EMBO/soda-nel huggingface dataset:
Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis (2023)
Figure 4

Figure 4 Ch25h deletion in CNS endothelial cells induces a remodeling of secreted lipids.

(A) Principal component analysis of 10 oxysterols measured by HPLC-MS in the supernatant of pMBMECs isolated from Ch25hfl/fl mice and Ch25hECKO mice. Cells were left unstimulated (NS) or stimulated by IL-1β.

(B) Loading plot showing the contribution of oxysterols to PC1 and PC2.

(C) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC concentration in the same conditions as in (B). Bars represent mean ± SD. n = 6 biological replicates/group except for Ch25fl/fl IL-1β were n = 5 biological replicates. The experiment was performed once.

(D) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC levels in spinal cord tissue extracted from Ch25hfl/fl (n = 8), Ch25hECKO (n = 3) and Ch25hBBBKO (n = 6) at the peak of EAE. Bars indicate mean ± SD.

(E) Left panel: Principal component analysis of 49 eicosanoids measured by HPLC-MS/MS in the supernatant of pMBMECs in the same conditions as in (A). n = 5 biological replicates/group. Right panel: Comparison of PC1 scores between the different conditions. Bars indicate mean ± SD.

(F) Upper panel: loading plot showing the contribution of each detected eicosanoid to PC1 and PC2. Lower panel: Relative contribution of prostaglandins to PC1.

(G) Prostaglandin concentration (nM) in the supernatant of pMBMECs comparing conditions mentioned in (A). n = 5 biological replicates/group and bars represent mean ± SD.

Data information: ns= non-significant, * P < 0.05, *** P ≤ 0.0005, **** P ≤ 0.00005. P values were determined by two-tailed unpaired t-test (D, E) or by two-way ANOVA with Sidak's post hoc test (C and G).
Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis (2023)
Figure 5

Figure 5 Ch25h deletion in endothelial cells favors PMN-MDSC expansion in the CNS during EAE.

(A) Flow cytometry analysis of bone marrow-derived cells cultured in MDSC polarizing conditions treated with vehicle control (EtOH), PGE2 (20nM) and 25-OHC (1µM). M-MDSC = Live cells CD11b+ Ly6Chigh Ly6G-, PMN-MDSC = Live cells CD11b+ Ly6Cint Ly6G+.

(B) Same conditions as in (A) except that bar graphs are shown. Symbols depict mean percentage of PMN-MDSC (upper panel) and M-MDSC (lower panel) in live cells. n = 5 biological replicates/group. Representative results of 2 independent experiments. Bars indicate mean ± SD.

(C) Impact of CNS PMN-MDSC and BM-PMN on CD4+ T cell proliferation assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the CNS (right panel) and BM (left panel) of WT mice at the peak of EAE. NS = non stimulated. n = 4 biological replicates/group. Bars indicate mean ± SD. CNS PMN-MDSC/CD4+ T cells: representative results of 2 independent experiments; BM-PMN: 1 experiment.

(D) Correlation of the percentage of PMN-MDSC in live cells with the percentage CD4+CD44+Ki67+ cells in live cells in the CNS at the peak of EAE assessed by flow cytometry. n = 17 biological replicates.

(E) Flow cytometry analysis of PMN-MDSC in the blood, BM, spleen and CNS for Arginase-1 staining.

(F) Quantification of (E) n = 12 biological replicates. The experiment was perfomed once. Bars indicate mean ± SD.

(G) Flow cytometry analysis of PMN-MDSC in the CNS at the peak of EAE in Ch25hfl/fl and Ch25hECKO mice.

(H) Percentage (upper left panel) and absolute number (lower left panel) of CNS live cells CD45+CD11b+Ly6CintLy6G+ PMN-MDSC; Percentage of live CD4+CD44+Ki67+ cells (upper right panel) and ratio (lower right panel) of absolute number of PMN-MDSC and proliferating CD4+ T cells in the CNS at the peak of the disease of Ch25hfl/fl (n = 9) and Ch25hECKO mice (n = 8). Symbols depict individual mice and bars indicate mean ± SD. Combined results of 2 independent experiments.

(I) EAE disease course in Ch25hBBBKO mice (n = 11) where the Cre-recombinase is expressed in endothelial cells of the CNS and Cre negative littermates (Ch25hfl/fl: n = 7 and Ch25hfl/wt : n = 2). Bars indicate mean ± SEM. Representative results of 2 independent experiments.

