Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Superficial Temporal Artery Posterior Branch Flap for Reconstruction of Composite Oral/Perioral Defects.

PURPOSE: Defects involving total lower and/or upper lip often extend to intraoral and/or perioral areas. Flaps based on superficial temporal artery, either as pedicled or free flaps, can be used for reconstruction of various head and neck defects. In this clinical study, we tried to demonstrate the application of a pedicled flap based on the posterior branch of superficial temporal artery in 3-dimensional reconstruction of these composite oral/perioral defects as a successful alternative in cases where microvascular tissue transfer cannot be performed. PATIENTS AND METHODS: Six male patients with composite perioral/oral defects who underwent reconstruction with a pedicled flap based on the posterior branch of the superficial temporal artery between April 2020 and December 2020 were evaluated retrospectively. Demographic data, topographic data of defects, and the dimensions of the flaps were gathered from patient files. All patients required reconstruction after tumor resection. RESULTS: All flaps survived without any signs of partial or total necrosis. Postoperatively, the patients did not report any oral incompetence or drooling, and they were able to fully close their mouths. CONCLUSIONS: Pedicled flaps based on the posterior branch of superficial temporal artery provide reliable results in composite perioral/oral reconstruction.

Periosteal Turnover Flap for Coverage and Salvage of Exposed Deep Brain Stimulation Device.

Implanted deep brain stimulation (DBS) devices are crucial in the treatment of movement disorders. Hardware extrusion is among the most frequent complications of the implantation process and requires reconstruction with well-vascularized tissues. The authors present a case of periosteal turnover flap for coverage of an exposed DBS device. An 11-year-old female patient with spastic cerebral palsy presented with an exposed DBS device located in the right parietal area. The exposed device was covered by a proximally based periosteal flap. Postoperative evaluations at months 1, 2, 3, and 8 revealed no signs of infection or dehiscence. This brief clinical study shows that reconstruction with periosteal turnover flaps is both an easy and excellent choice for secondary closure of exposed DBS devices.

Ceramide synthase 2-C24:1 -ceramide axis limits the metastatic potential of ovarian cancer cells.

Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C24:1 -ceramide for the formation of lamellipodia. These results suggested that C24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C24:1 -ceramide. Collectively, the CerS2-C24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer.

Targeting defective sphingosine kinase 1 in Niemann-Pick type C disease with an activator mitigates cholesterol accumulation.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.

Omentin-1 is associated with atrial fibrillation in patients with cardiac valve disease.

BACKGROUND: Epicardial adipose tissue (EAT) remodeling and adipocytokines are associated with structural remodeling in atrial fibrillation (AF). However, the role of omentin-1, a novel adipocytokine, in structural remodeling remains unknown. METHODS: Hematoxylin and eosin (H&E) and Masson's trichrome stains were used to investigate the histology of EAT and right atrial appendages. The expression levels of adipocytokines in these human samples were determined by immunohistochemical assay and western blotting. Models of transforming growth factor (TGF)-ß1-induced activation of cardiac fibroblasts (CFs) and TGF-ß1-induced endothelial-mesenchymal transition (EndMT) of human umbilical vein endothelial cell (HUVEC) were established to explore roles of omentin-1 in these processes. To determine changes in adipocytokines secretion under hypoxia conditions, adipocytes were treated with 5% O2 and 95% N2, and then CFs and HUVECs were co-cultured with the conditioned medium of adipocytes to determine the effects of hypoxia-treated adipocytes on these cells. RESULTS: Expression of omentin-1 was downregulated in the EAT and right atrial appendages from patients with AF compared to samples from patients without AF, while the TGF-ß1 level was upregulated in EAT from patients with AF. EAT from patients with AF exhibited adipocyte hypertrophy and severe interstitial fibrosis. Omentin-1 inhibited TGF-ß1-induced CF activation and reversed TGF-ß1-induced HUVEC EndMT. Adipocytes treated with hypoxia exhibited downregulation of omentin-1 and partly activated CFs. CONCLUSIONS: This study demonstrated that omentin-1 was an antifibrotic adipocytokine and was downregulated in patients with AF, which was partly mediated by hypoxia.

