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Niemann-Pick disease type C (NPC) is a rare neurovisceral disease caused mainly by mutations in the NPC1 gene. This autosomal recessive lysosomal disorder is characterised by the defective lysosomal secretion of cholesterol and sphingolipids. No effective therapy exists for the disease. We previously described a deep intronic point mutation (c.1554-1009 G > A) in NPC1 that generated a pseudoexon, which could be corrected at the cellular level using antisense oligonucleotides. Here, we describe the generation of two mouse models bearing this mutation, one in homozygosity and the other in compound heterozygosity with the c.1920delG mutation. Both the homozygotes for the c.1554-1009 G > A mutation and the compound heterozygotes recapitulated the hallmarks of NPC. Lipid analysis revealed accumulation of cholesterol in the liver and sphingolipids in the brain, with both types of transgenic mice displaying tremor and ataxia at 7-8 weeks of age. Behavioural tests showed motor impairment, hyperactivity, reduced anxiety-like behaviour and impaired learning and memory performances, features consistent with those reported previously in NPC animal models and human patients. These mutant mice, the first NPC models bearing a pseudoexon-generating mutation, could be suitable for assessing the efficacy of specific splicing-targeted therapeutic strategies against NPC. © The Author(s) 2017. ; The authors declare no competing financial interests. This study was partly funded by grants from the Spanish Ministry of Science and Innovation (SAF2011-25431, SAF2013-49129-C2-1-R and SAF2014-56562-R) and from the Catalan Government (2014SGR/932 and SGR2014/1125), as well as the Addi and Cassi Fund. We are particularly grateful for the support from the Addi and Cassi Fund, who also provided the heterozygous mice. The Centre for Genomic Regulation has received support as a Severo Ochoa Centre of Excellence SEV- 2012–0208. We thank Alexandre Garcia for technical assistance. The authors are also grateful for the support from the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), which is an initiative of the ISCIII. MGG was supported by a grant from the University of Barcelona (APIF). ; Peer reviewed

This study explores the effect of tributyltin (TBT) treatment on lipid homeostasis of D. magna using chemometric methods. The experimental design included two factors 'time' and 'dose' of TBT, and lipids were measured in replicate samples for each combination of factors. This study represents a case of multivariate multi-set data, obtained using a two factorial balanced design and explored using different multivariate chemometric techniques, to have an insight into data patterns, to separate main sources of variance and to identify lipid profiles associated with TBT dose. ANOVA- Simultaneous Component Analysis (ASCA) allowed the separation of the two sources of variation, suggesting thatthe TBT dose affected the evolution of the concentrations of lipids over time, although when the whole development process was considered, the interaction between time and dose factors was rather low. Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) helped to resolve four distinct profiles associated with the different phases of D. magna development, showing changes in their lipids composition, and in their relation with TBT dose. A discussion of the meaning of these changes on lipid profiles and of their time evolution is performed. © 2016 Elsevier B.V. ; This work was supported by the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 35 n. 32073.AM gratefully acknowledges the Juan de la Cierva Post-Doctoral research grant (JCI-2011-10895). ; Peer reviewed

Niemann-Pick disease type C (NPC) is a rare neurovisceral disease caused mainly by mutations in the NPC1 gene. This autosomal recessive lysosomal disorder is characterised by the defective lysosomal secretion of cholesterol and sphingolipids. No effective therapy exists for the disease. We previously described a deep intronic point mutation (c.1554-1009 G > A) in NPC1 that generated a pseudoexon, which could be corrected at the cellular level using antisense oligonucleotides. Here, we describe the generation of two mouse models bearing this mutation, one in homozygosity and the other in compound heterozygosity with the c.1920delG mutation. Both the homozygotes for the c.1554-1009 G > A mutation and the compound heterozygotes recapitulated the hallmarks of NPC. Lipid analysis revealed accumulation of cholesterol in the liver and sphingolipids in the brain, with both types of transgenic mice displaying tremor and ataxia at 7-8 weeks of age. Behavioural tests showed motor impairment, hyperactivity, reduced anxiety-like behaviour and impaired learning and memory performances, features consistent with those reported previously in NPC animal models and human patients. These mutant mice, the first NPC models bearing a pseudoexon-generating mutation, could be suitable for assessing the efficacy of specific splicing-targeted therapeutic strategies against NPC. ; This study was partly funded by grants from the Spanish Ministry of Science and Innovation (SAF2011-25431, SAF2013-49129-C2-1-R and SAF2014-56562-R) and from the Catalan Government (2014SGR/932 and SGR2014/1125), as well as the Addi and Cassi Fund. We are particularly grateful for the support from the Addi and Cassi Fund, who also provided the heterozygous mice. The Centre for Genomic Regulation has received support as a Severo Ochoa Centre of Excellence SEV- 2012-0208. We thank Alexandre Garcia for technical assistance. The authors are also grateful for the support from the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), which is an initiative of the ISCIII. MGG was supported by a grant from the University of Barcelona (APIF).

