Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides.

Phytosphingosine (PHS) is a sphingolipid component present mainly in epithelial tissues, including the epidermis and those lining the digestive tract. DEGS2 is a bifunctional enzyme that produces ceramides (CERs) containing PHS (PHS-CERs) via hydroxylation and sphingosine-CERs via desaturation, using dihydrosphingosine-CERs as substrates. Until now, the role of DEGS2 in permeability barrier functioning, its contribution to PHS-CER production, and the mechanism that differentiates between these two activities have been unknown. Here, we analyzed the barrier functioning of the epidermis, esophagus, and anterior stomach of Degs2 KO mice and found that there were no differences between Degs2 KO and WT mice, indicating normal permeability barriers in the KO mice. In the epidermis, esophagus, and anterior stomach of Degs2 KO mice, PHS-CER levels were greatly reduced relative to WT mice, but PHS-CERs were still present. We obtained similar results for DEGS2 KO human keratinocytes. These results indicate that although DEGS2 plays a major role in PHS-CER production, another synthesis pathway exists as well. Next, we examined the fatty acid (FA) composition of PHS-CERs in various mouse tissues and found that PHS-CER species containing very-long-chain FAs (≥C21) were more abundant than those containing long-chain FAs (C11-C20). A cell-based assay system revealed that the desaturase and hydroxylase activities of DEGS2 toward substrates with different FA chain lengths differed and that its hydroxylase activity was higher toward substrates containing very-long-chain FAs. Collectively, our findings contribute to the elucidation of the molecular mechanism of PHS-CER production.

Erratum: Diverse meibum lipids produced by Awat1 and Awat2 are important for stabilizing tear film and protecting the ocular surface.

[This corrects the article DOI: 10.1016/j.isci.2021.102478.].

Diverse meibum lipids produced by Awat1 and Awat2 are important for stabilizing tear film and protecting the ocular surface.

A lipid layer consisting of meibum lipids exists in the tear film and functions in preventing dry eye disease. Although the meibum lipids include diverse lipid classes, the synthesis pathway and role of each class remain largely unknown. Here, we created single and double knockout (KO and DKO, respectively) mice for the two acyl-CoA wax alcohol acyltransferases (Awat1 and Awat2) and investigated their dry eye phenotypes and meibum lipid composition. Awat2 KO and DKO mice exhibited severe dry eye with meibomian gland dysfunction, whereas Awat1 KO mice had mild dry eye. In these mice, specific meibum lipid classes were reduced: (O-acyl)-ω-hydroxy fatty acids and type 1ω wax diesters in Awat1 KO mice, wax monoesters and types 1ω and 2ω wax diesters in Awat2 KO mice, and most of these in DKO mice. Our findings reveal that Awat1 and Awat2 show characteristic substrate specificity and together produce diverse meibum lipids.

Direct uptake of sphingosine-1-phosphate independent of phospholipid phosphatases.

Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple KO (TKO) HAP1 cells that lacked all PLPPs (PLPP1-3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared with the WT cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPP-independent S1P uptake pathway. Tracer experiments using [32P]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of sphingolipid transporter 2 (SPNS2) or of major facilitator superfamily domain containing 2B (MFSD2B), both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas KO of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and capable of not only exporting but also importing S1P. Furthermore, we observed that erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration.

Catalytic residues, substrate specificity, and role in carbon starvation of the 2-hydroxy FA dioxygenase Mpo1 in yeast.

The yeast protein Mpo1 belongs to a protein family that is widely conserved in bacteria, fungi, protozoa, and plants, and is the only protein of this family whose function has so far been elucidated. Mpo1 is an Fe2+-dependent dioxygenase that catalyzes the α-oxidation reaction of 2-hydroxy (2-OH) long-chain FAs (LCFAs) produced in the degradation pathway of the long-chain base phytosphingosine. However, several biochemical characteristics of Mpo1, such as its catalytic residues, membrane topology, and substrate specificity, remain unclear. Here, we report that yeast Mpo1 contains two transmembrane domains and that both its N- and C-terminal regions are exposed to the cytosol. Mutational analyses revealed that three histidine residues conserved in the Mpo1 family are especially important for Mpo1 activity, suggesting that they may be responsible for the formation of coordinate bonds with Fe2+ We found that, in addition to activity toward 2-OH LCFAs, Mpo1 also exhibits activity toward 2-OH very-long-chain FAs derived from the FA moiety of sphingolipids. These results indicate that Mpo1 is involved in the metabolism of long-chain to very-long-chain 2-OH FAs produced in different pathways. We noted that the growth of mpo1Δ cells is delayed upon carbon deprivation, suggesting that the Mpo1-mediated conversion of 2-OH FAs to nonhydroxy FAs is important for utilizing 2-OH FAs as a carbon source under carbon starvation. Our findings help to elucidate the as yet unknown functions and activities of other Mpo1 family members.

