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Phosphatidylinositol 4-monophosphate [PtdIns(4)P] is a precursor for various phosphoinositides but also a membrane-embedded component crucial for membrane contact sites (MCSs). Several lipid transfer proteins are recruited to MCSs by recognizing PtdIns(4)P; however, it remains poorly elucidated how the production of PtdIns(4)P for lipid transport at MCSs is regulated. Following human genome-wide screening, we discovered that the PtdIns(4)P-related genes PI4KB, ACBD3, and C10orf76 are involved in endoplasmic reticulum-to-Golgi trafficking of ceramide by the ceramide transport protein CERT. CERT preferentially utilizes PtdIns(4)P generated by PI4KB recruited to the Golgi by C10orf76 rather than by ACBD3. Super-resolution microscopy observation revealed that C10orf76 predominantly localizes at distal Golgi regions, where sphingomyelin (SM) synthesis primarily occurs, while the majority of ACBD3 localizes at more proximal regions. This study provides a proof-of-concept that distinct pools of PtdIns(4)P are generated in different subregions, even within the same organelle, to facilitate interorganelle metabolic channeling for the ceramide-to-SM conversion.

Oxysterol-binding protein (OSBP), which transports cholesterol and phosphatidylinositol 4-monophosphate (PtdIns[4]P) between different organelles, serves as a conserved host factor for the replication of various viruses, and OSBP inhibitors exhibit antiviral effects. Here, we determined the crystal structure of the lipid transfer domain of human OSBP in complex with endogenous cholesterol. The hydrocarbon tail and tetracyclic ring of cholesterol interact with the hydrophobic tunnel of OSBP, and the hydroxyl group of cholesterol forms a hydrogen bond network at the bottom of the tunnel. Systematic mutagenesis of the ligand-binding region revealed that M446W and L590W substitutions confer functional tolerance to an OSBP inhibitor, T-00127-HEV2. Employing the M446W variant as a functional replacement for the endogenous OSBP in the presence of T-00127-HEV2, we have identified previously unappreciated amino acid residues required for viral replication. The combined use of the inhibitor and the OSBP variant will be useful in elucidating the enigmatic in vivo functions of OSBP.

Ceramide is a bioactive sphingolipid that mediates ionizing radiation- and chemotherapy-induced apoptosis. Neocarzinostatin (NCS) is a genotoxic anti-cancer drug that induces apoptosis in response to DNA double-strand breaks (DSBs) through ataxia telangiectasia mutated (ATM) activation. However, the involvement of ceramide in NCS-evoked nuclear events such as DSB-activated ATM has not been clarified. Here, we found that nuclear ceramide increased by NCS-mediated apoptosis through the enhanced assembly of ATM and the meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 (MRN) complex proteins in human lymphoblastoid L-39 cells. NCS induced an increase of ceramide production through activation of neutral sphingomyelinase (nSMase) and suppression of sphingomyelin synthase (SMS) upstream of DSB-mediated ATM activation. In ATM-deficient lymphoblastoid AT-59 cells compared with L-39 cells, NCS treatment showed a decrease of apoptosis even though ceramide increase and DSBs were observed. Expression of wild-type ATM, but not the kinase-dead mutant ATM, in AT-59 cells increased NCS-induced apoptosis despite similar ceramide accumulation. Interestingly, NCS increased ceramide content in the nucleus through nSMase activation and SMS suppression and promoted colocalization of ceramide with phosphorylated ATM and foci of MRN complex. Inhibition of ceramide generation by the overexpression of SMS suppressed NCS-induced apoptosis through the inhibition of ATM activation and assembly of the MRN complex. In addition, inhibition of ceramide increased by the nSMase inhibitor GW4869 prevented NCS-mediated activation of the ATM. Therefore, our findings suggest the involvement of the nuclear ceramide with ATM activation in NCS-mediated apoptosis. SIGNIFICANCE STATEMENT: This study demonstrates that regulation of ceramide with neutral sphingomyelinase and sphingomyelin synthase in the nucleus in double-strand break-mimetic agent neocarzinostatin (NCS)-induced apoptosis. This study also showed that ceramide increase in the nucleus plays a role in NCS-induced apoptosis through activation of the ataxia telangiectasia mutated/meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 complex in human lymphoblastoid cells.