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation.

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

Endogenous reduction of miR-185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K.

Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR-185-5p were differently expressed in plasma from patients with ACS by high-throughput RNA sequencing. The expressional levels of miR-185-5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT-PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR-185-5p. In HUVECs, miR-185-5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR-185-5p inhibitors performed the opposites. Further, the inhibitory effects of miR-185-5p up-regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus-mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain-function of miR-185-5p by agomir infusion down-regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR-185-5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR-185-5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.

Long-chain acyl-CoA synthetase 1 interacts with key proteins that activate and direct fatty acids into niche hepatic pathways.

Fatty acid channeling into oxidation or storage modes depends on physiological conditions and hormonal signaling. However, the directionality of this channeling may also depend on the association of each of the five acyl-CoA synthetase isoforms with specific protein partners. Long-chain acyl-CoA synthetases (ACSLs) catalyze the conversion of long-chain fatty acids to fatty acyl-CoAs, which are then either oxidized or used in esterification reactions. In highly oxidative tissues, ACSL1 is located on the outer mitochondrial membrane (OMM) and directs fatty acids into mitochondria for ß-oxidation. In the liver, however, about 50% of ACSL1 is located on the endoplasmic reticulum (ER) where its metabolic function is unclear. Because hepatic fatty acid partitioning is likely to require the interaction of ACSL1 with other specific proteins, we used an unbiased protein interaction technique, BioID, to discover ACSL1-binding partners in hepatocytes. We targeted ACSL1 either to the ER or to the OMM of Hepa 1-6 cells as a fusion protein with the Escherichia coli biotin ligase, BirA*. Proteomic analysis identified 98 proteins that specifically interacted with ACSL1 at the ER, 55 at the OMM, and 43 common to both subcellular locations. We found subsets of peroxisomal and lipid droplet proteins, tethering proteins, and vesicle proteins, uncovering a dynamic role for ACSL1 in organelle and lipid droplet interactions. Proteins involved in lipid metabolism were also identified, including acyl-CoA-binding proteins and ceramide synthase isoforms 2 and 5. Our results provide fundamental and detailed insights into protein interaction networks that control fatty acid metabolism.

Decreased ceramide underlies mitochondrial dysfunction in Charcot-Marie-Tooth 2F.

Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neurologic disorder, but its molecular mechanisms remain unclear. One variant of CMT, 2F, is characterized by mutations in heat shock protein 27 (Hsp27). As bioactive sphingolipids have been implicated in neurodegenerative diseases, we sought to determine if their dysregulation is involved in CMT. Here, we show that Hsp27 knockout mice demonstrated decreases in ceramide in peripheral nerve tissue and that the disease-associated Hsp27 S135F mutant demonstrated decreases in mitochondrial ceramide. Given that Hsp27 is a chaperone protein, we examined its role in regulating ceramide synthases (CerSs), an enzyme family responsible for catalyzing generation of the sphingolipid ceramide. We determined that CerSs colocalized with Hsp27, and upon the presence of S135F mutants, CerS1 lost its colocalization with mitochondria suggesting that decreased mitochondrial ceramides result from reduced mitochondrial CerS localization rather than decreased CerS activity. Mitochondria in mutant cells appeared larger with increased interconnectivity. Furthermore, mutant cell lines demonstrated decreased mitochondrial respiratory function and increased autophagic flux. Mitochondrial structural and functional changes were recapitulated by blocking ceramide generation pharmacologically. These results suggest that mutant Hsp27 decreases mitochondrial ceramide levels, producing structural and functional changes in mitochondria leading to neuronal degeneration.-Schwartz, N. U., Linzer, R. W., Truman, J.-P., Gurevich, M., Hannun, Y. A., Senkal, C. E., Obeid, L. M. Decreased ceramide underlies mitochondrial dysfunction in Charcot-Marie-Tooth 2F.

