoleic acid at the sn-2 position of seed TAG. This work provides insights to the mechanism of HFA-containing TAG assembly in lesquerella and directs future research to optimize this plant for HFA production.

Current progress towards the metabolic engineering of plant seed oil for hydroxy fatty acids production.

KEY MESSAGE: Hydroxy fatty acids produced in plant seed oil are important industrial material. This review focuses on the use of metabolic engineering approaches for the production of hydroxy fatty acids in transgenic plants. Vegetable oil is not only edible but can also be used for industrial purposes. The industrial demand for vegetable oil will increase with the continued depletion of fossil fuels and ensuing environmental issues such as climate change, caused by increased carbon dioxide in the air. Some plants accumulate high levels of unusual fatty acids in their seeds, and these fatty acids (FAs) have properties that make them suitable for industrial applications. Hydroxy fatty acids (HFAs) are some of the most important of these industrial FAs. Castor oil is the conventional source of HFA. However, due to the presence of toxin ricin in its seeds, castor is not cultivated on a large scale. Lesquerella is another HFA accumulator and is currently being developed as a new crop for a safe source of HFAs. The mechanisms of HFA synthesis and accumulation have been extensively studied using castor genes and the model plant Arabidopsis. HFAs accumulated to 17% in the seed oil of Arabidopsis expressing a FA hydroxylase gene from castor (RcFAH12), but its seed oil content and plant growth decreased. When RcFAH12 gene was coexpressed with additional castor gene(s) in Arabidopsis, ~30% HFAs were accumulated and the seed oil content and plant growth was almost restored to the wild-type level. Further advancement of our understanding of pathways, genes and regulatory mechanisms underlying synthesis and accumulation of HFAs is essential to developing and implementing effective genetic approaches for enhancing HFA production in oilseeds.

Enhanced production of hydroxy fatty acids in Arabidopsis seed through modification of multiple gene expression.

BACKGROUND: Castor (Ricinus communis L.) seeds contain unusual fatty acid, hydroxy fatty acid (HFA) used as a chemical feedstock for numerous industrial products. Castor cultivation is limited by the potent toxin ricin in its seeds and other poor agronomic traits, so it is advantageous to develop a suitable HFA-producing crop. Significant research efforts have been made to produce HFA in model Arabidopsis, but the level of HFA produced in transgenic Arabidopsis is much less than the level found in castor seeds which produce 90% HFA in seed oil. RESULTS: We designed a transformation construct that allowed co-expression of five essential castor genes (named pCam5) involved in HFA biosynthesis, including an oleate [Formula: see text] 12-hydroxylase (FAH12), diacylglycerol (DAG) acyltransferase 2 (DGAT2), phospholipid: DAG acyltransferase 1-2 (PDAT1-2), phosphatidylcholine (PC): DAG cholinephosphotransferase (PDCT) and Lyso-PC acyltransferase (LPCAT). Transgenic Arabidopsis pCam5 lines produced HFA counting for 25% in seed oil. By knocking out Arabidopsis Fatty acid elongase 1 (AtFAE1) in pCam5 using CRISPR/Cas9 technology, the resulted pCam5-atfae1 lines produced over 31% of HFA. Astonishingly, the pCam5-atfae1 line increased seed size, weight, and total oil per seed exceeding wild type by 40%. Seed germination, seedling growth and seed mucilage content of pCam5-atfae1 lines were not affected by the genetic modification. CONCLUSIONS: Our results provide not only insights for future research uncovering mechanisms of HFA synthesis in seed, but also metabolic engineering strategies for generating safe HFA-producing crops.

Genetic Engineering of Lesquerella with Increased Ricinoleic Acid Content in Seed Oil.

Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1-26.6% compared with that of 0.4-0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality.

Tobacco cessation in low- to middle-income countries: A scoping review of randomized controlled trials.