The obligate intracellular bacterium Chlamydia trachomatis is the major causative agent of bacterial sexually transmitted diseases worldwide. In infected cells, the ceramide transport protein (CERT) is recruited to inclusions, where C. trachomatis replicates using host-synthesized ceramide. The ceramide is converted to sphingomyelin (SM) by a chlamydial infection-dependent SM synthesis (cidSM-synthesis) pathway, which occurs even in the absence of the SM synthases (SMS)-1 and -2 of host cells. The ceramide mimetic compound (1R,3S)-HPA-12 and the nonmimetic compound E16A, both of which are potent inhibitors of CERT, repressed the proliferation of C. trachomatis in HeLa cells. Unexpectedly, (1R,3R)-HPA-12, a ceramide mimetic compound that lacks CERT inhibitory activity, also exhibited potent anti-chlamydial activity. Using endogenous SMS-knockout mutant HeLa cells, we revealed that (1R,3R)-HPA-12 mildly inhibited cidSM-synthesis. In addition, LC-MS analysis revealed that (1R,3R)-HPA-12 is converted to a phosphocholine-conjugated metabolite in an infection-dependent manner. Imaging analysis with a fluorescent analog of ceramide suggested that cidSM-synthesis occurs in the bacterial bodies and/or inclusions. Collectively, these results suggested that (1R,3R)-HPA-12 exerts its anti-chlamydia activity not only as an inhibitor of cidSM-synthesis, but also via putative toxic effects of its phosphocholine adduct, which is most likely produced by the cidSM-synthesis route.

Cavernous internal carotid artery (ICA) aneurysm complicated by simultaneous and spontaneous formation of thromboses in the aneurysm and the parent artery is a rare clinical condition. Although the majority of patients have good outcomes, some patients experience severe ischemic stroke. Here, we report a case of symptomatic large cavernous ICA aneurysm complicated by rapid growth of an intra-aneurysmal thrombosis with simultaneous parent artery thrombosis. A 68-year-old female presented with sudden-onset diplopia, right ptosis, right conjunctival hyperemia, and paresthesia of the right face. Magnetic resonance imaging (MRI) and digital subtract angiography (DSA) revealed the presence of a large partially thrombosed aneurysm in the cavernous portion of the right ICA. We planned endovascular embolization using a flow-diverting (FD) stent. Dual-antiplatelet therapy (DAPT) with aspirin and clopidogrel was started 2 weeks prior to treatment. Although the neurological state was stable, DSA conducted on the day of the endovascular treatment showed rapid growth of an intra-aneurysmal thrombosis and de novo thrombosis in the parent artery. Direct aspiration was performed via a distal support catheter with proximal blood flow arrest using a balloon-guide catheter, and the FD stent was successfully deployed. The patient’s symptoms improved postoperatively and DSA obtained 12 months after the procedure confirmed complete occlusion of the aneurysm. Although the exact mechanism of simultaneous thrombosis formation of the aneurysm and its parent artery remains unclear, it is important to recognize that rapid growth of the thrombosis increases the risk of ischemic stroke.

Endovascular treatment of unruptured intracranial aneurysms may increase cerebral microbleeds (CMBs) in postprocedural T2*-weighted MRIs, which may be a risk for future intracerebral hemorrhage. This study examined the characteristics of postprocedural CMBs and the factors that cause their increase. The patients who underwent endovascular treatment for unruptured intracranial aneurysms from April 2016 to February 2018 were retrospectively analyzed. Treatment techniques for endovascular treatment included simple coiling, balloon-assisted coiling, stent-assisted coiling, or flow diverter placement. To evaluate the increase in CMBs, a head MRI including diffusion-weighted imaging and T2*-weighted MRIs was performed on the preprocedural day; the first postprocedural day; and at 1, 3, and 6 months after the procedure. Among the 101 aneurysms that were analyzed, 38 (37.6%) showed the appearance of new CMBs. In the multivariate analysis examining the causes of the CMB increases, chronic kidney disease, a higher number of preprocedural CMBs, and a higher number of diffusion-weighted imaging–positive lesions on the first postprocedural day were independent risk factors. Furthermore, a greater portion of the increased CMBs was found in cortical and subcortical lesions of the treated vascular perfusion area within 1 month after the procedure. In endovascular treatment for unruptured intracranial aneurysms, CMBs tended to increase in patients with small vessel disease before the procedure, and it was also implicated in hemorrhagic changes after periprocedural microinfarction.