A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.

Perilipin 2 (PLIN2) is a lipid-droplet protein that is up-regulated in alcoholic steatosis and associated with hepatic accumulation of ceramides, bioactive lipids implicated in alcoholic liver disease pathogenesis. The specific role of ceramide synthetic enzymes in the regulation of PLIN2 and promotion of hepatocellular lipid accumulation is not well understood. We examined the effects of pharmacologic ceramide synthesis inhibition on hepatic PLIN2 expression, steatosis, and glucose and lipid homeostasis in mice with alcoholic steatosis and in ethanol-incubated human hepatoma VL17A cells. In cells, pharmacologic inhibition of ceramide synthase reduced lipid accumulation by reducing PLIN2 RNA stability. The subtype ceramide synthase (CerS)6 was specifically up-regulated in experimental alcoholic steatosis in vivo and in vitro and was up-regulated in zone 3 hepatocytes in human alcoholic steatosis. In vivo ceramide reduction by inhibition of de novo ceramide synthesis reduced PLIN2 and hepatic steatosis in alcohol-fed mice, but only de novo synthesis inhibition, not sphingomyelin hydrolysis, improved glucose tolerance and dyslipidemia. These findings implicate CerS6 as a novel regulator of PLIN2 and suggest that ceramide synthetic enzymes may promote the earliest stage of alcoholic liver disease, alcoholic steatosis.-Williams, B., Correnti, J., Oranu, A., Lin, A., Scott, V., Annoh, M., Beck, J., Furth, E., Mitchell, V., Senkal, C. E., Obeid, L., Carr, R. M. A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.

Sphingolipids in mitochondria.

Sphingolipids are bioactive lipids found in cell membranes that exert a critical role in signal transduction. In recent years, it has become apparent that sphingolipids participate in growth, senescence, differentiation and apoptosis. The anabolism and catabolism of sphingolipids occur in discrete subcellular locations and consist of a strictly regulated and interconnected network, with ceramide as the central hub. Altered sphingolipid metabolism is linked to several human diseases. Hence, an advanced knowledge of how and where sphingolipids are metabolized is of paramount importance in order to understand the role of sphingolipids in cellular functions. In this review, we provide an overview of sphingolipid metabolism. We focus on the distinct pathways of ceramide synthesis, highlighting the mitochondrial ceramide generation, transport of ceramide to mitochondria and its role in the regulation of mitochondrial-mediated apoptosis, mitophagy and implications to disease. We will discuss unanswered questions and exciting future directions. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Consistency over time of animal-based welfare indicators as a further step for developing a welfare assessment monitoring scheme: The case of the Animal Welfare Indicators protocol for dairy goats.

Consistency over time (COT) of animal-based indicators is key to a reliable and feasible welfare protocol, indicating that results are representative over long-term situations. High levels of consistency ensure fairness for the farmer and credibility of the system. In addition, indicator COT reduces recording costs, as having indicators that do not change over a long period of time will require less farm visits to achieve reliable estimates. To date, COT of animal-based indicators included in the welfare assessment of dairy goats has never been tested. Therefore, our aim was to investigate COT of animal-based indicators included in the Animal Welfare Indicators (AWIN) welfare assessment prototype protocol for dairy goats. To meet this goal, a study was designed where an average of 3 mo elapsed between 2 sets of visits to the same 20 dairy goat farms (10 in Portugal and 10 in Italy), with no major changes in management routines or housing conditions occurring during this period. Initially, we performed a Wilcoxon signed rank test to investigate whether the results obtained during the 2 visits were significantly different. After this preliminary screening, the indicators presenting nonsignificant differences between visits were submitted to a second step analysis, where discriminative and evaluative analyses were conducted to reach a final indicator lineup. The discriminative approach helped distinguishing among farms, whereas the agreement analysis showed us the range of differences between repeated assessments. Some particular conclusions could be drawn from this combined analysis, helping to the development of the final AWIN welfare assessment protocol for dairy goats and as a further step to develop a welfare assessment monitoring scheme for this and other species. In this sense, the AWIN welfare assessment protocol allows for the quick differentiation between farms based on the identification of persistent welfare problems, by recording highly consistent and feasible indicators. In a second step, a more comprehensive protocol, consisting of indicators more likely to be subject to variations along time, was applied. Repeated assessments and long-term studies of indicator consistency are needed to help determine the frequency of visits required to obtain a consistent and feasible welfare assessment scheme. This paper adds to the literature by providing guidance on the variability of animal-based indicators over time.