Abstract Background The early prediction of the severity of acute pancreatitis still represents a challenge for clinicians. Experimental studies have revealed the generation of specific halogenated lipids, in particular oleic acid chlorohydrin, in the early stages of acute pancreatitis. We hypothesized that the levels of circulating oleic acid chlorohydrin might be a useful early prognostic biomarker in acute pancreatitis in humans. Methods In a prospective, multicenter cohort study, plasma samples collected within 24 h after presentation in the emergency room from 59 patients with acute pancreatitis and from 9 healthy subjects were assessed for oleic acid chlorohydrin levels. Results Pancreatitis was mild in 30 patients, moderately severe in 16 and severe in 13. Oleic acid chlorohydrin levels within 24 h after presentation were significantly higher in patients that later progressed to moderate and severe acute pancreatitis. Using 7.49 nM as the cutoff point, oleic acid chlorohydrin distinguished mild from moderately severe-to-severe pancreatitis with high sensitivity/specificity (96.6/90.0%) and positive/negative predictive values (90.3/96.4%). Using 32.40 nM as the cutoff value sensitivity, specificity, positive and negative predictive values were all 100% for severe acute pancreatitis. It was found to be a better prognostic marker than BISAP score, hematocrit at 48 h, SIRS at admission, persistent SIRS or C-reactive protein at 48 h. Conclusions Oleic acid chlorohydrin concentration in plasma is elevated in patients with acute pancreatitis on admission and correlates with a high degree with the final severity of the disease, indicating that it has potential to serve as an early prognostic marker for acute pancreatitis severity. ; This work was supported by a research grant from the Carlos III Institute of Health with reference FIS PI13/00019 and co-funded with European Union ERDF funds (European Regional Development Fund). L. Bonjoch is supported by a predoctoral fellowship from Generalitat de Catalunya (AGAUR, FI DGR 2013).

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis. ; This research was funded by MCIN/AEI/10.13039/501100011033 and by the European Union ("ERDF A way of making Europe"), grants number CTQ2017-85378-R and PID2020-113813RB-I00 (to G.F.) and PID2019-107561RB-I00 (to J.M.L.). ; Peer reviewed

HIV‐1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751–854 in the cytoplasmic tail (CT751–854). Super‐resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751–854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering. ; The authors are grateful to Barbara Müller, N. Landau, and Tom Hope for providing the plasmids pCHIV and pCAGGS NL4‐3 Env, pMM310, and peGFP‐Vpr, respectively. Proteomic analysis was performed by the SGIKER service of the University of the Basque Country. The authors would like to thank Advanced Light Microscopy Unit at the Centre for Genomic Regulation (CRG), Barcelona, Spain for the access to Leica STED microscope. The following reagents were obtained through the NIH AIDS Reagent Program (Division of NIAID, NIH): Anti‐HIV‐1 gp41 Hybridome (Chessie 8) (Cat# 526) from Dr. George Lewis; Antiviral bicyclam JM‐2987 (hydrobromide salt of AMD‐3100) from NIAID, DAIDS (cat# 8128). This project was supported by the Basque Government (grant number IT1264‐19 to M.L. and F.‐X.C.) and the Spanish Ministry of Science, Innovation, and Universities (BFU‐2015‐68981‐P). This work was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. J.A.N.‐G. was supported by a FI predoctoral fellowship from the Basque Government and currently by Fundación Biofísica Bizkaia. A.A. was supported by Fundación Biofísica Bizkaia. S.O. was supported by an IKASIKER fellowship from the Basque Government. J.C. was supported by European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No. 793830. H.G.‐K. was supported by a grant from the Deutsche Forschungsgemeischaft within TRR86. ; Peer reviewed