Data information: ns= non-significant, *: P < 0.05, **: P ≤ 0.005, ****: P ≤ 0.00005. P values were determined by two-tailed unpaired t-test (B,C and H), Spearman correlation (D) and by two-way ANOVA with Sidak's post hoc test (F and I).
Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis (2023)
Figure 6

Figure 6 CD101+ neutrophils depletion protects Ch25hECKO mice from EAE and favors CNS PMN-MDSC accumulation.

(A) EAE disease course in Ch25hfl/fl and Ch25hECKO mice treated with isotype control antibody or Combo protocol (Ly6Ghigh cell depletion). Treatment was initiated on the day of first symptoms occurrence (day 12 post-immunization). Symbols depict mean clinical score and bars mean ± SEM. Ch25hfl/fl isotype: n = 7, Ch25hECKO isotype: n = 8, Ch25hfl/fl Combo: n = 8, Ch25hECKO Combo: n = 9. The experiment was perfomed once.

(B) Survival analysis in the same conditions as in (A). Ch25hfl/fl isotype: n = 7, Ch25hECKO isotype: n = 8, Ch25hfl/fl Combo: n = 8, Ch25hECKO Combo: n = 9.

(C) Representative contour plots of PMN-MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in (A).

(D) As in (C) except that statistical analysis and percentage of PMN-MDSC in live cells are shown. Symbols depict individual mice and bars mean ± SD. Ch25hfl/fl Isotype n = 4, Ch25hECKO isotype n = 4, Ch25hfl/fl Combo, n = 3, Ch25hECKO Combo n = 4.

(E) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry. Ch25hECKO and Ch25hfl/fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE (day 14 after immunization).

(F) As in (E) except that statistical analysis is shown. Symbols depict individual mice, bars mean ± SD. Ch25hfl/lfl isotype n = 7, Ch25hECKO isotype n = 5, Ch25hfl/lfl Combo n = 6, Ch25hECKO Combo n = 5. The experiment was performed once.

(G) Representative contour plots of CD101 expression in blood and CNS neutrophils of the same EAE mice as in (A) assessed by flow cytometry.

(H) As in (G) except that statistical analysis is shown. Symbols depict individual mice, bars indicate mean ± SD. Ch25hfl/lfl isotype n = 4 , Ch25hECKO isotype n = 4, Ch25hfl/lfl Combo n = 3, Ch25hECKO Combo n = 4. The experiment was performed once.

Data information: ns= non-significant, *P < 0.05, ** P ≤ 0.005,*** P ≤ 0.0005 **** P ≤ 0.00005. P values were determined by two-way ANOVA with Sidak's post hoc test (A, F and H), log-rank (Mantel-cox) test (B) and two-tailed unpaired t-test (D).

Tables

Table 1. Comparison of EAE disease course in Ch25hfl/fl, Ch25hECKO, Ch25hBECKO and Ch25hLECKO mice.

Table 1. Related to Figure 1. Ch25h deletion in blood ECs is protective during EAE.

AUC = Area under the curve. Mean ± SD or SEM for AUC are indicated. P values were determined by unpaired student t-test or log-rank (mantel-cox) test for diseases incidence. * : P <0.05. Representative results of 2 independent experiments per genotype with a minimum of n = 5 biological replicates/group.

Expanded View Figure Legends:
Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency (2016)
Figure 2

Figure 2. Scheme of SHOX and CYP26C1 proteins and luciferase assays.

A. SHOX protein representation and luciferase assay. The variants found in SHOX are indicated in red. Luciferase assays to test the impact of SHOX mutations on its transcriptional activity were performed on FGFR3 promoter in U2OS cells (n = 4). Experiments were performed in triplicates. pcDNA4-TO empty vector was used as control. R153L represents a mutation known to affect SHOX protein activity and was used as positive control (Schneider et al., 2005). Homeodomain, DNA binding domain; Src Homology 3 domain; OAR, OtpAristalessRax domain. RLU, Relative Light Units.

B. CYP26C1 protein representation and luciferase assay. Variants found in CYP26C1 are indicated in green. Cignal-RARE system luciferase assays to test the impact of CYP26C1 variants on its RA degradation activity were performed in U2OS cells treated with 250 nM all-trans retinoic acid (ATRA) for 24 hours (n = 4). Experiments were performed in triplicates.pIRES2-EGFP empty vector was used as control. The residue C459 represents the Iron binding residue (Q6VOL0, UniProtKB) and was mutated to Ala and used as a positive control. TM, Transmembrane helix; P450, cytochrome p450 domain. RLU, Relative Light Units.

