extfieldsofstudy: Biology Medicine

text:
Longikaurin A, a natural ent-kaurane, induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.

Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies reported worldwide, and accounts for 4.6% of all neoplasias. HCC is clinically resistant to conventional chemotherapy treatments, and is documented to have a 94% mortality rate. 1,2 There is thus an urgent need to develop more effective therapeutic agents to minimize both the incidence and severity of HCC.
Many anticancer agents dampen malignant growth by arresting the cell cycle at the G1, S or G2/M phases. 3 E3 ubiquitin ligases are a class of enzymes which regulate cell cycle progression, and changes in their activity can contribute to malignant cell proliferation. 4 S phase kinase-associated protein 2 (Skp2), is the F-box component of an E3 ubiquitin ligase, and may interact with several proteins, including p21 Cip1 , 5-7 p27 Kip1 , [8][9][10] p57 Kip2 and p130. 11,12 Skp2 function likely leads to degradation of tumor suppressor proteins, such as Cdk inhibitors p21 and p27, inducing formation of the Cdk complex and thereby accelerating cell cycle progression. Skp2 is detected at increased levels in several human cancers. 13 There is compelling evidence that cellular adaptation to reactive oxygen species (ROS) stress has a part in maintaining a cellular cancer phenotype and chemotherapy resistance. 14 Even a modest increase in ROS levels can stimulate cell growth and proliferation. 15,16 However, excessive ROS production surmounts cellular antioxidant defenses, triggering apoptosis. 17 Interestingly, cancer cells are more sensitive to rapid increases in ROS levels than are normal cells. Oncogenic transformation elevates basal ROS levels significantly so that any further acute increases can trigger reactivation of the apoptotic program in cancer cells. 18 Production of abnormal amounts of ROS interferes with cellular signaling pathways by free radical targeting of cellular macromolecules including proteins and DNA, as well as by triggering cell cycle arrest and apoptosis. 19,20 Diterpenoid compounds are established to have a crucial role in cancer chemotherapy. Isodon ternifolius (D. Don) Kudô , a prominent species of the Isodon genus, is known for producing bioactive ent-kaurane diterpenoids, [21][22][23] and has long been used as folk medicine for the treatment of icterohepatitis, enteritis and other inflammatory conditions. 24 I. ternifolius is also the major ingredient of a Chinese patent medicine 'FufangSanyexiangchacaiPian', which is used to treat acute and chronic hepatitis and hepatitis B. Longikaurin A (LK-A), shown in Figure 1a, is a major ent-kaurane diterpenoid produced by I. ternifolius. 25 To the best of our knowledge, the effectiveness of LK-A treatment on HCC has not been reported.
In the present study, we observed that LK-A inhibited both in vivo and in vitro proliferation of HCC. We further explored the mechanism by which LK-A may inhibit malignant proliferation, such as by downregulating Skp2 and inducing cell cycle arrest, and by causing apoptosis activated by ROS/ c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway induction. Collectively, our data suggests that the diterpenoid LK-A has significant potential as an antitumor agent for HCC.

Results
LK-A suppresses cell growth in HCC, and triggers cell cycle arrest at the G2/M phase. We used the HCC cell lines BEL-7402, SMMC-7721, Huh7 and HepG2 to investigate the effects of LK-A on HCC. As shown in Figure 1b, LK-A substantially inhibited HCC cell growth in a time and dose-dependent manner. In contrast, LK-A displayed only moderate cytotoxicity toward the normal liver cell line LO2. We decided to use SMMC-7721 and HepG2 cells for further investigation as part of this study. In addition, colony formation assays demonstrated that SMMC-7721 and HepG2 cells treated with LK-A for 36 h formed both fewer and smaller colonies than did control liver cells (Figure 1c), indicating that LK-A inhibits growth of the two HCC cell lines. To further examine the mechanism by which LK-A may inhibit proliferation of SMMC-7721 and HepG2 cells, we studied the effects of LK-A on cell cycle arrest. SMMC-7721 and HepG2 cells were incubated with varying concentrations of LK-A for 36 h, stained with propidium iodide, and analyzed by flow cytometry. Indeed, LK-A treatment led to a dose-dependent induction of cell cycle arrest in the G2/M phase arrest ( Figure 1d).

LK-A induces apoptosis of HCC cells.
To further probe LK-A inhibition of cell proliferation and colony formation, we used Hoechst 33342 staining to assess LK-A-dependent changes in cell morphology. SMMC-7721 and HepG2 cells treated with LK-A for 36 h displayed dramatically changed morphologies (Figure 2a). Arrowheads indicate cells exhibiting chromatin condensation, indicating the induction of apoptosis (Figure 2a). To determine if LK-A has a pro-apoptotic effect on HCC cells, flow cytometry analysis via Annexin V/PI staining was performed. Flow cytometry analysis indicated that LK-A-treated HCC cells undergo apoptosis at significantly higher rates than control cells ( Figure 2b). Furthermore, western blot analysis suggested a significant LK-A dose-dependent decrease in levels of procaspase-3, caspase-8, and an increase in cleaved Caspase-3, cleaved PARP (Figure 2c). Together, these results demonstrate that LK-A can induce apoptosis of HCC tumor cells.
Skp2 may have a major role in LK-A-induced SMMC-7721 G2/M phase arrest. To obtain further insight into the mechanisms of LK-A in SMMC-7721 cell cycle arrest, messenger RNA (mRNA) expression profiles of LK-A treated cells for 36h were compared with those of none treated cells using a human cell cycle RT 2 profiler PCR array containing 84 cell cycle-related genes. The results showed a total of three upregulated (CDKN1A, CDKN1B and GADD45A) and five downregulated (CCNB1, CDC2, CDK2, MKI67 and Skp2) genes, classified as such by a more than two-fold change in mRNA levels ( Figure 3a, Supplementary Table 1). These targets were subsequently validated by a western blotting assay. Consistent with mRNA expression levels determined by real-time PCR, SMMC 7721 cells treated with LK-A displayed a decrease in Skp2 protein levels ( Figure 3b). Skp2 is an E3 ubiquitin ligase, which may interact with several members of the Cdk complex and induce cell cycle progression by degradation of CDK inhibitors, such as p21 and p27. It has been previously shown that Skp2 downregulation leads to cell cycle arrest. [26][27][28] Here we further show by western analysis that Skp2 downregulation is accompanied by an increase in p21, p-cdc2 and Mty1 levels, and that p-Wee1 and CyclinB1 are decreased, but levels of p27 are nearly unchanged (Figure 3b).
To further explore the role of Skp2 in induction of cell cycle arrest, we constructed a pcDNA-SKP2 overexpression vector. Western blot analysis indicated that Skp2 overexpression leads to decreases in protein levels p21 and p-cdc2, but that the expression of CyclinB1 and cdc2 was elevated ( Figure 3c). As shown in Figure 3d, Skp2 overexpression nearly reverses the cell cycle arrest induced by LK-A. These results confirmed that Skp2 may have a major role in LK-Ainduced G2/M phase arrest.
The role of ROS generation in LK-A-induced cell apoptosis. Cellular ROS generation can significantly impact the effects of various anticancer agents on tumor cell apoptosis. 18,29 We therefore used the fluorescent probe 20,70-dichlorofluorescindiacetate (DCFH/DA) to monitor intracellular ROS levels in the presence and absence of LK-A. We found that LK-A-treated cells had significantly higher levels of ROS than did control cells (Figure 4a). We next set out to determine if increased ROS production impacts LK-A-induced apoptosis. Cells were treated with the antioxidant N-acetylcysteine (NAC) at 1 h before adding LK-A and incubation for an additional 36 h. We found that pretreatment with NAC caused a significant decrease in the levels of LK-A-induced cell apoptosis ( Figure 4b). Moreover, blocking ROS production by NAC resulted in an increase in pro-caspase-3, caspase-8 and a decrease in  LK-A-dependent ROS accumulation activates the JNK/c-Jun pathway. MAPK signaling cascades regulate not only cell growth, differentiation and development, but also apoptosis. 30,31 To examine the mechanism by which LK-A affects MAP kinase activation, the role of LK-A in the activation of extracellular signal-regulated kinase (ERK), JNK and p38 MAP kinase was investigated. As shown in Figure 4d, phosphorylation levels of both JNK and c-Jun gradually, and significantly, increased after LK-A treatment. However, there was no discernible change in phosphorylation levels of ERK and p38. As Figures 4e and f demonstrate, the JNK inhibitor SP600125 significantly restored cellular apoptosis in response to LK-A. As ROS production could determine the fate of cancer cells through regulating a number of cellular pathways, 32 we examined if ROS accumulation is involved in the activation of JNK/c-Jun pathway in our model system. Western blot analysis showed that pretreatment with NAC nearly reversed the phosphorylation of JNK and c-Jun (Figure 4g). These results illustrate that the apoptosis of SMMC-7721 cells induced by LK-A is mediated by the activation of the ROS/JNK/c-Jun pathway.
LK-A suppressed the tumor growth in mouse xenograft models. To further evaluate the role of LK-A in tumor proliferation in vivo, 3Â 10 6 SMMC-7721 cells were   (Figure 5b). The mean tumor volume for negative control mice increased from 12.81±3.11 to 868.21±258.48 mm 3 , whereas LK-A-treated mice at 3 and 6 mg/kg treatment increased from 13.78 ± 2.59 to 326.1 ± 182.65 mm 3 and 13.12 ± 3.26 to 101.17 ± 82.28 mm 3 , respectively, while the 5-FU mice increased from 12.72±2.93 to 102.04 ± 75.72 mm 3 (Figure 5c). By contrast, there was no significant loss in body weight in the experimental animals ( Figure 5d). The immunohistochemistry staining of excised tumor sections revealed a higher expression of cleaved Caspase-3, but the lower expression of Skp2 and Ki-67 in LK-A-treated tumors (Figure 5e) consistent with the in vitro results. To investigate any potential cytotoxic effects of LK-A on normal tissues, non-tumor-bearing mice were intraperitoneally treated with LK-A (6 mg/kg) and DMSO (negative control) every 3 days for 4 weeks and there was no significant loss in body weight (data not shown). Furthermore, H&E staining of the organs collected at the end of the study also suggested no major organ-related toxicities ( Figure 6).