The long-term outcomes of patients with intraprocedural aneurysm rupture (IPR) during endovascular coiling of unruptured intracranial aneurysms (UIAs) remain unclear. We investigated the long-term outcomes and predictors of neurological outcomes in patients who sustained IPR during coil embolization of UIAs.We retrospectively analyzed the medical record of 312 untreated UIAs in 284 patients who underwent endovascular coiling between April 2013 and July 2018.The mean follow-up period for the entire cohort was 25.6 months. Twelve patients (3.8%) experienced IPR. The mean aneurysm size in the IPR cohort was significantly smaller than that in the no-IPR cohort (P = 0.045). The IPR cohort had a higher percentage of earlier subarachnoid hemorrhage from another aneurysm (P = 0.019), anterior communicating artery (AComA) aneurysm (P0.001), and basilar artery (BA) aneurysm (P = 0.022) than the no-IPR cohort. Neurologic deterioration was observed in 3 patients. The morbidity and mortality rates of the IPR cohort were 25% and 8.3%, respectively. Patients with IPR during coil embolization for AComA aneurysm did not develop neurological deterioration. Two of the 3 patients (66.7%) with a BA aneurysm had neurological deterioration. The proportion of patients with an mRS score of 0-2 at the last follow-up did not differ between the 2 cohorts (P = 0.608).The proportion of functionally independent patients did not differ between patients with and without IPR. Patients with BA aneurysms who developed an IPR tended to exhibit more unfavorable clinical courses than patients with AcomA aneurysms.

57-year-old woman with sequelae of cerebral infarction was admitted to our hospital because her left-sided hemiparesis was worsened. The right internal carotid artery (ICA) was not visualized by carotid duplex sonography and brain MRA. Arterial spin labeling (ASL) perfusion MR images showed reduced signals in the bilateral ICA territories at post labeling delay 1,525 ms. Her neurological symptoms improved on the day after hospitalization. On day 3, the bilateral ICAs were well visualized on MRA, while cerebral perfusion in the ICA territories appeared to be normalized on ASL. We diagnosed cervical ICA vasospasm, based on the findings of cervical MRA and cerebral angiography. Three months later, the recurrence of ICA vasospasm occurred. ASL was useful for the serial non-invasive evaluation of cerebral hemodynamics from the onset to improvement in a patient with ICA vasospasm.

Lipid transfer proteins (LTPs) are recognized as key players in the inter-organelle trafficking of lipids and are rapidly gaining attention as a novel molecular target for medicinal products. In mammalian cells, ceramide is newly synthesized in the endoplasmic reticulum (ER) and converted to sphingomyelin in the trans-Golgi regions. The ceramide transport protein CERT, a typical LTP, mediates the ER-to-Golgi transport of ceramide at an ER-distal Golgi membrane contact zone. About 20 years ago, a potent inhibitor of CERT, named (1R,3S)-HPA-12, was found by coincidence among ceramide analogs. Since then, various ceramide-resembling compounds have been found to act as CERT inhibitors. Nevertheless, the inevitable issue remains that natural ligand-mimetic compounds might directly bind both to the desired target and to various undesired targets that share the same natural ligand. To resolve this issue, a ceramide-unrelated compound named E16A, or (1S,2R)-HPCB-5, that potently inhibits the function of CERT has recently been developed, employing a series of in silico docking simulations, efficient chemical synthesis, quantitative affinity analysis, protein–ligand co-crystallography, and various in vivo assays. (1R,3S)-HPA-12 and E16A together provide a robust tool to discriminate on-target effects on CERT from off-target effects. This short review article will describe the history of the development of (1R,3S)-HPA-12 and E16A, summarize other CERT inhibitors, and discuss their possible applications.

Peracetic acid (PAA) disinfectants are effective against a wide range of pathogenic microorganisms, including bacteria, fungi, and viruses. Several studies have shown the efficacy of PAA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, its efficacy in SARS-CoV-2 variants and the molecular mechanism of action of PAA against SARS-CoV-2 have not been investigated. SARS-CoV-2 infection depends on the recognition and binding of the cell receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the spike protein. Here, we demonstrated that PAA effectively suppressed pseudotyped virus infection in the Wuhan type and variants, including Delta and Omicron. Similarly, PAA reduced the authentic viral load of SARS-CoV-2. Computational analysis suggested that the hydroxyl radicals produced by PAA cleave the disulfide bridges in the RBD. Additionally, the PAA treatment decreased the abundance of the Wuhan- and variant-type spike proteins. Enzyme-linked immunosorbent assay showed direct inhibition of RBD-ACE2 interactions by PAA. In conclusion, the PAA treatment suppressed SARS-CoV-2 infection, which was dependent on the inhibition of the interaction between the spike RBD and ACE2 by inducing spike protein destabilization. Our findings provide evidence of a potent disinfection strategy against SARS-CoV-2.