Micro-CT evaluation of the quality of root fillings when using three root filling systems.

AIM: To evaluate the percentage volumes of filling materials and voids in single-rooted teeth filled with three different filling systems using micro-computed tomography (micro-CT). METHODOLOGY: Thirty single-rooted human teeth were used. After preparation of the root canals, the teeth were randomly divided into three groups (n = 10). The canals were filled with EndoREZ, ActiV GP or AH Plus/gutta-percha. Each specimen was scanned using a micro-CT device at a resolution of 13.68 µm. Percentage volumes of root filling materials and voids were calculated and data were analysed statistically using the Kruskal-Wallis test and the Mann-Whitney U-test with Bonferroni adjustment. Within each group, the Friedman test was performed with the Wilcoxon signed-rank test to detect discrepancies. RESULTS: The percentage volume of filling material was significantly lower in the ActiV GP group than in the other groups (P < 0.05), whilst the percentage volume of voids was significantly higher in the ActiV GP group than in the other groups (P < 0.05). CONCLUSION: None of the systems were associated with void-free root fillings. The ActiV GP system had a significantly higher percentage volume of voids than the other systems.

Tumor Necrosis Factor-α (TNFα)-induced Ceramide Generation via Ceramide Synthases Regulates Loss of Focal Adhesion Kinase (FAK) and Programmed Cell Death.

Ceramide synthases (CerS1-CerS6), which catalyze the N-acylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide in both the de novo and the salvage or recycling pathway of ceramide generation, have been implicated in the control of programmed cell death. However, the regulation of the de novo pathway compared with the salvage pathway is not fully understood. In the current study, we have found that late accumulation of multiple ceramide and dihydroceramide species in MCF-7 cells treated with TNFα occurred by up-regulation of both pathways of ceramide synthesis. Nevertheless, fumonisin B1 but not myriocin was able to protect from TNFα-induced cell death, suggesting that ceramide synthase activity is crucial for the progression of cell death and that the pool of ceramide involved derives from the salvage pathway rather than de novo biosynthesis. Furthermore, compared with control cells, TNFα-treated cells exhibited reduced focal adhesion kinase and subsequent plasma membrane permeabilization, which was blocked exclusively by fumonisin B1. In addition, exogenously added C6-ceramide mimicked the effects of TNFα that lead to cell death, which were inhibited by fumonisin B1. Knockdown of individual ceramide synthases identified CerS6 and its product C16-ceramide as the ceramide synthase isoform essential for the regulation of cell death. In summary, our data suggest a novel role for CerS6/C16-ceramide as an upstream effector of the loss of focal adhesion protein and plasma membrane permeabilization, via the activation of caspase-7, and identify the salvage pathway as the critical mechanism of ceramide generation that controls cell death.

Simultaneous head tissue conductivity and EEG source location estimation.