OBJECTIVE: The growing prevalence of tobacco use in low "to middle" income countries (LMICs) and the hurdles of conducting tobacco cessation in that context necessitates a focus on the scope of randomized controlled trials (RCTs) in LMICs to guide tobacco cessation in this environment. We conducted a scoping review to identify LMIC tobacco cessation RCTs. METHODS: Consistent with PRISMA-ScR guidelines and without language restrictions, we systematically searched peer-reviewed databases (MEDLINE, Embase, PsycINFO, articles published since inception, latest searches in March 2020) and gray literature (clinical trials registries, searches between September and December 2019). We searched for data on RCT type, outcome significance and intervention description. Inclusion: research conducted in LMICs; tobacco cessation; RCT. Exclusion: research conducted in high income countries; non-RCT; studies involving only those aged <18. Data was extracted from published reports. We generated narrative summaries of each LMIC's tobacco cessation RCT research environment. RESULTS: Of 8404 articles screened, we identified 92 studies. Tobacco cessation RCTs were recorded in 16 of 138 countries/territories in LMICs. Evidence was weak in quality and severely limited. Most RCTs were psychosocial, with limited behavioral and pharmacological variants. CONCLUSIONS: Tobacco control within LMICs is essential to reduce the tobacco mortality burden. Researchers should be cognizant that tobacco cessation in LMICs is still not an environment where best practice has been established. We suggest that developing solutions specific for LMICs is key to effective tobacco control in LMICs.

A transgene design for enhancing oil content in Arabidopsis and Camelina seeds.

BACKGROUND: Increasing the oil yield is a major objective for oilseed crop improvement. Oil biosynthesis and accumulation are influenced by multiple genes involved in embryo and seed development. The leafy cotyledon1 (LEC1) is a master regulator of embryo development that also enhances the expression of genes involved in fatty acid biosynthesis. We speculated that seed oil could be increased by targeted overexpression of a master regulating transcription factor for oil biosynthesis, using a downstream promoter for a gene in the oil biosynthesis pathway. To verify the effect of such a combination on seed oil content, we made constructs with maize (Zea mays) ZmLEC1 driven by serine carboxypeptidase-like (SCPL17) and acyl carrier protein (ACP5) promoters, respectively, for expression in transgenic Arabidopsis thaliana and Camelina sativa. RESULTS: Agrobacterium-mediated transformation successfully generated Arabidopsis and Camelina lines that overexpressed ZmLEC1 under the control of a seed-specific promoter. This overexpression does not appear to be detrimental to seed vigor under laboratory conditions and did not cause observable abnormal growth phenotypes throughout the life cycle of the plants. Overexpression of ZmLEC1 increased the oil content in mature seeds by more than 20% in Arabidopsis and 26% in Camelina. CONCLUSION: The findings suggested that the maize master regulator, ZmLEC1, driven by a downstream seed-specific promoter, can be used to increase oil production in Arabidopsis and Camelina and might be a promising target for increasing oil yield in oilseed crops.0.

Temporal and spatial expression of 2S albumin in castor (Ricinus communis L.).

We studied the temporal and spatial expression of the 2S albumin in castor (Ricinus communis L.) during seed development, germination, post-germination, and plant development. Quantitative polymerase chain reaction analysis showed that the 2S albumin transcript accumulated to a maximum level at the middle of seed development, showing a bell-shaped temporal pattern. Residual levels of the transcript were present in the mature seed and degraded rapidly upon germination. Immunodetection analysis was performed using an anti-2S albumin antibody under reducing conditions. During seed development, the 2S albumin precursor pro-protein began to be synthesized at 26 days after pollination (DAP); the pro-protein was thereafter processed to mature proteins at 40 DAP, suggesting that the post-translation modification of 2S albumin takes place during this time period. Both the 2S albumin precursor pro-protein and the mature proteins accumulated throughout seed maturation and desiccation stages. During seed germination, both forms of the 2S albumin proteins were present in endosperm and cotyledon until the completion of germination and degraded rapidly afterwards. However, the antibody also detected a group of proteins/peptides in endosperm and cotyledon when the seeds progressed to germination and post-germination stages. A 14 kDa protein in the leaves of fully developed seedlings and mature plants also reacted to the anti-2S albumin antibody. The identity of the proteins accumulated in germinating seed and leaf remains unknown.

Detection of castor contamination by real-time polymerase chain reaction.

Due to the potential for intentional contamination of food with crude preparations containing ricin, a real-time PCR method was developed for the detection of castor plant material in ground beef. One primer pair was identified and confirmed to be castor-specific and efficient for amplification of ricin in DNA extracts from castor or beef matrices. Of three different DNA extraction protocols compared, the hexadecyltrimethylammonium bromide (CTAB) method yielded the highest quality of DNA for QPCR assay. The detection limit for castor contamination in ground beef samples was <0.001% (<10 microg of castor acetone powder per gram of beef, corresponding to 0.5 microg of ricin), indicating excellent sensitivity for the assay, well below the threshold for oral toxicity.

Ricinus communis contains an acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester.