Discussion
As current HCC therapies have limited effectiveness and exhibit intolerable toxicities in most cases, natural products and their derivatives are pursued as new and ideal sources for anti-HCC drugs discovery. [33][34][35] The unique carbon skeleton and multiple pharmacological properties of diterpenes have recently gained attention as new candidates against human cancers. 36,37 I. ternifolius is the major ingredient of a Chinese patent medicine 'FufangSanyexiangchacaiPian', which is currently used to treat acute and chronic hepatitis and hepatitis B. LK-A (Figure 1a), an ent-kaurane diterpenoid obtained from I. ternifolius, has a similar structure to diterpenes, which anti-nasopharyngeal carcinoma has been reported recently. 38 However, in this study, we examined LK-A treatment in HCC models and revealed a significant suppression of tumor growth both in vivo and in vitro. Moreover, this compound triggered cell cycle arrest at G2/M phase and induced cell apoptosis. We also discovered that LK-A results in G2/M arrest via downregulation of Skp2 and inducing apoptosis through ROS/JNK/c-Jun apoptotic pathway in SMMC-7721 cells.
The decreased formation of cdc2/Cyclin B1 complex inhibits cell-cycle progression from the G2 phase to the M phase. 39 LK-A treatment in SMMC-7721 cells resulted in G2/M phase arrest in a dose-dependent manner, reduced cdc2, cyclinB1 protein levels and increased the expression of p-cdc2 and p-histone3. During G2 phase, the cdc2/Cyclin B1 complex is kept inactive via phosphorylation at Thr14 and Tyr15 of cdc2 by Myt1 and Wee1, respectively. [40][41][42] The Cip and Kip family of CDK inhibitors p21 also participates in the G2 checkpoint. P21 could inhibit CDK1 activity through binding to the CDK1/CyclinB1 complex and inducing G2 phase arrest. [43][44][45] In the present study, the expression of p-Wee1 decreased, but the protein levels of Mty1 and p21 were increased. Next, we focused on understanding how LK-A reduced the activation of cdc2/ CyclinB1 complex. Skp2, an F-box component of E3 ubiquitin ligases, has an essential role in the regulation of cell cycle progress. 27,46,47 Moreover, many studies demonstrate that Skp2 can serve as a therapeutic target in various cancers by mediating the cytostatic and cytotoxic effects of different chemotherapeutic drugs. 48 In this study, we discovered that LK-A downregulation of Skp2 subsequently increased the expression of p21. Consistent with this data, the overexpression of Skp2 nearly blocked any LK-A-induced upregulation of p21 and p-cdc2 protein levels and decreased cdc-2 and CycilnB1 protein levels. Our data suggest that downregulation of Skp2 by LK-A is essential for LK-A-induced cell cycle arrest.
Apoptosis, a fundamental process essential for development and maintenance of tissue homeostasis, is also a major route to eradicate cancer cells. 49 Nowadays, an effective strategies for cancer prevention and treatment is targeting of Figure 6 No obviously organ-related toxicity was found. Non-tumor-bearing mice were intraperitoneally treated with LK-A (6 mg/kg) or DMSO (dissolved in sodium chloride, control) every 3 days for 4 weeks. After the experiment, animals were sacrificed and the organs were fixed in formalin overnight and processed for paraffin embedding. The paraffin-embedded blocks were sectioned and stained by hematoxylin and eosin the signaling intermediates in apoptosis. 50 In this work, we revealed that LK-A treatment induced a dose-dependent apoptosis in HCC cells. We also saw that LK-A treatment resulted in a significant decrease of pro-caspase-3, caspase-8 and increase of proteolytic cleavage of Caspase-3 and PARP.
Recently, studies have showed that excessive ROS production, induced by exogenous agents, will cause cellular apoptosis through the activation of signaling pathway, which results in selectively killing cancer cells. [51][52][53] LK-A induced a significant increase in ROS production in SMMC-7721 cells, while pretreatment with the ROS inhibitor NAC partially abrogates the LK-A-induced increase of apoptosis. Interestingly, NAC similarly abolished the LK-A effects on pro-Caspase3, cleaved Caspase-3, Caspase-8 and cleaved PARP. These results demonstrate that the induction of ROS is a critical upstream event for LK-A-induced SMMC-7721 apoptosis. Furthermore, we examined the levels of DNA damage-related proteins, such as phosphorylated ataxia telangiectasia mutated (p-ATM), phosphorylated ataxia telangiectasia and Rad3-related (p-ATR), CDC25A, CDC25C and Cyclin F, before and after LK-A treatment in SMMC-7721 cells. We found that after the treatment of LK-A, the levels of p-ATR and p-ATM were all substantially increased, and meanwhile, the levels of CDC25C decreased (Supplementary Figure S1). These data, taken together, suggest that LK-A treatment might activate DNA damage in HCC cells, and ultimately leads cancer cells to apoptosis. Clearly, further work is needed to clarify the mechanisms of LK-A to regulate DNA damageinduced HCC cell apoptosis in detail.
Various apoptotic stimuli can rapidly activate MAPKs, which include JNK, ERK and p38MAPK. [54][55][56][57] In SMMC-7721 cells, treatment with LK-A did not change p38 and ERK phosphorylation but the levels of phospho-JNK increased in a dosedependent manner. Surprisingly, JNK activation is involved in the events of LK-A-mediated apoptosis, which was confirmed by the use of the JNK inhibitor SP600125. Until now, considerable evidences suggested that JNK is primarily activated by various environmental stresses including: UV radiation, osmotic shock, heat shock, oxidative stress, chemotherapeutic agents and protein synthesis inhibitors, among these, oxidative stress is particularly important. 58 So we investigated the activation of JNK with ROS elevation during LK-A treatment. Interestingly, our results showed that pretreatment with NAC in SMMC-7721 cells abolished the JNK/c-Jun activation by LK-A treatment. From these data, we concluded that LK-A induced apoptosis through ROSdependent JNK/c-Jun apoptotic pathway.
In conclusion, this study presented an ent-kaurane diterpenoid LK-A from I. ternifolius, which inhibited the proliferation of HCC cells and SMMC-7721 xenograft tumor growth. In vitro, LK-A significantly induce cell cycle arrest at G2/M phase and apoptosis. Moreover, in SMMC-7721 cells, the agent arrests cell cycle at the G2/M phase through downregulation of Skp2, and induces apoptosis via the ROS-dependent JNK /c-Jun pathway. In the SMMC-7721 cells xenograft model, LK-A showed significant antitumor activity with low levels of toxicity. This compelling evidence suggests that LK-A is great candidate for the development of new chemotherapy agents for the treatment of human HCC.