Accurate electroencephalographic (EEG) source localization requires an electrical head model incorporating accurate geometries and conductivity values for the major head tissues. While consistent conductivity values have been reported for scalp, brain, and cerebrospinal fluid, measured brain-to-skull conductivity ratio (BSCR) estimates have varied between 8 and 80, likely reflecting both inter-subject and measurement method differences. In simulations, mis-estimation of skull conductivity can produce source localization errors as large as 3cm. Here, we describe an iterative gradient-based approach to Simultaneous tissue Conductivity And source Location Estimation (SCALE). The scalp projection maps used by SCALE are obtained from near-dipolar effective EEG sources found by adequate independent component analysis (ICA) decomposition of sufficient high-density EEG data. We applied SCALE to simulated scalp projections of 15cm(2)-scale cortical patch sources in an MR image-based electrical head model with simulated BSCR of 30. Initialized either with a BSCR of 80 or 20, SCALE estimated BSCR as 32.6. In Adaptive Mixture ICA (AMICA) decompositions of (45-min, 128-channel) EEG data from two young adults we identified sets of 13 independent components having near-dipolar scalp maps compatible with a single cortical source patch. Again initialized with either BSCR 80 or 25, SCALE gave BSCR estimates of 34 and 54 for the two subjects respectively. The ability to accurately estimate skull conductivity non-invasively from any well-recorded EEG data in combination with a stable and non-invasively acquired MR imaging-derived electrical head model could remove a critical barrier to using EEG as a sub-cm(2)-scale accurate 3-D functional cortical imaging modality.

Convex Instrumented Hemiepiphysiodesis With Concave Distraction: A Treatment Option for Long Sweeping Congenital Curves.

BACKGROUND: Convex hemiepiphysiodesis has been reported to have mixed results in the treatment of congenital spine deformities. Multiple modifications of the original technique were suggested to improve the results. The purpose of this study is to report the results and complications of an instrumented convex growth arrest procedure modified with concave distraction. METHODS: The records of 11 patients with long sweeping congenital curves (involving multiple anomalous vertebrae) who underwent convex instrumented hemiepiphysiodesis with concave distraction were evaluated. Mean age at index operation was 58 months (range, 29 to 101 mo). Lengthening of the concave distraction rod was done every 6 months. The magnitude of coronal/sagittal deformity and T1-T12 height were measured on the preoperative, postoperative, and latest follow-up radiographs. Average follow-up was 44.9 months (range, 24 to 89 mo). RESULTS: In the coronal plane, the convex hemiepiphysiodesis segment was corrected from an average of 60.5 to 40.4 degrees postoperatively and further improved to 35.5 degrees at the latest follow-up. The distracted segment was corrected from 33.4 to 15.2 degrees postoperatively and to 12.7 degrees at the latest follow-up. Sagittal plane alignment was minimally affected. The average T1-T12 height was 157.1 mm in the early postoperative period and 181.1 mm at last follow-up. During follow-up, we identified partial pull-out of screws on the distraction side in 5 of the 11 patients and rod breakages in 3 patients. These were revised during planned lengthenings. There were no unplanned surgeries, deep wound infections, nor neurological complications. CONCLUSIONS: Convex instrumented hemiepiphysiodesis with concave distraction resulted in good curve correction while maintaining the growth of thorax. The correction of the anomalous segment improved over time, proving the effectiveness of the hemiepiphysiodesis. Addition of a concave distraction construct appears to enhance spinal growth, thereby augmenting the hemiepiphysiodesis effect.

Sustained PKCßII activity confers oncogenic properties in a phospholipase D- and mTOR-dependent manner.