Lipid transfer proteins mediate inter-organelle transport of membrane lipids at organelle contact sites in cells, playing fundamental roles in the lipidome and membrane biogenesis in eukaryotes. We previously developed a ceramide-mimetic compound as a potent inhibitor of the ceramide transport protein CERT. Here we develop CERT inhibitors with structures unrelated to ceramide. To this aim, we identify a seed compound with no ceramide-like structure but with the capability of forming a hydrogen-bonding network in the ceramide-binding START domain, by virtual screening of ~3 × 106 compounds. We also establish a surface plasmon resonance-based system to directly determine the affinity of compounds for the START domain. Then, we subject the seed compound to a series of in silico docking simulations, efficient chemical synthesis, affinity analysis, protein-ligand co-crystallography, and various in vivo assays. This strategy allows us to obtain ceramide-unrelated compounds that potently inhibited the function of CERT in human cultured cells.

Casein kinase 1 γ (CK1G) is involved in the regulation of various cellular functions. For instance, the ceramide transport protein (CERT), which delivers ceramide to the Golgi apparatus for the synthesis of sphingomyelin (SM), is inactivated when it receives multiple phosphorylation by CK1G. Using human genome-wide gene disruption screening with an SM-binding cytolysin, we found that loss of the

The lipid molecule ceramide is transported from the endoplasmic reticulum to the Golgi apparatus for sphingomyelin production via the ceramide transport protein (CERT), encoded by CERT1. Hyperphosphorylation of CERT's serine-repeat motif (SRM) decreases its functionality. Some forms of inherited intellectual disability (ID) have been associated with a serine-to-leucine substitution in the SRM (S132L mutation) and a glycine-to-arginine substitution outside the SRM (G243R mutation) in CERT; however, it is unclear if mutations outside the SRM disrupt the control of CERT functionality. In the current investigation, we identified a new CERT1 variant (dupAA) in a patient with mild ID that resulted from a frameshift at the C-terminus of CERT1. However, familial analysis revealed that the dupAA variant was not associated with ID, allowing us to utilize it as a disease-matched negative control for CERT1 variants that are associated with ID. Biochemical analysis showed that G243R and S132L, but not dupAA, impair SRM hyperphosphorylation and render the CERT variants excessively active. Additionally, both S132L and G243R mutations but not dupAA caused the proteins to be distributed in a punctate subcellular manner. On the basis of these findings, we infer that the majority of ID-associated CERT variants may impair SRM phosphorylation-dependent repression, resulting in an increase in sphingomyelin production concurrent with CERT subcellular redistribution.

ABO blood antigens on the human red blood cell membrane as well as different cells in various human tissues have been thoroughly studied. Anti-A and -B antibodies of IgM are present in serum/plasma, but blood group-specific glyco-antigens have not been extensively described. In this study, we performed comprehensive and quantitative serum glycomic analyses of various glycoconjugates and free oligosaccharides in all blood groups. Our comprehensive glycomic approach revealed that blood group-specific antigens in serum/plasma are predominantly present on glycosphingolipids on lipoproteins rather than glycoproteins. Expression of the ABO antigens on glycosphingolipids depends not only on blood type but also on secretor status. Blood group-specific glycans in serum/plasma were classified as type I, whereas those on RBCs had different structures including hexose and hexosamine residues. Analysis of free oligosaccharides revealed that low-molecular-weight blood group-specific glycans, commonly containing lacto-N-difucotetraose, were expressed in serum/plasma according to blood group. Furthermore, comprehensive glycomic analysis in human cerebrospinal fluid showed that many kinds of free oligosaccharides were highly expressed, and low-molecular-weight blood group-specific glycans, which existed in plasma from the same individuals, were present. Our findings provide the first evidence for low-molecular-weight blood group-specific glycans in both serum/plasma and cerebrospinal fluid.