Lipid polarity gradient formed by ω-hydroxy lipids in tear film prevents dry eye disease.

Meibum lipids form a lipid layer on the outermost side of the tear film and function to prevent water evaporation and reduce surface tension. (O-Acyl)-ω-hydroxy fatty acids (OAHFAs), a subclass of these lipids, are thought to be involved in connecting the lipid and aqueous layers in tears, although their actual function and synthesis pathway have to date remained unclear. Here, we reveal that the fatty acid ω-hydroxylase Cyp4f39 is involved in OAHFA production. Cyp4f39-deficient mice exhibited damaged corneal epithelium and shortening of tear film break-up time, both indicative of dry eye disease. In addition, tears accumulated on the lower eyelid side, indicating increased tear surface tension. In Cyp4f39-deficient mice, the production of wax diesters (type 1ω and 2ω) and cholesteryl OAHFAs was also impaired. These OAHFA derivatives show intermediate polarity among meibum lipids, suggesting that OAHFAs and their derivatives contribute to lipid polarity gradient formation for tear film stabilization.

Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS.

Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC/MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from human and mouse SC. Phytosphingosine- and 6-hydroxy sphingosine-type ceramides, which both contain an additional hydroxyl group, were abundant in the human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, phytosph-ingosine- and 6-hydroxy sphingosine-type ceramides were present at ∼1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for ∼90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing ß-hydroxy or ω-hydroxy FA were abundant in mice. The hydroxylated ß-carbon in ß-hydroxy ceramides was in the (R) configuration. Genetic knockout of ß-hydroxy acyl-CoA dehydratases in HAP1 cells increased ß-hydroxy ceramide levels, suggesting that ß-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the ER, is a substrate for ß-hydroxy ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.

Severe Skin Permeability Barrier Dysfunction in Knockout Mice Deficient in a Fatty Acid ω-Hydroxylase Crucial to Acylceramide Production.

The skin permeability barrier is indispensable for maintaining water inside the body and preventing the invasion of pathogens and allergens; abnormalities lead to skin disorders such as atopic dermatitis and ichthyosis. Acylceramide is an essential lipid for skin barrier formation, and CYP4F22 is a fatty acid ω-hydroxylase involved in its synthesis. Mutations in CYP4F22 cause autosomal recessive congenital ichthyosis, although the symptoms vary among mutation sites and types. Here, we generated knockout mice deficient in Cyp4f39, the mouse ortholog of human CYP4F22, to investigate the effects of completely abrogating the function of the fatty acid ω-hydroxylase involved in acylceramide production on skin barrier formation. Cyp4f39 knockout mice died within 8 hours of birth. Large increases in transepidermal water loss and penetration of a dye from outside the body were observed, indicating severe skin barrier dysfunction. Histologic analyses of the epidermis revealed impairment of lipid lamella formation, accumulation of corneodesmosomes in the stratum corneum, and persistence of periderm. In addition, lipid analyses by mass spectrometry showed almost complete loss of acylceramide and its precursor ω-hydroxy ceramide. In conclusion, our findings provide clues to the molecular mechanisms of skin barrier abnormalities and the pathogenesis of ichthyosis caused by Cyp4f39 and CYP4F22 by association.

Yeast Mpo1 Is a Novel Dioxygenase That Catalyzes the α-Oxidation of a 2-Hydroxy Fatty Acid in an Fe2+-Dependent Manner.