Protein kinase C (PKC) is a family of serine/threonine kinases implicated in a variety of physiological processes. We have shown previously that sustained activation of the classical PKCα and PKCßII induces their phospholipase D (PLD)-dependent internalization and translocation to a subset of the recycling endosomes defined by the presence of PKC and PLD (the pericentrion), which results in significant differences in phosphorylation of PKC substrates. Here, we have investigated the biological consequences of sustained PKC activity and the involvement of PLD in this process. We find that sustained activation of PKC results in activation of the mammalian target of rapamycin (mTOR)/S6 kinase pathway in a PLD- and endocytosis-dependent manner, with both pharmacologic inhibitors and siRNA implicating the PLD2 isoform. Notably, dysregulated overexpression of PKCßII in A549 lung cancer cells was necessary for the enhanced proliferation and migration of these cancer cells. Inhibition of PKCßII with enzastaurin reduced A549 cell proliferation by >60% (48 h) and migration by >50%. These biological effects also required both PLD activity and mTOR function, with both the PLD inhibitor FIPI and rapamycin reducing cell growth by >50%. Reciprocally, forced overexpression of wild-type PKCßII, but not an F666D mutant that cannot interact with PLD, was sufficient to enhance cell growth and increase migration of noncancerous HEK cells; indeed, both properties were almost doubled when compared to vector control and PKC-F666D-overexpressing cells. Notably, this condition was also dependent on both PLD and mTOR activity. In summary, these data define a PKC-driven oncogenic signaling pathway that requires both PLD and mTOR, and suggest that inhibitors of PLD or mTOR would be beneficial in cancers where PKC overexpression is a contributing or driving factor.

Safety and Efficacy of Apical Resection Following Growth-friendly Instrumentation in Myelomeningocele Patients With Gibbus: Growing Rod Versus Luque Trolley.

INTRODUCTION: Thoracolumbar/lumbar kyphosis in myelomeningocele patients is a common and severely debilitating condition, amenable only to surgical correction. Several surgical techniques have been proposed. Growth-friendly techniques should be preferred in this patient population due to an already compromised trunk height. The growing rod (GR) and Luque trolley (LT) with Galveston instrumentation are well-known growth-friendly techniques. We compared results and complications in 2 groups of patients who have undergone kyphectomy and fixation, either with the GR (group 1) or the LT with Galveston pelvic fixation (group 2). METHODS: Ten patients undergoing GR fixation and 5 patients undergoing LT with Fackler fixation following kyphectomy (vertebral column resection or multiple eggshell) were included. GRs were lengthened every 6 months. Unplanned surgery in group 1 was defined as an unscheduled operation due to complication; all subsequent operations in group 2 were considered unplanned. Thoracic and local kyphosis and T1-S1 and T1-12 heights were measured preoperatively, postoperatively, and at final follow-up. RESULTS: Mean age at initial surgery was 6 years and 6.5 years for groups 1 and 2, respectively. Mean age at the last follow-up was 12.5 years for group 1 and 13.1 years for group 2. Mean follow-up was 72.7 months for group 1 and 68.6 months for group 2. Preoperative, postoperative, and final follow-up kyphosis angles in that order for group 1 were 72.3 degrees (10 to 110 degrees), 16.9 degrees (-50 to +55 degrees), and 21.6 degrees (-41 to +97 degrees), and for group 2 106.6 degrees (81 to 132 degrees), 15.6 degrees (-37 to +50 degrees), and 19.2 degrees (-42 to +38 degrees), respectively. Postoperative and final follow-up in that order for mean T1-T12 and T1-S1 heights for group 1 were 14 (11.2 to 18.7) cm, 20.4 (19.3 to 25.7) cm and 21 (17.2 to 23.2) cm, 31.6 (23.6 to 41.5) cm. Postoperative and final follow-up in that order for mean T1-T12 and T1-S1 heights for group 2 were 15.9 (14.3 to 19.7) cm, 20.1 (15.5 to 24.6) cm and 24.4 (17.7 to 27.8) cm, 29.5 (25.3 to 31.3) cm. Growth per year was 1.05 and 0.84 cm for groups 1 and 2, respectively (P=0.297). Fourteen versus 4 unplanned surgeries were performed in groups 1 and 2, respectively, and an additional 4 implant revisions were performed in group 1 during planned lengthenings. CONCLUSIONS: Both the LT and the GR system are reasonable alternatives of fixation postkyphectomy, both of which preserve growth to differing degrees. In this patient population with an already severely stunted trunk height, the surgeon must choose whether the amount of extra growth achieved by the GR is worth the risk of an increased number of surgeries.

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 ß-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.