Background:Dietary sphingolipids have various biofunctions, including skin barrier improvement, anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are responsible for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear.Methods:In this study, five structurally different LCBs were isolated and their absorption ratios and levels of their metabolites were analyzed using a lymph-duct-cannulated rat model by liquid chromatography tandem mass spectrometry (LC/MS/MS) with a novel multistage fragmentation method.Results:The five orally administered LCBs were absorbed and detected in lymph as free LCBs and several metabolites including ceramides, glucosylceramides, and sphingomyelins. The absorption rates of LCBs were 0.10-1.17% depending on their structure. The absorption rate of 4-trans,8-cis-sphingadienine was highest (1.17%), whereas that of 4-trans,8-trans,10-trans-sphingatrienine was lowest (0.10%). The amount of 4-trans,8-cis-sphingadienine-bound sphingomyelin in lymph was particularly higher than other four LCB-bound sphingomyelins.Conclusion:Structural differences among LCBs, particularly the geometric isomerism at the C8–C9 position, significant affect the absorption rate and amounts of metabolites. This is the first report revealed that the absorption rate and metabolism of sphingolipids are dependent on their LCB structure.

Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses. IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

Starfish Asterias amurensis produces sphingoid bases d18:3, 9-methyl-d18:3 (9Me-d18:3), and d22:2, which possess unique structural features. In this study, sphingoid bases prepared from A. amurensis glucosylceramides displayed unexpected elution behaviors from a general octadecyl silyl high-performance liquid chromatography (HPLC) column. For separation and isolation, sphingoid bases were fractionated by octadecyl silyl HPLC after N-acetylation, yielding d18:3, 9Me-d18:3, and two d22:2 isomers. To compare the biological activities of individual sphingoid bases, their effects on sphingolipid production in normal human keratinocytes were evaluated. Treatment with sphingoid bases increased the content of ceramides, glucosylceramides, and sphingomyelins in keratinocytes. Moreover, ceramides, which contain saturated ultra-long-chain fatty acids (C30-34), were significantly increased by treatment with d18:3, but not with other A. amurensis sphingoid bases. The mRNA level of the early differentiation marker keratin 10 was markedly decreased and sphingolipid synthesis-related genes were slightly increased in keratinocytes exposed to A. amurensis-derived d18:3, 9Me-d18:3, and d22:2 isomers. These results suggest that A. amurensis-derived sphingoid bases induce differentiation to varying degrees, sphingolipid production depends on their chemical structures, and d18:3 is the most promising functional sphingoid base.

The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER–Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER–Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM.

Glycolipids are now known to be rapidly converted to mediators for inflammatory reactions or to signaling molecules that control inflammatory events in the nervous system. The present study aimed to explore whether disturbed glycolipids metabolism in the nervous system is present in patients with a neuroinflammatory disorder, encephalo-myelo-radiculo-neuropathy (EMRN), because most EMRN patients have been reported to exhibit autoantibodies against neutral glycolipids. Although molecular pathogenesis of this disorder remains unknown, we tried to search the immunochemical abnormalities in this disorder. ELISA for activated peripheral C5 complement and mass spectrometry analysis of cerebrospinal fluid clearly disclosed a significant upregulation of active C5 complement, C5a levels in sera as well as a significant accumulation of species-specific ceramides but not sphingomyelin in cerebrospinal fluid from EMRN patients. Furthermore, we confirmed the occurrence of anti-neutral glycolipids antibodies in all EMRN patients. Thus, the present study might indicate the pathophysiology of this disorder is the dysregulation of glycolipids metabolism and abnormal production of autoantibodies against neutral glycolipids resulting in the abnormal complement activation, although molecular basis for these sphingolipids dysregulation and the occurrence of autoantibodies against glycolipids remains to be elucidated at present. The present study implicates a new therapeutic strategy employing anti-ceramide and/or anti-complement therapy for this disorder.

•The pathophysiology of the stroke-like episodes of MELAS has not completely been elucidated.•Here we report a case of stroke-like episodes, successfully treated with levetiracetam.•Neuronal hyperexcitability can be the underlying mechanism of stroke-like episodes in MELAS.

The obligate intracellular bacterium Chlamydia trachomatis proliferates in the membranous compartment inclusion formed in host cells. The host ceramide transport protein CERT delivers ceramide from the endoplasmic reticulum to the Golgi complex for the synthesis of sphingomyelin (SM). Chlamydia trachomatis has been suggested to employ CERT to produce SM in the inclusion by host SM synthases (SMSs). Here, we found that C. trachomatis proliferates and produces infective progeny even in SMS1 and SMS2 double-knockout HeLa cells, but not in the SMS1/SMS2/CERT triple-knockout cells. Interestingly, infected cells convert ceramide to SM without host SMSs. These results suggest that C. trachomatis-infected cells can convert ceramide to SM without host SMSs after CERT-mediated transfer of ceramide to the inclusions.