Phytosphingosine (PHS) is the major long-chain base component of sphingolipids in Saccharomyces cerevisiae The PHS metabolic pathway includes a fatty acid (FA) α-oxidation reaction. Recently, we identified the novel protein Mpo1, which is involved in PHS metabolism. However, the details of the FA α-oxidation reaction and the role of Mpo1 in PHS metabolism remained unclear. In the present study, we revealed that Mpo1 is involved in the α-oxidation of 2-hydroxy (2-OH) palmitic acid (C16:0-COOH) in the PHS metabolic pathway. Our in vitro assay revealed that not only the Mpo1-containing membrane fraction but also the soluble fraction was required for the α-oxidation of 2-OH C16:0-COOH. The addition of Fe2+ eliminated the need for the soluble fraction. Purified Mpo1 converted 2-OH C16:0-COOH to C15:0-COOH in the presence of Fe2+, indicating that Mpo1 is the enzyme body responsible for catalyzing the FA α-oxidation reaction. This reaction was also found to require an oxygen molecule. Our findings indicate that Mpo1 catalyzes the FA α-oxidation reaction as 2-OH fatty acid dioxygenase, mediated by iron(IV) peroxide. Although numerous Mpo1 homologs exist in bacteria, fungi, protozoa, and plants, their functions had not yet been clarified. However, our findings suggest that these family members function as dioxygenases.

The 3-hydroxyacyl-CoA dehydratases HACD1 and HACD2 exhibit functional redundancy and are active in a wide range of fatty acid elongation pathways.

Differences among fatty acids (FAs) in chain length and number of double bonds create lipid diversity. FA elongation proceeds via a four-step reaction cycle, in which the 3-hydroxyacyl-CoA dehydratases (HACDs) HACD1-4 catalyze the third step. However, the contribution of each HACD to 3-hydroxyacyl-CoA dehydratase activity in certain tissues or in different FA elongation pathways remains unclear. HACD1 is specifically expressed in muscles and is a myopathy-causative gene. Here, we generated Hacd1 KO mice and observed that these mice had reduced body and skeletal muscle weights. In skeletal muscle, HACD1 mRNA expression was by far the highest among the HACDs However, we observed only an ∼40% reduction in HACD activity and no changes in membrane lipid composition in Hacd1-KO skeletal muscle, suggesting that some HACD activities are redundant. Moreover, when expressed in yeast, both HACD1 and HACD2 participated in saturated and monounsaturated FA elongation pathways. Disruption of HACD2 in the haploid human cell line HAP1 significantly reduced FA elongation activities toward both saturated and unsaturated FAs, and HACD1 HACD2 double disruption resulted in a further reduction. Overexpressed HACD3 exhibited weak activity in saturated and monounsaturated FA elongation pathways, and no activity was detected for HACD4. We therefore conclude that HACD1 and HACD2 exhibit redundant activities in a wide range of FA elongation pathways, including those for saturated to polyunsaturated FAs, with HACD2 being the major 3-hydroxyacyl-CoA dehydratase. Our findings are important for furthering the understanding of the molecular mechanisms in FA elongation and diversity.

Uterus didelphys with obstructed hemivagina and contralateral multicystic dysplastic kidney.

Müllerian anomalies are often associated with urinary tract anomalies. Herlyn-Werner-Wunderlich syndrome or obstructed hemivagina and ipsilateral renal anomaly are rare Müllerian anomaly comprising uterine didelphys, obstructed hemivagina, and ipsilateral renal anomaly. We report an unusual case of uterus didelphys with obstructed hemivagina and contralateral (not ipsilateral) multicystic dysplastic kidney. An early diagnosis is critical to prevent complications such as adhesions with subsequent infertility. A careful evaluation of the female reproductive tract may be necessary in children with not only renal aplasia but also lateral renal anomalies such as multicystic dysplastic kidney.

[The use of bevacizumab in refractory peritoneal dissemination of gastric cancer with malignant ascites-two case reports].

Vascular endothelial growth factor (VEGF) plays an important role in proliferation of cancer cells, angiogenesis and vascular permeability in peritoneal dissemination. In addition, the release of VEGF by tumor cells has been identified as a main factor promoting the intraperitoneal secretion of fluid. Accordingly, recent evidence suggests that targeting VEGF may have the potential to suspend the ascites production resulting from peritoneal metastasis. We previously reported the effects of bevacizumab (BEV) on the growth inhabitation of peritoneal nodules and the reduction of ascites in peritoneal metastatic models. Here we present two cases of women (62 and 42 years old) with refractory peritoneal dissemination of scirrhous gastric cancer and server symptomatic ascites. They required frequent paracenteses for ascites which was resistant to systemic and intraperitoneal chemotherapy of S-1 and taxane. We treated them with intravenous BEV (5-10 mg/kg) with dramatic improvement in their ascites. BEV may have a role in the management of malignant ascites in the patient with refractory gastric cancer which should be confirmed in a larger series of well selected patients.

Isolated congenital megacystis without intestinal obstruction: a mild variant of chronic intestinal pseudoobstruction syndrome?