Materials and Methods
Materials. I. ternifolius (D. Don) Kudô leaves were collected in Jinxiu, Guangxi, China. Ten kilogram of dried and milled plant material was subject to extraction at room temperature four times with 100 l 70% aqueous Me 2 CO, for 3 days each time, and then filtered. The filtrate was then evaporated under reduced pressure and partitioned four times with 60 l of EtOAc. The substrate from the EtOAc partition (938.5 g) was applied to silica gel (200-300 mesh) and eluted with CHCl 3 -Me 2 CO (1 : 0-0 : 1), to give six fractions, A-F. Fraction B (618.5 g) was decolorized on an MCI gel and eluted with 90% MeOH-H 2 O to yield fractions B1-B4. Fractions B1 (116 g) and B2 (135 g) were further separated by repeated silica gel column chromatography to produce LK-A (20 g). The final LK-A concentrate was dissolved in DMSO to a concentration of 50 mM and stored at À 20 1C. Working concentrations of 1.5, 3.0 and 4.5 mM LK-A were prepared freshly before each experiment by dilution in media. All cell culture reagents were purchased from Gibco Cell culture. The HCC cell lines, HepG2 and Huh7, and the normal hepatic cell line LO2, were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum (FBS) and incubated at 37 1C with 5% CO 2 , and 95% humidity. HCC cell lines SMMC-7721, and BEL-7402 were maintained in RPMI 1640 supplemented with 10% FBS.
Cell viability assay. The effect of LK-A on cell viability was assessed by using the PrestoBlue indicator. A total of 3 Â 10 3 cells/well were seeded in 96-well plates overnight and then treated with varying concentrations of LK-A (0, 2, 4, 6, 8, 10 mM). In order to minimize any effects of the solvent on cell viability, the final concentration of DMSO was kept to o0.05% in all wells. Cells were either incubated for 24, 36 or 48 h at 37 1C in a humidified incubator, and then 10 ml PrestoBlue reagent was added into each well and incubated for another 40 min. Absorbances were measured at 570 and 600 nm in each well by a microplate spectrophotometer (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA).
Colony formation assay. SMMC-7721 and HepG2 cells were plated as 10 3 cells/well in six-well plates and maintained with or without LK-A for 1 week. After growth, colonies were fixed with methanol for 30 min and stained with 0.1% crystal violet for visualization and counting.
Detection of the cell cycle stage. To determine the distribution of cell cycle stages, control and treated cells were collected into polypropylene tubes and pelleted at 2000 rpm for 5 min. Cells were then washed with phosphate-buffered saline (PBS) and fixed with chilled 70% ethanol for 48 h at 4 1C. Fixed cells were washed with PBS and incubated with RNase A (50 mg/ml) for 30 min followed by incubation with propidium iodide (50 mg/ml) for 30 min at room temperature. Cell cycle analysis was performed on a Coulter Epics XL flow cytometry system (Beckman Coulter, Miami, FL, USA). In total, 15 000 events were recorded in each analysis. The percentage of cells either at G0/G1, S or G2/M stages was calculated using MultiCycle AV for Windows Version 295 (Beckman Coulter).
Hoechst 33342 staining for morphological evaluation. Approximately 5 Â 10 4 cells/well were plated in six-well plates, and the cells were then incubated with (0, 1.5, 3.0 or 4.5 mM) LK-A for 36 h. After incubation, cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min and then stained with 20 mg/ml Hoechst 33342 for 15 min at room temperature in the dark. Cells were then assessed by fluorescence microscopy for morphological changes after LK-A treatment.
Apoptosis detection by flow cytometry. The percentage of apoptotic cells was determined by using an Annexin V-FITC apoptosis detection kit, as per the manufacturer's protocol. After LK-A or mock treatment, cells were harvested, washed in cold PBS and then pelleted. Cells were stained in the dark for 15 min at room temperature, and analyzed by flow cytometry within 1 h of ending the staining procedure. Data were analyzed by CXP Analysis Software Version 2.0 (Beckman Coulter).
Real-time PCR gene array. RNA was extracted from LK-A treatment and none treatment SMMC-7721 cells using Trizol (Invitrogen, Carlsbad, CA, USA) and we cleaned them using the RNeasy MinElute cleanup kit (Qiagen, Valencia, CA, USA). Subsequently, total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) and cDNA was amplified by PCR using 2 Â Super Array PCR master mix (SABiosciences, Frederick, MD, USA). Real-time PCR was then performed on each sample using the Human Cell Cycle RT 2 Profiler PCR array (PAHS-020A; SABiosciences) on an ABI 7900HT real-time PCR system (Applied Biosystems, Grand Island, NY, USA) according to the manufacturer's instructions. Data were normalized for GAPDH levels by the DDCt method.
Measurement of ROS production. Intracellular ROS production was detected by using the peroxide-sensitive fluorescent probe DCFH-DA. In brief, cells were first incubated with DCFH-DA (10 mmol/l) at 37 1C for 30 min and then treated with LK-A for 3 h. Cells were then washed twice and resuspended in PBS to measure ROS accumulation by flow cytometry.
Transfection of SMMC-7721 cells with pcDNA-Skp2. Cells were transfected using Lipofectamine 2000, as per the manufacturer's protocol. Twentyfour hours post transfection, cells were treated with LK-A and cultured for an additional 36 h, prior to collection for western blot and flow cytometric analyses.
Western blotting assay. Cells were harvested from plates by gentle scraping lysed, and stored at À 80 1C. Protein concentrations were measured by the BCA Protein Assay. Fifty milligram of protein from each sample was subject to SDS-PAGE, transferred electrophoretically to 0.45 mm polyvinylidene fluoride (PVDF) membrane and subsequently incubated in blocking buffer (5% nonfat dry milk) for 1 h at room temperature. PVDF membranes were incubated with appropriate primary antibody overnight at 4 1C, washed and incubated with secondary antibody for 2 h at room temperature. Specific antibody binding was detected by chemiluminescence detection.
Vivo tumor xenograft study. Six-week-old male BALB/c nude mice were purchased from the Experimental Animal Center at Sun Yat-sen University. A total of 3 Â 10 6 SMMC-7721 cells were subcutaneously inoculated into the right flank of nude mice, to initiate tumor growth. When the tumor sizes reached B3 Â 3 mm, mice were randomly divided into four groups of six animals and treated intraperitoneally with LK-A (3 mg/kg and 6 mg/kg), 5-Fu (10mg/kg) or DMSO (negative control) dissolved in NaCl every 3 days for 4 weeks. Body weights and tumor volumes were measured every 3 days for 4 weeks. At the end of the study, animals were sacrificed and tumors were removed and weighed for use in immunohistochemistry experiments. The following formula was used to determine tumor volumes: tumor volume ¼ L Â W 2 /2, where L is the length and W the width. All animal handling and procedures were approved by the Ethical Committee of the Sun Yat-Sen University.
Tumor histology and immunohistochemistry. Isolated tumors and organ tissues were fixed in formalin and embedded in paraffin. Five micrometer sections were cut and stained with H&E. For immunohistochemical staining, sections were deparaffinized in xylene and hydrated through with graded alcohol, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Antigen retrieval was completed by cooking tissue sections for 3 min with citrate buffer (pH 6.0) for cleaved Caspase-3 and Skp2, and in EDTA buffer (pH 8.0) for Ki-67. Slides were incubated with 10% normal goat serum for 10 min at room temperature to block nonspecific binding, followed by incubation overnight with rabbit monoclonal antibody, anti-cleaved Caspase-3, Skp2 and mouse monoclonal antibody anti-Ki-67 at 4 1C in a moist chamber. Slides were washed twice with PBS and sequentially incubated with a secondary antibody for 30 min at room temperature, before staining with DAB (3, 3-diaminobenzidine). Finally, slides were counterstained with hematoxylin, dehydrated and mounted.
Statistical analysis. All data were expressed as mean±standard deviation (S.D.) and statistically compared by one-way analysis of variance with Dunnett's test or unpaired Student's t-test in different experiments. A P-value of o0.05 was considered to be statistically significant.