A 79-year-old woman came to us because of sudden onset of dysarthria. She had taken apixaban due to her non-valvular atrial fibrillation. A neurological examination revealed mild facial palsy of her right side, and magnetic resonance imaging showed acute brain infarction at the left frontal lobe. There were no stenotic lesions on intracranial or extracranial magnetic resonance angiography, and she was diagnosed with cardioembolic stroke. Intravenous infusion of heparin and edaravone was initiated, and her neurological symptoms improved. However, she gradually developed jaundice and anemia. Gastro-intestinal bleeding was not observed, and her blood test met the diagnostic criteria for hemolytic anemia. Because both the direct Coombs test and cold agglutinin were positive, she was diagnosed with mixed-type autoimmune hemolytic anemia. Although her serum hemoglobin level decreased to 7.0 g/dl on the 12th hospital day, her anemia gradually improved after steroidal therapy with transfusion. It was revealed that she had shown mild anemia (hemoglobin: 9.2-10.9 g/dl) and hyperbilirubinemia (total bilirubin: 1.8-2.6 mg/dl) for 6 months. Therefore, her latent autoimmune hemolytic anemia became activated with the occurrence of cardioembolic stroke. Autoimmune hemolytic anemia might have promoted cardiac thrombus formation despite the administration of an anticoagulant in this case. It should be noted that autoimmune hemolytic anemia can develop as thrombotic disease. In the present case, autoimmune hemolytic anemia was diagnosed based on the development of cardioembolic stroke.

Background Antiplatelet agents are typically administered before and after treatment using flow-diverter stents (FDS) to prevent thrombotic complications, but the effects of anticoagulants are unclear. We present a patient with a giant aneurysm treated with an FDS. The thrombus within the aneurysm was dissolved when a direct factor Xa inhibitor was administered to treat lower limb venous thrombosis that occurred secondary to steroid use. Case Description A 60-year-old woman with a 30-mm giant thrombosed aneurysm in the cavernous segment of the right internal carotid artery presenting with headache and right abducens nerve palsy was treated by placing an FDS. Diplopia and increased pain in her right eye appeared on postoperative day 7, and both were alleviated by continuous oral administration of prednisolone. Angiography 3 months postoperatively revealed that the aneurysm thrombosis had progressed, and there were signs of healing. However, at the same time, lower limb venous thrombosis occurred, which was treated by continuous edoxaban. Six months after surgery, her headaches worsened and angiography showed that the aneurysm was again contrast enhanced and that the thrombus within the aneurysm had dissolved. After discontinuing edoxaban 9 months after surgery, the aneurysmal thrombosis had again rapidly progressed. Conclusions Administration of a direct factor Xa inhibitor during healing after placing an FDS may cause dissolution of an existing thrombus; therefore factor Xa inhibitors must be used with caution.

Introduction: Intraprocedural rupture(IPR) is one of the most severe complications during coil embolization of intracranial aneurysms. The incidence of IPR during endovascular coiling has been estimated to be 1-8.7%; however, few studies have focused exclusively on unruptured aneurysms. The aim of the present study is to evaluate the predisposing factors for IPR during coil embolization of unruptured intracranial aneurysms. Methods: This study included 254 consecutive patients with 281 unruptured intracranial aneurysms treated with endovascular coiling between April 2013 to March 2018 at our institution (mean age 61.7, range 26-88). Dissecting aneurysms and repeated endovascular procedures in recurrent aneurysms were excluded. The medical records were reviewed retrospectively and the following data were collected: age; gender; medical history; smoking habit; family history of intracranial aneurysm; presence or absence of multiple aneurysms; history of earlier subarachnoid hemorrhage from another aneurysm; aneurysm location, size and morphology; and procedural details. The mechanisms of IPR were also investigated. Univariate and multivariate analyses were conducted to determine independent predictors of IPR. Results: The incidence of IPR was 4.2%(12 of 281 endovascular procedures). The most common mechanism of IPR was perforation by coils. In the univariate analysis, IPR was associated with aneurysm size(less than 5mm)[odds ratio(OR) 4.8, 95% confidence interval(CI) 1.44-15.88, p=0.005], aneurysms of the anterior communicating artery(OR 4.6, CI 1.39-15.35, p=0.007), aneurysms of the basilar artery(OR 4.4, CI 1.10-17.56, p=0.024) and history of earlier subarachnoid hemorrhage from another aneurysm(OR 5.8, CI 1.10-30.30, p=0.020). There was no significant association between IPR and treatment modality. Multivariate logistic regression analysis proved that the size of aneurysm(less than 5mm) was an independent factor associated with IPR(OR 6.9, CI 1.24-38.52, p=0.027). Conclusion: This study suggests the association of the size of aneurysm with incidence of IPR during coil embolization for unruptured intracranial aneurysms. Small aneurysms less than 5mm are associated with increased risk of IPR during coil embolization.