Inhibition and aging of neuropathy target esterase (NTE) by exposure to neuropathic organophosphorus compounds (OPs) can result in OP-induced delayed neuropathy (OPIDN). In the present study we aimed to build a model of OPIDN in adult zebrafish. First, inhibition and aging of zebrafish NTE activity were characterized in the brain by using the prototypic neuropathic compounds cresyl saligenin phosphate (CBDP) and diisopropylphosphorofluoridate (DFP). Our results show that, as in other animal models, zebrafish NTE is inhibited and aged by both neuropathic OPs. Then, a neuropathic concentration inhibiting NTE activity by at least 70% for at least 24 h was selected for each compound to analyze changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and glycerolphosphocholine (GPC) profiles. In spite to the strong inhibition of the NTE activity found for both compounds, only a mild increase in the LPCs level was found after 48 h of the exposure to DFP, and no effect were observed by CBDP. Moreover, histopathological evaluation and motor function outcome analyses failed to find any neurological abnormalities in the exposed fish. Thus, our results strongly suggest that zebrafish is not a suitable species for the development of an experimental model of human OPIDN. © 2018 The Author(s). ; This study was funded by the NATO SfP project MD.SFPP 984777 (D.R.), the European Research Council under European Union's Seven Framework Programme (FP/2007–2013)/ERC Grant Agreement n. 320737, and the Spanish Government (CTM2017-83242-R; D.R.). M.F. acknowledges the financial support from the Government of Catalonia through a Beatriu de Pinos fellowship (2016 BP-B 00233). I.F. was supported by the Ministry of Economy and Competitivity of Spain (FPI grant BES-2015-075023). The authors thank Mr. Marc Mañas for his valuable assistance building the Open Field Test setup. ; Peer reviewed

Niemann-Pick type C (NPC) disease, a lysosomal storage disorder caused by defective NPC1/NPC2 function, results in the accumulation of cholesterol and glycosphingolipids in lysosomes of affected organs, such as liver and brain. Moreover, increase of mitochondrial cholesterol (mchol) content and impaired mitochondrial function and GSH depletion contribute to NPC disease. However, the underlying mechanism of mchol accumulation in NPC disease remains unknown. As STARD1 is crucial in intramitochondrial cholesterol trafficking and acid ceramidase (ACDase) has been shown to regulate STARD1, we explored the functional relationship between ACDase and STARD1 in NPC disease. Liver and brain of Npc1 mice presented a significant increase in mchol levels and STARD1 expression. U18666A, an amphiphilic sterol that inhibits lysosomal cholesterol efflux, increased mchol levels in hepatocytes from Stard1 mice but not Stard1 mice. We dissociate the induction of STARD1 expression from endoplasmic reticulum stress, and establish an inverse relationship between ACDase and STARD1 expression and LRH-1 levels. Hepatocytes from Npc1 mice treated with U18666A exhibited increased mchol accumulation, STARD1 upregulation and decreased ACDase expression, effects that were reversed by cholesterol extraction with 2-hydroxypropyl-β-cyclodextrin. Moreover, transfection of fibroblasts from NPC patients with ACDase, decreased STARD1 expression and mchol accumulation, resulting in increased mitochondrial GSH levels, improved mitochondrial functional performance, decreased oxidative stress and protected NPC fibroblasts against oxidative stress-mediated cell death. Our results demonstrate a cholesterol-dependent inverse relationship between ACDase and STARD1 and provide a novel approach to target the accumulation of cholesterol in mitochondria in NPC disease. ; We acknowledge the support from grants SAF2017-85877R, PID2019-111669RB-100 and PID2020-115055RB-I00 from Plan Nacional de I+D funded by the Agencia Estatal de Investigacion ´ (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER) and from the CIBEREHD; the center grant P50AA011999 Southern California Research Center for ALPD and Cirrhosis funded by NIAAA/NIH; as well as support from AGAUR of the Generalitat de Catalunya SGR-2017-1112, European Cooperation in Science & Technology (COST) ACTION CA17112 Prospective European Drug-Induced Liver Injury Network, the "ER stressmitochondrial cholesterol axis in obesity-associated insulin resistance and comorbidities"-Ayudas FUNDACION BBVA and the Red Nacional 2018-102799-T de Enfermedades Metabolicas ´ y Cancer ´ and the Project 201916/31 Contribution of mitochondrial oxysterol and bile acid metabolism to liver carcinogenesis 2019 by Fundacio ´ Marato TV3. E.S. V. was supported by a contract from the "Ministerio de Educacion, ´ Cultura y Deporte" (FPU15/04537) of the Spanish Government.