Associations of CTLA4 Gene Polymorphisms with Graves' Ophthalmopathy: A Meta-Analysis

Many studies have established that T-lymphocyte antigen-4 (CTLA4) is a susceptible gene for Graves' disease (GD). Also many studies showed the association between the CTLA4 exon-1 49A/G polymorphism and the risk of developing Graves' ophthalmopathy (GO) in GD patients. But those results were inconsistent. In recent years many new studies were published which helped to shed light on the relationship of CTLA4 SNP49 with GO. So we performed the meta-analysis to explore the association between the SNP49 and GO susceptibility in GD patients. Studies up to February 29, 2012, were searched by using PubMed. The odds ratio was used to evaluate the strength of the association. Altogether 12 case-control studies involving 2,505 participants were included in the meta-analysis. Results showed that the G allele was related to the increased risk of GO compared with the A allele under allelic genetic model (OR = 1.14, 95% CI: 1.14–1.72, P = 0.001) in European subgroup. No publication bias was detected. Our results showed that the SNP49 polymorphism of CTLA4 gene was related to increased risk of GO.

Introduction
Graves' disease is a thyroid autoimmune disorder with 25-50% individuals having ophthalmopathy which is called thyroid-associated ophthalmopathy (TAO) or GO. The clinical manifestations of GO can mostly be explained by the increased volume of extraocular muscles and orbital connective tissues [1]. The symptoms of GO progress in some patients which eventually will lead to blindness. Though many studies have established the role of T cells in GO development [2][3][4], the understanding of the mechanism of GO is still poor. More effective therapy may develop on a better understanding of the T cell-fibroblast interaction.
Human CTLA-4 gene, located on 2q33, encodes a molecule which plays an important role in the downregulation of CD28 interaction with the ligands on the surface of antigenpresenting cells (APCs). The important inhibitory role of CTLA-4 in T-cell function has made it be one candidate gene when exploring autoimmune diseases.
One meta-analysis exploring the association of CTLA-4 gene polymorphism with GO was performed in 2006 which found no definite result [21]. Many new researches have been done since 2006 which will help to clarify the association. So we performed the meta-analysis with more case-control study results.

Search Strategy.
We searched PubMed up to February 29, 2012, for all possible publications on the association between the CTLA-4 49A/G polymorphism and GO. Search terms were used as follows: "Cytotoxic T-lymphocyte associated antigen-4 or CTLA4, " "Graves' disease, " and "ophthalmopathy. " We also manually searched all reference lists of the relevant articles for additional papers. Language was limited to English. All eligible studies met the following criteria: (1) case-control design investigating the associations of CTLA4 2 International Journal of Genomics  [15] 49A/G polymorphism and GO; (2) providing genotype distribution information in both cases and controls or provided odds ratio (OR) with 95% confidence interval (95% CI) (or sufficient data that allowed us to calculate these). When a study reported results on different subpopulations, we treated each subpopulation as a separate comparison in the metaanalysis. For overlapping data, the most complete or recent study was included.

Data Extraction. Two authors (Pengfei Du and
Changjiang Wang) independently extracted data and reached consensus on all items. The following information was sought from each report: authors, journal and year of publication, country of origin, selection and characteristics of GO cases and controls, demographics, ethnic group of the study population, eligible and genotyped cases and controls, and number of cases and controls for the genotype. For studies including subjects of different ethnic groups, data were extracted separately for each ethnicity whenever possible.

Statistical Analysis.
The primary analysis compared GO cases with controls for the frequency of G versus A alleles. This analysis aims to detect overall differences. Pooled OR with 95% CI was used to evaluate the strength of the associations of CTLA-4 49A/G polymorphism and GO. When zero events occurred, we treated this problem by adding 0.5 to all the 2 × 2 cells of the contingency table to calculate the study-specific OR just as implemented in RevMan software. We examined the association of 49A/G polymorphism and GO for allelic contrast (G versus A). Furthermore, we conducted subgroup analyses by stratifying ethnicity into Europeans and Asians separately. A test was used to determine the significance of the pooled OR and value < 0.05 was considered significant. The between-study heterogeneity across all eligible comparisons was estimated using the chi-square based Q statistic [25].
Heterogeneity was considered significant for < 0.10. Data were combined using both fixed-effects (Mantel-Haenszel) and random effects (DerSimonian and Laird) models [26]. Random effects incorporate an estimate of the between-study variance and tend to provide wider confidence intervals, when the results of the constituent studies differ among themselves. In the absence of between-study heterogeneity, the two methods provide identical results. Random effects are more appropriate when heterogeneity is present [26]. Inverted funnel plots and the Begg-Mazumdar publication bias diagnostic (nonparametric correlation coefficient) [27] evaluated whether the magnitude of the observed association was related to the variance of each study; that is, whether large studies gave different results compared with smaller ones. All the statistical analyses were performed using RevMan5.0 software (the Cochrane Collaboration, Oxford, England). All values were two-sided.

Association of the 49A/G Polymorphism with GO.
A total of 12 studies concerning the 49A/G polymorphism included 2,505 individuals (1,082 cases and 1423 controls). The pooled frequency of G allele was 68.1% and 51.8% for cases and controls, respectively. Two studies suggested an at-risk effect of G allele [11,24] and the others did not produce significant results. The overall analysis for evaluating the association of 49A/G polymorphism with GO under allelic model showed that there was large heterogeneity between these 13 studies ( heterogeneity = 0.01), and the random effects pooled OR was not significant: OR = 1.14, 95% CI: 0.94-1.38, = 0.18 ( Figure 1). One study was excluded from the metaanalysis because of the contribution to the heterogeneity in the European subgroup (random OR = 1.40, 95% CI 1.14-1.72, = 0.001, heterogeneity = 0.19) [17]. Sensitivity analyses evaluating the influence of single studies on overall risk estimate by omitting one study in each turn did not materially alter the overall effect size, with a range from OR = 1.32 (95% CI 1.09-1.60) to OR = 1.48 (95% CI 1.19-1.85). The GG genotype is associated with increased risk of GO in European subgroup (random OR = 1.89, 95% CI 1.26-2.83, = 0.002, heterogeneity = 0.32) (Figure 2). The G carrier was more likely to have GO than A carrier in European subgroup (random OR = 1.31, 95% CI 1.07-1.60, = 0.01, heterogeneity = 0.01) (Figure 3).