Data information: Data are shown as means ± SD.* p-value = 0.0286, two-tailed Mann-Whitney non-parametric t test.
Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency (2016)
Figure 3

Figure 3. CYP26C1 is expressed in human primary chondrocytes and retinoic acid affects SHOX expression.

A. Left panel, RT-PCR showing the expression of CYP26C1 mRNA in human primary chondrocytes. SOX9 was used as chondrocyte marker. Right panel, western blot showing the expression of CYP26C1 in human primary chondrocytes on protein level. Overexpression of CYP26C1 in U2OS cells was carried out to compare protein sizes. +, cDNA; -, water; M, marker; HPC, human primary chondrocytes.

B. Relative expression of SHOX mRNA normalized to the reference genes SDHA and HPRT in human primary chondrocytes treated with ATRA 100 nM for 6h (n = 5). One outlier with high relative expression (2.3 fold) was excluded from ATRA 100 nM treatment (two-sided Grubb's test, Z value 2.34, p-value < 0.05).

C. SHOX promoter was cloned in pGL3basic for luciferase reporter experiments as previously described (Verdin et al, 2015). In silico analysis of SHOX promoter identified three putative RXRa binding sites which were mutated to test their direct effect on SHOX expression upon treatment with ATRA 250 nM in U2OS cells (n = 4). Experiments were performed in triplicates. RLU, Relative Light Units.

D. CYP26C1 and SHOX are members of the retinoic acid pathway. Vitamin A, retinol (ROL), enters the cell and is oxidized to retinaldehyde (RAL). RAL is then oxidized to retinoic acid (RA). RA can enter the nucleus and regulate the expression of its targets. CYP26C1 controls RA intracellular levels by oxidizing this molecule in more hydrosoluble retinoid molecules like 4'-hydroxy-retinoic acid (4'-OH-RA), which can be readily excreted. High levels of RA downregulate SHOX expression.

Data information: Data are shown as means ± SD. (B) ** p = 0.0079, (C) * p-value = 0.0286, n.s., not significant, (B, C) two-tailed Mann-Whitney non-parametric t test.
Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency (2016)
Figure 4

Figure 4. Pattern of defects in zebrafish embryos injected with anti-shox morpholino.

A-C. Wild type embryos injected with control MO or with shox MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins. shox morphants show smaller fins compared to controls (n = 30 embryos).

C. Expression of col2a1 at 55 hpf was examined by in situ hybridization in wild type embryos injected with control MO and with shox MO. Arrow and dotted line indicate the pectoral fin.

D. Pectoral fin area was measured by imageJ (n = 30 embryos).

Data information: Data are shown as means ± SD. *** p-value = 0.0001, two-tailed unpaired Student's t test.
Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency (2016)
Figure 5

Figure 5. Pattern of defects in zebrafish embryos injected with anti-cyp26c1 morpholino.

A-D. Wild type embryos injected with control MO or with cyp26c1 MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins; *, otic vesicle. cyp26c1 morphants show smaller fins compared to controls (n = 30 embryos). (C, D) Expression of col2a1 (C) and shox (D) at 55 hpf was examined by in situ hybridization in embryos injected with control MO or with cyp26c1 MO. (C) Dorsal view and magnification on the pectoral fins of col2a1 expression. Arrow and dotted line indicate the pectoral fin. (D) Dorsal view and magnification on the pectoral fins of shox expression. Arrow and dotted line indicate the pectoral fin.

E. Pectoral fin area was measured by imageJ (n = 30 embryos).

F. Cignal-RARE system luciferase assay testing cyp26c1 MO on RA acid levels in zebrafish embryos (n = 5 replicates). Embryos were separated in groups of 20-30 and luciferase assayed. RLU, Relative Light Units.

G. Relative shox mRNA expression normalized to reference genes eef1a and b-actin in zebrafish embryos injected with control MO or cyp26c1 MO (n = 4). RNA was extracted from 10-15 injected embryos at 55 hours post fertilization.

H. Relative shox mRNA expression normalized to reference genes eef1a and b-actin in zebrafish embryos treated with 100 nM ATRA (n = 4). Embryos were collected at 24 hpf and treated with mock control or ATRA. RNA was extracted from 10-15 embryos after 6 hours treatment.