Recently, Vaz et al. reported four families with complex hereditary spastic paraplegia (cHSP) and biallelic variants in PCYT2 encoding CTP: phosphoethanolamine cytidylyltransferase (ET), the rate-limiting enzyme for phosphatidylethanolamine biosynthesis. Patient-derived fibroblasts and plasma had significant abnormalities in both neutral etherlipid and etherphospholipid metabolism (Vaz et al., 2019). We wish to broaden the phenotypic and genetic spectrum of PCYT2-related disorders with two additional patients. Clinical features are detailed in Table 1. ; This study was supported by the Centre for Biomedical Research on Rare Diseases (CIBERER) [ACCI19-759], the URDCat program (PERIS SLT002/16/00174), the Hesperia Foundation and the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia [2017SGR1206] to A.P., and Instituto de Salud Carlos III [PI14/00581] (Co-funded by European Regional Development Fund. V.V., E.V. and L.P.S. were funded by grants from Instituto de Salud Carlos III, co-funded by European Social Fund. ESF investing in your future (Rio Hortega, CM18/00145; Sara Borrell, CD19/00221; PFIS, FI18/00141) ; Peer reviewed

Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system. ; This work was supported by the Ministry of Spain (MINECO) (SAF2015-71026-R and BFU2015-70454-REDT/ Adipoplast to ML, SAF2013-45887-R to LH, SAF2014-52223-C2-1-R to DS, and SAF2014-52223-C2-2-R to NC, cofunded by the European Regional Development Fund [ERDF]), the Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN) (Grant CB06/03/0001 to DS), the Government of Catalonia (2014SGR465 to DS), the Fundació La Marató de TV3 (201627-30 to DS), the Xunta de Galicia (2015-CP079 to ML) and the European Community's Seventh Framework Programme (FP7/2007- 2013) under grant agreement no. 281854 – the ObERStress project to ML. ; Peer reviewed

The prevalence of nonalcoholic fatty liver disease (NAFLD) has increased drastically due to the global obesity pandemic but at present there are no approved therapies. Here, we aimed to revert high‐fat diet (HFD)‐induced obesity and NAFLD in mice by enhancing liver fatty acid oxidation (FAO). Moreover, we searched for potential new lipid biomarkers for monitoring liver steatosis in humans. We used adeno‐associated virus (AAV) to deliver a permanently active mutant form of human carnitine palmitoyltransferase 1A (hCPT1AM), the key enzyme in FAO, in the liver of a mouse model of HFD‐induced obesity and NAFLD. Expression of hCPT1AM enhanced hepatic FAO and autophagy, reduced liver steatosis, and improved glucose homeostasis. Lipidomic analysis in mice and humans before and after therapeutic interventions, such as hepatic AAV9‐hCPT1AM administration and RYGB surgery, respectively, led to the identification of specific triacylglyceride (TAG) specie (C50:1) as a potential biomarker to monitor NAFFLD disease. To sum up, here we show for the first time that liver hCPT1AM gene therapy in a mouse model of established obesity, diabetes, and NAFLD can reduce HFD‐induced derangements. Moreover, our study highlights TAG (C50:1) as a potential noninvasive biomarker that might be useful to monitor NAFLD in mice and humans. ; We thank Gloria González‐Aseguinolaza for kindly supplying the EalbAATp promoter. Financial support statement: This study was supported by the Ministry of Spain (MINECO) (SAF2014‐52223‐C2‐1‐R to DS, SAF2017‐83813‐C3‐1‐R to DS and LH, SAF2017‐82813‐C3‐3R to NC co‐financed by the European Regional Development Fund [ERDF], a doctoral fellowship to JFM, and a Juan de la Cierva—Incorporación research fellowship [IJCI‐2016‐28313] to PM and DS), the Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN) (CB06/03/0001 to DS), the Government of Catalonia (2014SGR465 to DS), Instituto de Salud Carlos III (FISPI18/01584 to VL‐C, FIS‐PI17/00412 to RB, FIS PI18/01484 to SA and FIS17/01455 to DS‐I, Miguel Servet Fund CP15/00106 to DS‐I and Rio Hortega grant (CM19/00039) to CM) and co‐financed by the European Regional Development Fund (ERDF), Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) to JC‐EG, the Fundació La Marató de TV3 (201627‐30 to DS and 201627‐31 to NC), the European Foundation for the Study of Diabetes (EFSD)/Lilly and EFSD/Janssen‐Rising Star and L'Oréal‐UNESCO "For Women in Science" research fellowships to LH, a doctoral fellowship from the Ciência sem Fronteiras‐CNPq (237976/2012‐9) to MW. Figure 4F was created with BioRender.com. ; Peer reviewed