Discussion
The current meta-analysis including 12 case-control studies was in an effort to clarify the relationship between CTLA-4 gene polymorphism and GO susceptibility. The overall results indicated that the CTLA-4 49A/G polymorphism was associated with susceptibility of GO which was more significant in European population. Although obvious heterogeneity was detected for 49A/G associations, sensitivity analyses did not materially alter the overall and subgroup results under different genetic models, indicating that the results were stable and reliable.
The polymorphism of CTLA-4 had been suggested in many diseases which included Addison's disease, autoimmune hypothyroidism, and rheumatoid arthritis. The CTLA-4 49A/G SNP in exon-1 leads to the substitution of Ala with Thr in the signal peptide part which was reported to cause misprocessing of CTLA-4 in the ER resulting in less efficient glycosylation and diminished surface expression of CTLA-4 protein [30]. Studies have demonstrated that the longer repeats of the 3 UTR microsatellite are associated with reduced CTLA-4 inhibitory function [31]. Some workers have shown association between the G allele of 49A/G and reduced control of T-cell proliferation [9,32]. One study found an association between the protective allele A of the 49A/G SNP, which is associated with increased CTLA-4 function/expression, and interferon induced thyroiditis [33]. Hence, we postulated that, under most cases of spontaneous autoimmunity, alleles associated with reduced CTLA-4 function and/or expression could lead to increased activation of T cells thereby triggering autoimmunity.
The association of CTLA-4 49A/G SNP with GO was more significant in European population but not in Asian population. The CTLA4 49A/G SNP shows the same effect on the GO development with different significance. The results are confusing but not conflicting. This can be explained by the following reasons. Firstly, both GD and GO are multigenic conditions. It is unlikely to have one major susceptibility gene contributing to the development of GO. In the studies of Xu et al., they transiently transfected a T-cell line with a CTLA-4 construct harboring either the G or the A allele of the 49A/G SNP and found no difference in CTLA-4 expression and/or function harboring the A or the G allele [34]. It is 4 International Journal of Genomics Heterogeneity: 2 = 0.08; 2 = 19.54, df = 7 (P = 0.007); I 2 = 64% Heterogeneity: 2 = 0.00; 2 = 0.41, df = 4 (P = 0.98); I 2 = 0% Heterogeneity: 2 = 0.06; 2 = 25.50, df = 12 (P = 0.01); I 2 = 53% Test for subgroup differences: 2 = 4.77, df = 1 (P = 0.03), I 2 = 79.0% Zhang (2006) [22] Han (2006) [21] (a)  Test for subgroup differences: 2 = 8.84, df = 1 (P = 0.03), I 2 = 88.7% Villanueva (2000) [ [22] Han (2006) [21] Chong (2008) [23] (b) Figure 1: Meta-analysis for the effect of the G allele versus the A allele on the risk of GO in GD patients. Each comparison is presented by the name of the first author and the year of publication. The point estimate of the odds ratio and the accompanying 95% confidence interval (CI) are shown. "Total" represents the summary random effects estimation for the comparison along with the respective 95% confidence interval. Values above 1 denote an increased risk for GO with the G allele.
likely that the 49A/G SNP did not directly affect the T-cell activation but may in LD with some major effect alleles. Secondly, the impact of smoking on GO was not specifically considered in the association studies. As was reported, the odds ratio for both current smokers and exsmokers was high and the GO was more severe in smokers than in nonsmokers [35][36][37].
In conclusion, our analysis first intensified the association between 49A/G and GO susceptibility, which showed that the G allele is a risk factor for GO. More risk factors should be considered in future association studies of the CTLA-4 SNP with GO. The gene-gene and gene-environment interaction should also be taken into consideration in future research. The identification of the candidate gene and understanding  Figure 2: Meta-analysis for the effect of the GG versus the AA genotype on the risk of GO in GD patients in European subgroup. Each comparison is presented by the name of the first author and the year of publication. The point estimate of the odds ratio and the accompanying 95% confidence interval (CI) are shown. "Total" represents the summary random effects estimation for the comparison along with the respective 95% confidence interval. Values above 1 denote an increased risk for GO with the GG genotype.  Figure 3: Meta-analysis for the effect of the CTLA4 49A/G allele in G carriers and in A carriers on the risk of GO in GD patients. Each comparison is presented by the name of the first author and the year of publication. The point estimate of the odds ratio and the accompanying 95% confidence interval (CI) are shown. "Total" represents the summary random effects estimation for the comparison along with the respective 95% confidence interval. Values above 1 denote an increased risk for GO with the G allele. of the mechanisms by which they cause disease will identify those individuals at risk for developing GO in the future. Treatment will evolve towards more preventive strategies. The treatment will be personalized by targeting the specific pathways that are most contributory to GO development. We believe that the future will witness the engagement of novel molecules, such as altered peptide ligands [38] and specific monoclonal antibodies, to modulate specific autoimmune pathways in affected individuals, thus treating the cause of the disease.


text:
A comprehensive insight into the lipid composition of Myxococcus xanthus by UPLC-ESI-MS.

Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism’s response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.

were of LC-MS grade. HPLC grade ammonia (Sigma) was used as a 10% aqueous solution and formic and acetic acid (both Carl Roth) were of analytical grade. Glyceryl tristearate ف 99% {TG (18:

Extraction procedures
Cells were harvested by centrifugation at 15,000 g for 15 min at room temperature and washed once with the same volume of distilled H 2 O.
A modifi ed method of Bligh and Dyer ( 22 ) was applied to extract all lipids. The cell pellets were thoroughly resuspended in 2 ml of distilled H 2 O and 7.5 ml of a mixture of MeOH/DCM 2:1 (v/v) was added. After incubation for 20 min under agitation, phase separation was induced by the addition of 2.5 ml of DCM and H 2 O, respectively. The lower phase was removed after centrifugation at 750 g for 20 min and the cells and upper phase was re-extracted twice with 2 ml DCM. The combined lower phases were fi ltered through solvent-resistant 0.22 M polytetrafl uoroethylene membrane fi lters and dried under a stream of nitrogen at 40°C in weighted vials for the exact determination of the mass of the extracts. For analysis, the lipid extracts were dissolved in a monoacylglycerophosphoethanolamines and ether lipids, as well as glycerolipids, needed to be monitored ( 12 ). The availability of a single analytical method, able to detect all lipid species, will facilitate rapid analogous investigations.
Results from TLC resolved 32 PO 4 3 Ϫ metabolic labeling studies available for Stigmatella aurantiaca ( 13 ), Myxococcus fulvus ( 14 ), and M. xanthus ( 15 ) provide some information about the nature and molar ratio of phosphate-containing lipids present in those strains. However, there is as yet no comprehensive information on the complete lipidome of myxobacteria. This is a prerequisite to visualize and evaluate the changes in lipid composition of mutant strains during vegetative growth or fruiting body formation, as well as providing a basis for further investigations on lipid biomarkers or signals for fruiting body formation.
We sought to develop a LC-MS method that: i ) is able to resolve as many lipid species as possible, covering lysophospholipids, glycerophospholipids, and glycerolipids; ii ) gives MS and MS 2 spectra for most of the molecular species in a single run for structure elucidation and identifi cation; iii ) involves the standard setup of ultra-performance (UP)LC-MS instrumentation used for routine analysis; iv ) solely uses readily available software; and v ) needs only a very short measuring time.
Many lipidomic methods that involve the direct measurement of lipid species from lipid extracts are based on direct infusion into ESI sources or ionization by MALDI ( 16,17 ), in which the detection of less abundant molecular species is typically impeded by ion suppression. This effect can be circumvented by separation of the analytes using chromatography ( 18 ). The development of a comprehen sive LC method for lipids is challenging due to their highly diverse physical properties, ranging from partly water (H 2 O) soluble (lysophospholipids) over amphiphilic (glycerophospholipids) to highly nonpolar (glycerolipids), nonionic [glycerolipids, ceramides (Cers)], and strongly pH dependent zwitter, cationic, and anionic molecules (glycerophospholipids). All chromatographic parameters (different eluents, nature, and concentration of additives, as well as temperature and pH) have to be carefully controlled over the whole chromatographic process. The use of eluent additives is limited, as they have to be compatible with MS coupling.
To date, many LC methods have been introduced that involve normal phase chromatography, hydrophilic/ lipophilic interaction chromatography, and reversed phase (RP) chromatography, in most cases to address particular analytical challenges (19)(20)(21). RP chro mato graphy, which can be regarded as "the" gold standard in routine small molecule analytics, is ideal for resolving molecules with even small differences in polarity, and it is also our method of choice, not least because it is part of our system's standard instrumentation.   +140 V (positive mode); nitrogen fl ow, 11.83 l/min; drying gas temperature, 300°C; and nebulizing gas pressure, 44 psi. Both MS and MS/MS data were recorded in the same run using the "ultra" scan mode with a trap drive of 64.3 (negative mode) and 53.3 (positive mode), respectively, a scan speed of m/z 25,000 per second and a mass resolution of m/z 0.6 over a range of m/z 100-2,000, with one spectrum having an average of seven scans in the MS mode and three scans in the MS 2 mode. For autoMS/MS fragmentation, an absolute threshold of 25,000 cts was chosen, and already fragmented precursor ions were excluded for further fragmentation after two spectra for 0.5 min. The isolation width was m/z 1.5. In a fi rst step, a LibraryEditor library was established to enable automatic identifi cation of lipid species from the lipid extracts. MS 2 spectra that were acquired during the runs were automatically extracted from the data set using the "Compound Ϫ autoMS(n)" algorithm with the following settings: intensity threshold positive, 1,000,000; intensity threshold negative, 10,000; maximum number of compounds, 350; retention time window (min), 0.25; spectrum type, line spectra only; background subtraction, none. Lipid species were identifi ed from the MS 2 spectra and added to the library together with their retention times. This library was applied to all HPLC-MS runs using the "Identify Mass Spectra" function of data analysis with the following settings: desired score, 900; minimum score, 600; minimum parameter matching score, 250; maximum number of spectra, 1; accepted retention time deviation, 0.1 min; maximum retention time deviation, 0.2 min.