Background Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear. Methods In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method. Results The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10–1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties. Conclusions Structural differences among LCBs, particularly geometric isomerism at the C8–C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.

Arachidonic acid (AA) plays a pivotal role in the development of edema via its oxidized metabolites derived from cyclooxygenase (COX) and lipoxygenase (LOX), and is recently recognized as an activator of TRPV3. However, it is not clear whether AA plays some TRPV3-mediated pathological roles in the development of edema. Pharmacological and histological studies using ICRTRPV3+/+ and ICRTRPV3-/- mice indicated that higher ear edema responses to topical application of AA were observed in ICRTRPV3+/+ mice compared with ICRTRPV3-/- mice. However, there was no difference in the ear edema response to 12-O-tetradecanoylphorbol 13-acetate, skin histology, and skin barrier function between these mouse strains. Furthermore, oxidized fatty acids from the lesional site were analyzed to elucidate the TRPV3-mediated pathological roles of AA, and the results revealed that there were no differences in the level of COX or LOX metabolites derived from AA between both mouse strains. We concluded that AA plays a role in the development of TRPV3-mediated ear edema and that this result may contribute to better understanding of the pathophysiological mechanisms involved in the development of a certain type of edema.

International audience; We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cβ1 (PLCβ1) as a new candidate. PLCβ1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCβ1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCβ1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCβ1: plasma membrane remodeling, and in particular, caveolae formation.

Dietary sphingolipids such as glucosylceramide (GlcCer) are potential nutritional factors associated with prevention of metabolic syndrome. Our current understanding is that dietary GlcCer is degraded to ceramide and further metabolized to sphingoid bases in the intestine. However, ceramide is only found in trace amounts in food plants and thus is frequently taken as GlcCer in a health supplement. In the present study, we successfully prepared konjac ceramide (kCer) using endoglycoceramidase I (EGCase I). Konjac, a plant tuber, is an enriched source of GlcCer (kGlcCer), and has been commercialized as a dietary supplement to improve dry skin and itching that are caused by a deficiency of epidermal ceramide. Nerve growth factor (NGF) produced by skin cells is one of the itch factors in the stratum corneum of the skin. Semaphorin 3A (Sema 3A) has been known to inhibit NGF-induced neurite outgrowth of epidermal nerve fibers. It is well known that the itch sensation is regulated by the balance between NGF and Sema 3A. In the present study, while kGlcCer did not show an in vitro inhibitory effect on NGF-induced neurite outgrowth of PC12 cells, kCer was demonstrated to inhibit a remarkable neurite outgrowth. In addition, the effect of kCer was similar to that of Sema 3A in cell morphological changes and neurite retractions, but different from C2-Ceramide. kCer showed a Sema 3A-like action, causing CRMP2 phosphorylation, which results in a collapse of neurite growth cones. Thus, it is expected that kCer is an advanced konjac ceramide material that may have neurite outgrowth-specific action to relieve uncontrolled and serious itching, in particular, from atopic eczema., Highlights • We prepared konjac ceramide (kCer) from konjac GlcCer using endoglycoceramidase I. • kCer inhibits NGF-induced neurite outgrowth by semaphorin 3A-like action. • Konjac GlcCer does not show NGF-induced neurite outgrowth such as kCer.

Asterias amurensis starfish provide several bioactive species in addition to being fishery waste. Glucosyl ceramides (GlcCers) were extracted from the viscera of these starfish and were isolated by silica gel column chromatography. Degraded GlcCers generated A. amurensis sphingoid bases (ASBs) that mainly consisted of the triene-type bases d18:3 and 9-methyl-d18:3. The effect of these bases on ceramide synthesis and content were analyzed using normal human epidermal keratinocytes (NHEKs). The bases significantly enhanced the de novo ceramide synthesis and gene expression in NHEKs for proteins, such as serine-palmitoyltransferase and ceramide synthase. Total ceramide, GlcCer, and sphingomyelin contents increased dramatically upon ASB treatment. In particular, GlcCer bearing very-long-chain fatty acids (≥C28) exhibited a significant content increase. These ASB-induced enhancements on de novo ceramide synthesis were only observed in undifferentiated NHEKs. This stimulation of the de novo sphingolipid synthesis may improve skin barrier functions.