Data information: Data are shown as means ± SD. (E) ** p-value = 0.0006 (F) * p-value = 0.0119, (G) * p-value = 0.0286, (H) * p-value = 0.0286. (E) Two-tailed unpaired Student's t test. (F-H) Two-tailed Mann-Whitney non-parametric t test.
Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency (2016)
Figure 6

Figure 6. Co-injection of titrated subphenotypic doses of anti-shox and anti-cyp26c1 morpholinos impairs limb development.

A-C. Wild type embryos injected with control MO, subphenotypic doses of shox MO, cyp26c1 MO or a combination of shox + cyp26c1 MOs. (A) Dorsal views of the embryos at 55 hours post fertilization (hpf). Dotted line, pectoral fins. shox + cyp26c1 double morphants show smaller fins compared to control and single MOs (n = 30 embryos). (B, C) Dorsal view and magnification on the pectoral fins of col2a1 expression at 55 hpf. Arrow and dotted line indicate the pectoral fin.

D. Pectoral fins area was measured by imageJ (n = 30 embryos).

E. Relative expression of shox mRNA normalized to reference genes eef1a and b-actin in zebrafish embryos injected with control MO, shox MO, cyp26c1 MO, or shox + cyp26c1 MOs (n = 4). RNA was extracted from 10-15 injected embryos at 55 hours post fertilization.

Data information: Data are shown as means ± SD. (D) *** p-value = 0.0001, (E) ** p-value = 0.0048. (D) One-way ANOVA Bonferroni's multiple comparison test. (E) Klustal-Wallis Dunn's multiple comparison test.
The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication (2020)
Figure 1

Figure 1. Identification of human batokines by proteomics and transcriptomics.

A Schematic illustration of the experiment. Clonal human brown and white adipocytes were subjected to RNA sequencing and mass spectrometry.

B Bioinformatic analysis defined 16 secreted candidates (fold change ≥5) from brown compared to white adipocytes.

C Schematic illustration of the experiment. C57Bl/6 mice (11 weeks old) received adenovirus (2x109 PFU/mouse) for each candidate via tail-vein. GFP adenovirus was used as a control. Seven days after adenovirus infection, mice were euthanized and inguinal (iWAT) and epididymal (eWAT) white fat were weighed. n=5 for all the candidates and control.

D Changes (Δ g) in inguinal WAT (iWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant.

E Changes (Δ g) in epididymal WAT (eWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01, ***P<0.001 relative to GFP by Student's t-test. N.S., not significant.

F Expression of Pltp mRNA in indicated tissues. Data are shown by relative Pltp mRNA levels normalized by 18s expression. n=3 for all tissues (except BAT and WAT n=5, Testis n=2). *P<0.05 Pltp expression in BAT vs other tissues (except testis and lung) by Student's t-test.

G Plasma PLTP activity from control (Ppargflox/flox) and BAT-less mice (Pparg UCP1-KO, Ucp1-Cre x Ppargflox/flox). Following 6-h fasting plasma was obtained from PpargUCP1-KO mice and littermate control. n=3 for both groups. *P < 0.05 relative to control mice by Student's t-test.

H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type (WT) and aP2-PRDM16 mice [24]. Peptide levels was determined by quantitative proteomics. n=2 for both groups.

I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ. ChIP-seq data for PRDM16 and PPARγ were obtained from [31,32], respectively.

Data information: All the data were represented as mean ± s.e.m.
The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication (2020)
Figure 2

Figure 2. PLTP increases whole-body energy expenditure and protects against diet-induced body weight gain.

A Schematic illustration of the experiment. C57Bl/6 mice (7 weeks old) received AAV expressing PLTP or GFP (as control) at the viral titer of 5x1012 GC/mouse via tail-vein. High fat diet (HFD) started one week after AAV administration for both groups. Metabolic studies and euthanasia were performed after 10 weeks of HFD.

B Changes in body weight of mice in (A) during 10 weeks of HFD. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05, **P<0.01, ***P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.

C Changes in food intake of mice in (A) during 10 weeks of HFD (cohort 1). n=9 for GFP (cage 1 = 5 mice; cage 2 = 4 mice) and n=8 for PLTP (cage 1 = 4 mice; cage 2 = 4 mice)

D Body composition of mice in (A) at 10 weeks of HFD determined by EchoMRI. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05 relative to GFP by two-way ANOVA followed by Tukey's test. N.S., not significant.