As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain complete coding regions corresponding to 694 and 690 amino acids, respectively. The third cDNA, RcACS 1, encodes a truncated gene sequence. The RcACS 2 and RcACS 4 share 77% identity at the amino acid sequence level. Complementation tests showed that both RcACS 2 and RcACS 4 successfully restored growth of a yeast mutant strain (YB525) deficient in ACS. Lysates from yeast cells expressing RcACS 2 and 4 were enzymatically active when using 14C-labeled oleic acid as a substrate. A cell fractionation study indicates that RcACS 2 and 4 are mainly associated with membranes. Substrate specificity assays indicate that the RcACS 2 preferentially activates ricinoleate, while the RcACS 4 has a preference for nonhydroxy fatty acids.

Expression profiles of genes involved in fatty acid and triacylglycerol synthesis in castor bean (Ricinus communis L.).

Castor seed triacylglycerols (TAGs) contain 90% ricinoleate (12-hydroxy-oleate) which has numerous industrial applications. Due to the presence of the toxin ricin and potent allergenic 2S albumins in the seed, it is desirable to produce ricinoleate from temperate oilseeds. To identify regulatory genes or genes for enzymes that may up-regulate multiple activities or entire pathways leading to the ricinoleate and TAG synthesis, we have analyzed expression profiles of 12 castor genes involved in fatty acid and TAG synthesis using quantitative reverse transcription-polymerase chain reaction technology. A collection of castor seeds with well-defined developmental stages and morphologies was used to determine the levels of mRNA, ricinoleate and TAG. The synthesis of ricinoleate and TAG occurred when seeds progressed to stages of cellular endosperm development. Concomitantly, most of the genes increased their expression levels, but showed various temporal expression patterns and different maximum inductions ranging from 4- to 43,000-fold. Clustering analysis of the expression data indicated five gene groups with distinct temporal patterns. We identified genes involved in fatty acid biosynthesis and transport that fell into two related clusters with moderate flat-rise or concave-rise patterns, and others that were highly expressed during seed development that displayed either linear-rise or bell-shaped patterns. Castor diacylglycerol acyltransferase 1 was the only gene having a higher expression level in leaf and a declining pattern during cellular endosperm development. The relationships among gene expression, cellular endosperm development and ricinoleate/TAG accumulation are discussed.

Diacylglycerol acyltransferase activity and triacylglycerol synthesis in germinating castor seed cotyledons.

The central importance of storage lipid breakdown in providing carbon and energy during seed germination has been demonstrated by isolating the genes encoding the enzymes involved in FA beta-oxidation. In contrast, little is known about the ability of germinating seeds to synthesize TAG. We report that castor cotyledons are capable of TAG synthesis. The rate of incorporation of ricinoleic acid into TAG reached a peak at 7 d after imbibition (DAI) (1.14 nmol/h/mg) and decreased rapidly thereafter, but was sustained at 20 DAI in cotyledons and true leaves. The castor DAG acyltransferase (RcDGAT) mRNA and protein were expressed throughout seed germination at levels considerably enhanced from that in the dormant seed, thus indicating new expression. Significant degradation of the RcDGAT protein was observed after 7 DAI. The DGAT activity was found to be predominantly a function of the level of the intact RcDGAT protein, with the rate of TAG synthesis decreasing as degradation of the RcDGAT protein proceeded. A possible mechanism for the degradation of the RcDGAT protein is discussed. The induction of DGAT mRNA and protein, the capacity for TAG synthesis in vitro and in tissue slices, and the differing TAG composition of dormant seed TAG vs. cotyledonary TAG provide strong circumstantial evidence for active TAG synthesis by cotyledons. However, we have not yet determined the physiological significance of this capability.

A simple and sensitive assay for distinguishing the expression of ricin and Ricinus communis agglutinin genes in developing castor seed (R. communis L.).

Castor oil is the only commercial source of ricinoleic acid and has numerous industrial applications. Among the factors limiting domestic production of castor oil is the presence of the toxin ricin and its less toxic homologue Ricinus communis agglutinin (RCA) in seeds. Although the sequences of ricin and RCA genes are known, their transcriptional expression patterns have not been distinguished due to their high degree of sequence similarity. As the information is critical for assessing success in developing a ricin-free castor crop using genetic silencing, we have designed a gene specific reverse transcription-polymerase chain reaction (RT-PCR) assay to examine the expression of the ricin and RCA genes in developing seeds. The results show that the ricin and RCA mRNA are highly abundant in seeds during the development of endosperm, and the expression pattern is similar to that observed in the Northern analysis. The RT-PCR results can be confirmed by a simple RT-PCR-based restriction fragment analysis.