Summary Background & aims Acute lung injury (ALI) is less severe in obese than in nonobese patients, but the mechanism is unclear. Secretory leukocyte protease inhibitor (SLPI) is the key anti-inflammatory protein in various lung diseases. We have previously reported changes of the surgical stress in obese rats using lard-based high-fat diet (HFD). The purpose of this study was to elucidate the effect of lard-based HFD on the pathophysiology of lipopolysaccharide (LPS)-induced ALI, and the role of SLPI expression. Methods Male Wistar rats were fed lard-based HFD (60 kcal% fat) or control diet (CD) for either 4 or 12 weeks and were killed after intraperitoneal LPS injection. Analyses included messenger RNA expression of TNF-α, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), IL-10 and SLPI in the lung tissue and bronchoalveolar lavage fluid, and histology of the lungs. Results Rats fed HFD for 12 weeks showed suppression of the lung injury and oxidative stress after LPS injection, as indicated by reduction of pulmonary TNF-α, MIP-2 and iNOS mRNA expression and 8-hydroxy-2′-deoxyguanosine immunostaining. The increased pulmonary SLPI caused by lard was associated with decreased pro-inflammatory cytokines and oxidative stress, which eventually resulted in the prevention of ALI. Those effects of lard on LPS-induced ALI were greater after 12 weeks than after 4 weeks feeding, as indicated by the reduction of TNF-α, MIP-2 and iNOS levels. Conclusions Feeding lard-based HFD for 12 weeks attenuated LPS-induced ALI with increased pulmonary SLPI expression in rats.

Background and Aims: Recombinant tissue plasminogen activator (rt-PA) is often withheld for mild or rapidly improving acute ischemic stroke. The aim of this study is to determine risk factors for poor outcomes in such cases. Method: This study included 772 consecutive acute ischemic stroke patients from the Fukuoka Stroke Registry who did not receive rt-PA therapy because of mild or rapidly improving symptoms. Logistic regression analysis was performed to determine the predictors of poor outcomes (modified Rankin Scale 3-6 at 3 months after onset). Results: Eighty-six patients (11.1%) showed a poor outcome. Compared to patients with a good outcome (n=686), those with a poor outcome were older (78±10 vs. 68±13 years, p Conclusion: In patients with older age, chronic kidney disease, cortical symptoms, or limb paralysis, rt-PA administration should be considered even for mild or rapidly improving stroke.

Background/Objectives: Adipose tissue (AT) has an important role in energy homeostasis. The dysfunction of AT or the hyper-accumulation of neutral lipids leads to various metabolic diseases. Recent studies indicate that sphingo lipid metabolism associates with the development of metabolic diseases. Sphingomyelin, a major sphingolipid in mammals, requires sphingomyelin synthase (SMS) for biosynthesis. Previously, we reported that Sms2 deficiency inhibited diet-induced obesity, fatty liver, and insulin resistance in mice. However, the contribution of Sms2 to obesity and insulin resistance in AT is largely unknown. In this study, we investigated whether Sms2 deficiency in ATs affects obesity and insulin resistance.Subjects/Methods: Wild-type and Sms2 knockout (KO) mice were fed a high-fat for 12 weeks. Body and AT weights, and the food intake, were recorded. The AT status and macrophage infiltration were evaluated by histological analysis. The expression levels of genes and proteins involved in adipogenesis, inflammation, energy expenditure, and fatty acid metabolism were examined.Results: In white adipose tissue (WAT) from Sms2 KO mice, the number of small adipocytes increased but the adipocyte size decreased. In epididymal WAT, Sms2 deficiency inhibited inflammation and macrophage infiltration. Moreover, adipogenesis was moderately suppressed. In subcutaneous WAT from Sms2 KO mice, the expression of genes involved in energy expenditure and browning (Ucp1, Cidea, Tbx1) was elevated. In brown adipose tissue (BAT) from Sms2 KO mice, the lipid droplet surface area was lower than that of WT mice and the expression of genes involved in fatty acid synthesis (Fasn, Scd1) decreased.Conclusion: These results demonstrate that Sms2 deficiency leads to moderate adipogenesis and inflammatory suppression in epididymal WAT, increased energy expenditure by the browning of subcutaneous WAT, and suppression of fatty acid synthesis in BAT, suggesting that these synergetic effects in ATs from Sms2 KO mice contribute to the suppression of diet-induced obesity and insulin resistance.