Megacystis is frequently involved with chronic intestinal pseudoobstruction syndrome; however, isolated megacystis without intestinal obstruction is extremely rare. We present the case of a female patient with isolated congenital megacystis without severe intestinal obstruction. In this case, barium enema did not reveal any significant findings; however, histologic evaluation of her rectum showed hypoganglionosis of the submucous and myenteric plexuses. These findings indicate that this case may be a mild variant of chronic intestinal pseudoobstruction syndrome. The presence of megacystis should alert the physician to the possibility of chronic intestinal pseudoobstruction syndrome.

Chromatographic characterization of hydrophilic interaction liquid chromatography stationary phases: hydrophilicity, charge effects, structural selectivity, and separation efficiency.

Fourteen commercially available particle-packed columns and a monolithic column for hydrophilic interaction liquid chromatography (HILIC) were characterized in terms of the degree of hydrophilicity, the selectivity for hydrophilic-hydrophobic substituents, the selectivity for the regio and configurational differences in hydrophilic substituents, the selectivity for molecular shapes, the evaluation of electrostatic interactions, and the evaluation of the acidic-basic nature of the stationary phases using nucleoside derivatives, phenyl glucoside derivatives, xanthine derivatives, sodium p-toluenesulfonate, and trimethylphenylammonium chloride as a set of samples. Principal component analysis based on the data of retention factors could separate three clusters of the HILIC phases. The column efficiency and the peak asymmetry factors were also discussed. These data on the selectivity for partial structural differences were summarized as radar-shaped diagrams. This method of column characterization is helpful to classify HILIC stationary phases on the basis of their chromatographic properties, and to choose better columns for targets to be separated. Judging from the retention factor for uridine, these HILIC columns could be separated into two groups: strongly retentive and weakly retentive stationary phases. Among the strongly retentive stationary phases, zwitterionic and amide functionalities were found to be the most selective on the basis of partial structural differences. The hydroxyethyl-type stationary phase showed the highest retention factor, but with low separation efficiency. Weakly retentive stationary phases generally showed lower selectivity for partial structural differences.

Piezoelectric extracorporeal shockwave lithotripsy for bile duct stone formation after choledochal cyst excision.

We report a case of bile duct stones in which piezoelectric extracorporeal shockwave lithotripsy (ESWL) was highly effective for the clearance of stones. A 16-year-old girl, who had undergone excision of a choledochal cyst when she was 3 years old, presented a spiking fever and colic abdominal pain. Radiological investigations showed two large stones incarcerating to the proximal end of hepatico-jejunostomy anastomosis. Massive debris was also present in intrahepatic bile duct proximal to the anastomosis. She underwent piezoelectric ESWL with an EDAP LT02 lithotripter. An average of 40 min ESWL session was repeated at intervals of 2 or 3 days. Neither anesthetic nor sedative treatment was required. By the end of the sixth session, the stones incarcerated were fragmented and the debris in the intrahepatic bile duct was completely eliminated. We conclude that piezoelectric ESWL is a less invasive, effective and repeatable method, therefore, it could be a treatment of choice for bile duct stone formation after choledochal cyst excision.

Hydrogel formation between enantiomeric B-A-B-type block copolymers of polylactides (PLLA or PDLA: A) and polyoxyethylene (PEG: B); PEG-PLLA-PEG and PEG-PDLA-PEG.

A mixed suspension of the enantiomeric B-A-B triblock copolymers, polyoxyethylene-block-poly(L-lactide)-block-polyoxyethylene (PEG-PLLA-PEG) and polyoxyethylene-block-poly(D-lactide)-block-polyoxyethylene (PEG-PDLA-PEG), was found to induce reversible gel-to-sol transition depending on the polymer concentration and temperature. The storage and loss moduli of the gel formed at lower temperature were much higher than those of the gel prepared from the corresponding ABA-type triblock copolymers because of the higher polymer concentration in the former. Although the stereo-complexation of the PLLA and PDLA blocks occurred at higher temperature also in the B-A-B copolymers, it was not responsible for the gelation of the mixed suspension. The PEG chains, involved in the helix formation of the PLLA and PDLA, should form helices with opposite helical senses to aggregate and lead the gelation of the system.

Vesicoureteral junction obstruction associated with unilateral renal agenesis.

A 7-month-old boy with a solitary kidney showed recurrent urinary tract infection. Magnetic resonance urography helps in the identification of vesicoureteral junction obstruction associated with unilateral renal agenesis.