E Whole-body energy expenditure of mice at 3 weeks of HFD. Area under the curve (AUC) of VO2 is shown in the right graph. n=7 for GFP and n=8 for PLTP (cohort 2). *P<0.05 relative to GFP by Student's t-test.

F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis. n=7 for GFP and n=8 for PLTP (cohort 2). N.S., not significant by Student's t-test

G OCR in the interscapular BAT and iWAT of control and PLTP-treated mice. One week after the treatment, BAT and iWAT were isolated from mice (7 weeks old) that received adenovirus for GFP or PLTP. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.

H Gene expression of indicated thermogenic genes in (A) from interscapular BAT for GFP (n=8) and PLTP (n=8) groups (cohort 1) except n=7 for Ucp1 of the PLTP group. Tbp was used as an internal control. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant.

I H&E staining of interscapular BAT from mice in (A) at 10 weeks of HFD (cohort 1). Representative images from n=3 mice for both groups. Scale Bar, 50µm.

Data information: All the data were represented as mean ± s.e.m.
The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication (2020)
Figure 3

Figure 3. PLTP improves systemic glucose homeostasis.

A Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 10 weeks of HFD. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=9 for GFP and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.

B Insulin tolerance test of mice in (A) at 10 weeks of HFD. Mice were fasted for four hours and administered insulin (0.75 U/kg body weight). n=9 for GFP control and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.

C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test.

D Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 6 weeks of HFD. Note that no difference was found in body weight between the groups by two-way ANOVA repeated measures followed by Bonferroni's test.. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=7 for GFP control and n=10 for PLTP. AUC of glucose is shown in the right graph. *P<0.05 by Student's t-test.

E 18F-FDG PET/CT scan images of mice that received adenovirus GFP (control) or PLTP at 14 weeks of HFD. Mice received CL316,243 at dose of 1 mg/kg of body weight 30 min before the PET/CT images. Representative images of n=5 for both groups. Yellow arrows indicate anatomical references as bladder, heart and interscapular BAT.

F Quantification of 18F-FDG uptake in indicated tissues from mice in (E). After the 18F-FDG PET/CT imaging, indicated tissues were harvested and the radioactivity was measured by scintillation counter. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.

Data information: All the data were represented as mean ± s.e.m.
The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication (2020)
Figure 4

Figure 4. PLTP improves lipid homeostasis and increases bile acids in vivo

A Lipidomics-based quantification of indicated lipid species in mice that received tail-vein injection of adenovirus expressing PLTP or GFP (control). Blood and tissues were harvested after 10 weeks of HFD. n=4 for GFP and n=5 for PLTP in mice on HFD. LPC = (lyso)phosphatidylcholine, LPE = (lyso)phosphatidylethalonamine, PA = phosphatidic acid, SM = sphingomyelin, TAG = triglycerides, FFA = free fatty acid. Statistical analysis is demonstrated (P value) relative to GFP by Student's t-test.

B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test.

C Relative mRNA levels of indicated genes in the liver of mice on HFD. Tbp was used as an internal control. n=4 for GFP and n=6 for PLTP. *P<0.05, **P<0.01 relative to GFP by Student's t-test.

D Bile acid levels in the liver of mice in (B). n=8 in mice on RD and n=6 in mice on HFD. *P<0.05, **P<0.01 relative to GFP by Student's t-test.

E Bile acid levels in the plasma of mice in (B). n=8 in mice on RD and n=7 in mice on HFD. ***P<0.001 relative to GFP by Student's t-test.

F Bile acid levels in the feces of mice in (B). Feces were collected from mice on RD and HFD after seven days of treatment. n=7 in mice on RD and HFD. **P<0.01, ***P<0.001 relative to GFP by Student's t-test.

G Plasma bile acids composition of mice in (B). Pool of samples from mice on HFD that received GFP or PLTP. n=5 for both groups.

H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid (CA). Cells were treated with CA at indicated concentrations for 24 h. 0.1% ETOH was used as vehicle control (Ctr). n=3. Ucp1 mRNA expression was normalized by Tbp as an internal control. **P<0.01 relative to Ctr by one-way ANOVA followed by Tukey's test. N.S., not significant.

Data information: All the data were represented as mean ± s.e.m.