Regulation of diacylglycerol acyltransferase in developing seeds of castor.

We have previously reported the cloning of castor diacylglycerol acyltransferase (RcDGAT) based on its homology to other plant type 1 diacylglycerol acyltransferases (DGATs). To elucidate the physiological role of the RcDGAT, we have investigated the regulation of RcDGAT expression in developing seeds of castor. The RcDGAT transcript appeared at 12 d after pollination (DAP), reached the highest level at 26 DAP, and declined rapidly after that. However, the RcDGAT protein started to accumulate at 26 DAP, reached its peak at 47 DAP, then remained at this high level until 54 DAP. The significant difference between the expression of mRNA and protein indicates that gene expression of RcDGAT in maturing castor seeds is controlled at the posttranscriptional level. We found that DGAT activity measured in microsomal membranes isolated from seed at different stages of development was parallel to RcDGAT protein level, suggesting DGAT activity is mainly a function of the level of RcDGAT protein. We monitored the triacylglycerol (TG) composition and content during seed development. Compared with the overall rate of TG accumulation, DGAT activity appeared coincidently with the onset of lipid accumulation at 26 DAP; the highest DGAT activity occurred during the rapid phase of lipid accumulation at 40 DAP; and a decline in DGAT activity coincided with a decline in the accumulation rate of TG after 40 DAP. The ricinoleate-containing TG content was very low (only about 7%) in oil extracted from seeds before 19 DAP; however, it increased up to about 77% of the oil at 26 DAP. The relative amount of triricinolein in oil at 26 DAP was 53 times higher than that at 19 DAP, and it was about 76% of the amount present in oil from mature castor seeds. The close correlation between profiles of RcDGAT activity and oil accumulation confirms the role of RcDGAT in castor oil biosynthesis.

Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean.

The oil from castor seed (Ricinus communis) contains 90% ricinoleate, a hydroxy FA that is used in producing numerous industrial products. Castor diacylglycerol acyltransferase (RcDGAT) is a critical enzyme, as it catalyzes the terminal step in castor oil biosynthesis in which the products contain two or three ricinoleoyl moieties. We have isolated a cDNA encoding RcDGAT from developing castor seeds. Analysis of the sequence reveals that this cDNA encodes a protein of 521 amino acids with a molecular mass of 59.9 kDa. Although there are regions of high similarity to other plant DGAT coding sequences, there are sequences that distinguish it as well. Southern blot analysis suggests that the castor genome contains a single copy of RcDGAT. Analysis by reverse transcription-PCR reveals that the accumulation of the mRNA reaches its highest level at 19 d after pollination and declines thereafter. Expression of the full-length cDNA for RcDGAT in the yeast Saccharomyces cerevisiae, strain INVSc1 results in sevenfold higher DGAT activity compared with controls. When different molecular species of DAG were provided as substrates to the microsomal mixture, the RcDGAT showed a greater preference to catalyze the transfer of oleate from [14C]oleoyl-CoA to diricinolein than to diolein and dipalmitolein. With the addition of 0.25 mM substrates, diricinolein gave 318 pmol/mg/min diricinoleoyloleoylglycerol (RRO), while diolein and dipalmitolein gave only about 195 pmol/mg/min of triolein (OOO) and 120 pmol/mg/min dipalmitoyleoylglycerol (PoPoO), respectively. This work will facilitate investigation of the role of RcDGAT in castor oil biosynthesis.

Identification of diacylglycerol and triacylglycerol containing 11,12,13-trihydroxy-9,14-octadecadienoic acid in castor oil.

Castor oil has many industrial uses. Molecular species of acylglycerols containing monohydroxy, dihydroxy and trihydroxy fatty acids in castor oil have been reported. We report here the identification of acylglycerols containing a triOH18:2 fatty acid in castor oil. The structure of this novel fatty acid was proposed as 11,12,13-trihydroxy-9,14-octadecadienoic acid by the mass spectrometry of the lithiated adducts of acylglycerols in the HPLC fractions of castor oil. The fragmentation pathways of the lithiated adduct of 11,12,13-trihydroxy-9,14-octadecadienoic acid were proposed. We also proposed the biosynthetic pathways of polyhydroxy fatty acids in castor.