Data processing
For the calculation of the relative percent of lipid species, extracted ion chromatograms (EICs) were created for every single precursor ion of the identifi ed lipid species. All chromatographic traces were smoothed using a Gaussian algorithm (1 pt width, 2 cycles) to enhance peak detection. Those EICs from the identifi ed lipid species were integrated by applying the "Compounds Ϫ Chromatograms" algorithm with the following settings: signal-tonoise threshold, 5; area threshold, 10%; intensity threshold, 10,000 for the negative ion mode and 1,000,000 for the positive ion mode; skim ratio, 0; smoothing width, 1. Charts containing the identifi ed autoMSn compounds and the areas under the curve (AUCs) of the EICs were exported into Excel charts. Those charts were manually inspected for false identifi cations and missing EIC peak integrations.

GC-MS FAME analysis
Derivatization procedures and GC-MS conditions are described elsewhere ( 23 ).

HPLC-MS conditions
For every data set, 5 l of lipid extract was separated on an ACQUITY UPLC TM BEH C18 column (Waters) with a length of 50 mm, an inner diameter of 2.1 mm, and a mean particle size of 1.7 M connected to an UltiMate 3000 (Thermo) HPLC system coupled to an AmaZon X (Bruker) mass spectrometer at a constant fl ow rate of 0.6 ml/min and a temperature of 50°C using a binary gradient. Eluent A consisted of H 2 O/ACN 50/50 (v/v) and solvent system B of ACN/isopropanol 10/90 (v/v). Both eluents contained 10 mM NH 4 AcO as a buffering additive and were adjusted to pH 5.5 using formic acid in order to maintain the analytes in the same charge state during the separation process. The gradient was programmed as follows: start at 1% B, increased to 95% within 10 min in a linear manner, hold at 95% for 2 min and re-equilibrate for 2 min at 1% B.
The ESI parameters for the negative ion mode were: capillary voltage, +4.5 kV (negative mode) Ϫ 4.5 kV (positive mode); end plate offset, Ϫ 500 V; capillary exit, Ϫ 140 V (negative mode) Relative AUCs given as per thousand of the total AUC of all identifi ed lipid molecular species determined separately for positive and negative ionization modes. MM (molecular mass) is the calculated molecular weight of monoisotopic mass of respective lipid species. See supplementary Tables I, II for details.  [PE(P)s], and CLs from the HPLC-MS runs in the negative ionization mode and the DGs and TGs in the positive ion mode were separately summed and normalized to 1,000, and the peak area of each individual EIC peak was calculated as per milliliter of the total integral of all peaks (see supplementary Table I and II for the complete data set and Table 1 for a summary). MATLAB release 2013b (The MathWorks, Inc., Natick, MA) was used for the visualization of the base peak full scan UPLC-ESI-MS chromatograms and plotting the lipid profi les. The base peak full scan UPLC-ESI-MS chromatograms were created by using the functions "meshgrid" and "surf". The "stem3" function was applied to create the 3D lipid profi les.

Identifi cation of lipids by MS
The experiments of the positive ionization and the negative ionization mode were combined in order to identify a maximum possible number of lipids. The above-mentioned standards were used in order to evaluate the chromatographic and fragmentation behavior of those lipid species. This information plus the reference spectra and software tools from LIPID MAPS ( 24 ) were used as the bases for the MS-based structure elucidation of the lipid species . The fragmentation behavior of PI-Cers have been described previously ( 25,26 ). ESI-MS data for the identifi cation and structure elucidation of PGs, PEs, and CLs, as well as glycerolipids (DGs and TGs) are also available (27)(28)(29)(30)(31)(32). The presence of PGs, PEs, and N -acylsphinganines (Cers), as well as the presence of distinctive ␣ -hydroxylated FAs together with the complete FA profi les of the investigated strains have been published ( 9,11,14,15  In general, isobaric molecular species could not be resolved by HPLC, and thus yielded one MS 2 spectra (see Tables 1, 3 ; supplementary Figs. I-VII ) For the creation of the lipid profi les, only the integrals from the negative ion mode for the PEs, PGs, PI-Cers, and Cers and from the positive ion mode for the DGs and TGs were used. PE and Cer ions were also clearly detectable in positive ion mode, but not used for the creation of lipid profi les.

High resolution MS analyses
Determination of the exact mass of Cer molecular species from lipid extracts was carried out using a Dionex Ultimate 3000 RSLC coupled to a Bruker micrOTOF-Q II equipped with an ESI source set to negative ionization mode. HPLC settings were identical to those described above, though the column bed was 100 mm in length and the gradient length was doubled to 20 min accordingly. The mass spectrometer was calibrated using a sodium formate calibrant solution (10 mM). MS data were acquired within the mass range of m/z 200-2,000.

Nomenclature
For the sake of uniformity and comprehensibility, we used the nomenclature of the LIPID MAPS Lipid Classifi cation System ( 24 ) for the names and abbreviations . RESULTS A binary eluent system consisting of H 2 O and ACN in solvent A and ACN and isopropanol in solvent B (for details see the Experimental section) proved to be useful for the separation and elution of an extensive range of lipid standards on a RP-18 column, including glycerolipids, glycerophospholipids, and sphingolipids with different degrees of acylation, as well as acidic, basic, and amphoteric polar head groups ( Fig. 1 ). The use of a 10 mM ammonium acetate buffer adjusted with formic acid to pH 5.5, a pH at which all major phospholipids have an equable charge state ( 34,35 ), gave an acceptable separation of the lipid extract of M. xanthus DK1622 ( 36 ) as depicted in

Lipid profi le of M. xanthus
Applying the described method to normalized lipid extracts resulted in two sets of LC-MS data for each sample: one recorded in the positive ionization mode and the other one in the negative ionization mode. Figure 2 shows Ϫ ( 45,46 ). Molecular composition was confi rmed by high-resolution MS (see Table 3 ). the resulting base peak chromatograms of the LC-MS analysis of lipid extracts from vegetatively grown DK1622 cells in negative ( Fig. 2A ) and positive ( Fig. 2B ) ionization modes, respectively. The most intense signals are discernable in the region of R t = ف 6.0-8.5 min and have m/z values of approximately 550-850 in negative ion mode, with another large group of signals occurring at R t = ف 9.0-10.5 min and having m/z values of 700-880 in positive ionization mode. Additionally, the most intense glycerophospholipid molecular species are discernable as dimeric ions, and there is an unknown compound class with a strong abundance at R t = ف 9.8 min with about m / z 1,008, which could not be identifi ed.
Evaluation of autoMS 2 data resulted in the identifi cation of a large number of PE and PG species, as well as the previously identifi ed PE(P)s ( 4 ) (supplementary Figs. I-III), hydroxylated and nonhydroxylated N -acylsphinganines (Cers) (supplementary Fig. IV), and interestingly, for the fi rst time in a prokaryote, PI-Cers [ Fig. 3 ; for a summary see Table 1 and supplementary Table I and II (the full data set obtained from data analysis)]. Consistent with the relative low abundance and limited diversity of sphingoid bases, as determined by GC-MS analysis of lipid methanolyzed cell extracts ( Table 2 ), they are detectable as less abundant compounds. Finally, CLs are detectable as late eluting compounds with rather low abundances (supplementary Fig. V).
Fragmentation spectra of the late eluting molecules in positive ionization mode are consistent with ammonium adducts of DGs and TGs ( 37 ) (supplementary Figs. VI, VII), as well as protonated Cers. PEs are the only detectable glycerophospholipid species and are distinguished by the [M-141+H] + MS 2 fragment resulting from the neutral loss of the polar headgroup. They were not further evaluated, as these fragment spectra do not contain any additional structural information.
None of the evaluated MS 2 spectra displayed evidence for other glycerophospholipids, glycerolipids, or precursors of identifi ed lipids such as lysophospholipids, diacylglycerophosphoserines, or PIs.
Concerning the elution pattern of these lipid species, the following regularities can be observed (see Fig. 2 ; Table 1 ;  supplementary Tables I, II): i ) As expected, retention time increases with increasing length of the acyl chains attached to the glycerophospholipids, as hydrophobic interaction with the RP-18 stationary phase becomes more intense. ii ) Elution order for glycerophospholipids is PGs → PEs → PE(P)s → CLs, as long as the number of carbon atoms and the degree of saturation in the acyl side chains are identical . iii ) PI-Cers elute earlier than Cers, whereas ␣ -hydroxylation in the acyl side chain further decreases the retention time. iv ) For DGs and TGs, analogously to the glycerophospholipids, retention time increases with acyl chain length and degree of saturation. v ) Lipids from the same class with an identical overall carbon and double bond number generally coelute. However, data from MS 2 fragmentation of glycerophospholipids in the negative ion mode does allow the assignment of fatty acyl residues to sn -1 or sn -2 positions of the glycerol backbone of those lipid species (supplementary Figs. I-III). In total, 3 Cers, 4 PI-Cers, 52 PEs, 2 PE(P)s, 20 PGs, 32 glycerophosphoglycerophosphoglycerols, and 7 DGs were identifi able. Additionally, 46 TG molecular species were identifi ed, but the exact assignment of all three fatty acyl moieties to the three hydroxyl functions of the glycerol backbone was not possible under these conditions, as only MS 3 fragments of the [M-NH 3 -R n COOH] + ions allow the assignment of the fatty acyl residues to a particular hydroxy group of the glycerol backbone. Nevertheless, the fragment spectra of TGs indicated a high diversity of those molecules comprising almost all saturated and unsaturated FAs present in M. xanthus . Beyond that, all TG ion species consisted of several isobaric TG species which coeluted, just as observed for the PGs and PEs (supplementary Fig. VIIC). We did not test the use of adduct-forming additives such as Li + and Na + because this would only further increase the number of ionic species and would not solve the problem of overlapping tandem mass spectra; the same applies to the CLs. Only MS 3 experiments allow the exact determination of the acyl residues present in the  V).

UPLC-MS analysis allows the assignment of a particular polar headgroup to Cers and detects loss of lipid species in mutant strains
We applied the UPLC-MS method and data evaluation procedure to lipid extracts of vegetatively grown cells from various strains with mutations in different genes involved in lipid biosynthesis. Strain ⌬ 0191 lacks a FA hydroxylase, which is responsible for the formation of 2-OH FAs ( Table 2 ). Strain Ϫ MXAN_3748 bears a mutation in a serine-palmitoyl-CoA acyltransferase homolog and is impaired in sphingoid base formation ( 9 ) ( Table 2 ). Figure 4A, B shows EICs of various Cer species from wild-type, ⌬ 0191, and Ϫ MXAN_3748 extracts. Strain ⌬ 0191 shows the selective loss of hydroxylated Cers ( Fig. 4A ). Strain Ϫ MXAN_3748 no longer forms either Cers or PI-Cers, as determined by both low resolution MS and confi rmative high resolution MS experiments ( Fig. 4B , Table 3 ).  Analogously, in strain Ϫ MXAN_1528 bearing a mutation in elbD , a multidomain enzyme involved in ether lipid biosynthesis ( 12 ), the relative abundance of vinyl ether species is strongly reduced ( Table 2 , Fig. 4C ).

Lipid profi les of various mutants defi cient in lipid biosynthesis
Integration of EIC peak areas of the monoisotopic masses of the respective lipid molecular species allows the construction of lipid profi les. If only the relative proportion of all lipid species are compared among the various investigated strains, no major differences are observable, apart from the above mentioned loss of certain lipid species and the expected relative increase of the remaining molecular species (supplementary Fig. VIII).
In order to gain an in-depth look into the lipid profi le of M. xanthus , we constructed a scatter plot comprising mass, retention time, and relative abundances of all analyzed lipid molecular species given as the sum of all peak areas per thousand ( Fig. 5 ; Table 1 ; supplementary Tables I, II). The observed pattern refl ects previous results from GC-MS analysis of FAMEs and neutral lipids with a high proportion of lipid molecular species containing 15:0 and 16:1. The occurring FAs are somewhat equally distributed among the various lipid classes with an almost "Gaussian"-like distribution among the TGs and PEs ( Fig. 5 ). Like Curtis et al. ( 11 ), we identifi ed numerous PE and also PG molecular species with unsaturated FAs attached to the sn -1 position of the glycerol backbone ( Table 1 ), indicating a less specifi c glycerol-3-phosphate 1-O-acyltransferase activity compared with Escherichia coli , where those lipids are less prevalent ( 32 ). Vinyl ether-containing phospholipids were exclusively detected within the PE molecules, whereas O -alkylglycerols could not be detected under these conditions, which is consistent with previous results ( 4 ). The relatively low proportion of PGs is consistent with results from Orndorff and Dworkin ( 15 ), who specifi ed the amount of PGs as 9% of total 32 PO 4 3 Ϫ -labeled M. xanthus membranes . Analogously, CLs are minor compounds, but exhibit a high molecular diversity, as each molecule is potentially made up of four different FAs ( Table 1 ). Neutral lipids, especially TGs, gave strong signals as ammonium adducts, although they are less prevalent in vegetative cells ( 10 ). Their molecular diversity seems to be even higher than that of the polar lipids.
In an additional step, we created scatter plots that compare the relative changes in the abundance of the individual lipid species between the mutant lipid profi les and the wild-type profi le. This refl ects the changes in the abundance of individual molecular species in the analyzed mutant strains compared with the wild-type, displayed in a 3D way. Now, shifts and trends in the mutant profi les are able to be visually appraised ( Fig. 6A-C , supplementary Table I).
The lipid profi le of strain ⌬ 0191 versus wild-type shows the expected relative increase of nonhydroxylated Cers in addition to the aforementioned loss of hydroxylated Cer molecular species and a general increase in the abundance of PG molecular species ( Fig. 6A , Table 1 ). There is a conspicuous increase in PE(15:0/17:0) and a simultaneous strong decrease in the abundance of PE(15:0/15:0) and PE(15:1/15:0). These fi ndings match well with the FAME pattern of strain ⌬ 0191, which shows a higher proportion of i17:0 FA compared with the wild-type ( Table 2 ). The changes in abundance within the TG molecular species seem to be rather random, but could be attributed to a higher ratio of iso17:0 to iso15:0 FAs . Figure 6B visualizes the changes in the lipid profi le of Ϫ MXAN_3748 compared with the wild-type. A general increase in higher molecular weight lipid species within the PE, PG, and TG molecular species, as well as a greater proportion of PE(P), is observable, meaning that there is a general increase in longer and/or less saturated FAs that could compensate for the loss of the lipophilic Cer species.

DISCUSSION
Such a comprehensive data set about molecular lipid species in myxobacteria has not been published before. Curtis et al. ( 38 ) reported the analysis of lipid molecular species from the PE fraction of M. xanthus by HPLC-ESI-MS, resulting in the identifi cation of 26 PE molecular species; though for four of these, no assignment of the fatty acyl residues to the sn-1 and sn-2 position of the glycerol backbone was achieved. Our UPLC-MS analysis confi rmed that ␣ -hydroxylation of FAs and sphingolipid formation are indeed metabolically linked, as the lipid extract of the ␣ -hydroxylase deletion mutant ⌬ 0191 lacks the hydroxylated sphingolipids and the spt -disruption mutant Ϫ MXAN_3748 is devoid of any detectable sphingolipids and does not contain other lipids with an ␣ -hydroxylated FA. When the results concerning the abundance of neutral lipids are compared with those obtained by high temperature GC-MS analysis ( 10 ), it seems that the latter method results in overestimation of the presence of MGs and DGs. This is very likely due to the derivatization procedure with N-Methyl-N-(trimethylsilyl) trifl uoroacetamide , which converts the free hydroxy groups of the MGs and DGs into easily ionizable trimethylsilyl derivates. In contrast, identical amounts of TGs yield more intense ESI-MS signals than underivatized MGs or DGs ( Fig. 1 ). In the end, the creation of calibration curves using molecular standards for every lipid class will be necessary to determine their individual response factors. This will be the only way to establish the exact ratio between different lipid classes. Although not further evaluated, it is interesting that the TG molecular species seem to contain fatty acyl species that are not detectable in FAME-GC-MS analysis, as the [M-NH 3 -R n COOH] + fragment at m/z 521.6 in the mass spectrum shown in supplementary We did not detect any glycerophosphoserines or lysophospholipids during our analysis, although these molecules are putative precursors of the highly abundant PEs or their degradation products, respectively. These lipids were detected in 32 PO 4 3 Ϫ -labeled TLC-resolved membrane fractions of M. xanthus , but not in its whole cell lipid extract ( 15 ), leading us to conclude that they are products of ongoing enzymatic reactions during the lengthy membrane fractionation procedure. We cannot exclude the presence of traces of these compounds with abundances lower than the limit of detection. The use of a column with a higher resolution and different MS experiments and instrumentation may lead to the identifi cation of more lipid species. However, trace component analysis was not the intention of this study.
At the same time, the low abundance of CLs, as determined by our ESI-MS analyses, is consistent with the low abundance of CLs found in the 32 PO 4 3 Ϫ -labeled cells ( 15 ). The presence of PI-Cers is an interesting fi nding of this study, as this is a class of lipid known to be present in fungi such as Saccharomyces cerevisiae ( 39 ) and Aspergillus niger  ( 40 ). They have also been found in protozoan pathogens like Trichomonas brucei ( 41 ), Trypanosoma brucei ( 41 ), Leishmania major ( 42 ), and plants ( 43 ). However, to our knowledge, the occurrence of bacterial PI-Cers has never been previously described. It seems that sphingolipids are a common component of myxobacterial membranes, as Cers were found in Cystobacter fuscus ( 44 ), Cer phosphoethanolamines in Myxococcus stipitatus ( 45 ), and Cer phosphoethanolamines, ␤ -D-glucosylsphingenines, and even phosphonosphingolipids in Sorangium cellulosum So ce56 ( 46 ), where they serve together with ornithine-containing lipids and glycerol ether lipids as substitutes for missing lipopolysaccharides in the outer membrane ( 46 ).
In S. cerevisiae , PI-Cers are synthesized by the inositol phosphorylceramide synthase catalytic subunit (AUR1), an enzyme that transfers a phosphoinositol headgroup to ␣ -hydroxyceramides from a diacylglycerophosphoinositol moiety ( 47 ). Protein MXAN_0451 (annotated as a PAP2 family protein) is 29% homologous to AUR1 (E-value = 2 Ϫ 20 ), a further indication of the capability of M. xanthus to synthesize this lipid species. Interestingly, no diacylglycerophosphoinositols were detected in the lipid extracts, though it has been previously noted that a myo -inositol-1-phosphate synthase homolog (MXAN_0452) as well as a CDP-alcohol phosphatidyltransferase homolog (MXAN_0450) are encoded next to MXAN_0451 ( 48 ). This may be due to a fast conversion of diacylglycerophosphoinositols to PI-Cers as the occurrence of diacylglycerophosphoinositols are indeed described for M. fulvus and S. aurantiaca ( 13 ).
In general, glycosylated Cers are important signaling molecules in multicellular organisms ( 49 ), but no specifi c functions for PI-Cers are known in lower eukaryotes, except that inhibition of AUR1 is lethal to S. cerevisiae ( 50 ) and that PI-Cer is at least indirectly associated with the pathogenicity of Cryptococcus neoformans ( 51 ). A possible signaling function of PI-Cers in myxobacterial fruiting body formation or sporulation remains to be investigated. Although there have been no systematic investigations on putative signaling functions of Cers in bacteria, it was noted that sphingolipids (as determined by the sphingobases) accumulate in developing M. xanthus cells, but are dispensable for fruiting body formation ( 9 ).
In summary, the changes in the lipid profi les between wild-type and mutant strains can be used to unveil genes and their protein products that are involved in lipid biosynthesis. An overview about gene candidates with homology to genes involved in lipid biosynthesis from other organisms exists in the literature ( 48 ). Even when there is functional redundancy between paralogous genes, it is possible to deduce protein functions from shifts in the lipid profi les of the respective mutant as shown in Fig. 6A for the ⌬ 0191 mutant, in which the biosynthesis of a polar headgroup is not affected.
We are aware that fi nal statements concerning changes in lipid profi les are not possible without statistically analyzable data, although some observable changes, like the previously established decrease or loss of selected molecular species and the easily explained general increase in degree of saturation and chain length of FAs within the lipids suggest that our data analysis procedure is generally a valid approach. Moreover, the visualization of the data using scatter plots resolved by the lipid class strongly facilitates data inspection and interpretation. This becomes even more important with increasing numbers of analytes. We envision that refi nement of this method using appropriate internal standards and statistics will yield lipid profi les that can be linked to transition profiles, diffusibility of membranes, activities of selected membrane proteins and membrane proteomes, and allow us to unravel the multifactorial links between those properties and the lipid composition of membranes.