Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

In Alzheimer's disease (AD), tau, a microtubule-associated protein (MAP), becomes hyperphosphorylated, aggregates, and accumulates in the somato-dendritic compartment of neurons. In parallel to its intracellular accumulation in AD, tau is also released in the extracellular space, as revealed by its increased presence in cerebrospinal fluid (CSF). Consistent with this, recent studies, including ours, have reported that neurons secrete tau, and several therapeutic strategies aim to prevent the intracellular tau accumulation. Previously, we reported that late endosomes were implicated in tau secretion. Here, we explore the possibility of preventing intracellular tau accumulation by increasing tau secretion. Using neuronal models, we investigated whether overexpression of the vesicle-associated membrane protein 8 (VAMP8), an R-SNARE found on late endosomes, could increase tau secretion. The overexpression of VAMP8 significantly increased tau secretion, decreasing its intracellular levels in the neuroblastoma (N2a) cell line. Increased tau secretion by VAMP8 was also observed in murine hippocampal slices. The intracellular reduction of tau by VAMP8 overexpression correlated to a decrease of acetylated tubulin induced by tau overexpression in N2a cells. VAMP8 staining was preferentially found on late endosomes in N2a cells. Using total internal reflection fluorescence (TIRF) microscopy, the fusion of VAMP8-positive vesicles with the plasma membrane was correlated to the depletion of tau in the cytoplasm. Finally, overexpression of VAMP8 reduced the intracellular accumulation of tau mutants linked to frontotemporal dementia with parkinsonism and α-synuclein by increasing their secretion. Collectively, the present data indicate that VAMP8 could be used to increase tau and α-synuclein clearance to prevent their intracellular accumulation.

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1β secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.

Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates, limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use, and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user-defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percentage of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates, and (v) localization of aggregates (cytosol, perinuclear, or nuclear). A data table of aggregate information on a per cell basis, as well as a summary table, is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules, and inclusion bodies caused by huntingtin polyglutamine expansion.

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

Acute lung injury (ALI), is a rapidly progressing heterogenous pulmonary disorder that possesses a high risk of mortality. Accumulating evidence has implicated the activation of the p65 subunit of NF-κB [NF-κB(p65)] activation in the pathological process of ALI. microRNAs (miRNAs), a group of small RNA molecules, have emerged as major governors due to their post-transcriptional regulation of gene expression in a wide array of pathological processes, including ALI. The dysregulation of miRNAs and NF-κB activation has been implicated in human diseases. In the current study, we set out to decipher the convergence of miR-99b and p65 NF-κB activation in ALI pathology. We measured the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) in bronchoalveolar lavage fluid using ELISA. MH-S cells were cultured and their viability were detected with cell counting kit 8 (CCK8) assays. The results showed that miR-99b was up-regulated, while PRDM1 was down-regulated in a lipopolysaccharide (LPS)-induced murine model of ALI. Mechanistic investigations showed that NF-κB(p65) was enriched at the miR-99b promoter region, and further promoted its transcriptional activity. Furthermore, miR-99b targeted PRDM1 by binding to its 3'UTR, causing its down-regulation. This in-creased lung injury, as evidenced by increased wet/dry ratio of mouse lung, myeloperoxidase activity and pro-inflammatory cytokine secretion, and enhanced infiltration of inflammatory cells in lung tissues. Together, our findings indicate that NF-κB(p65) promotion of miR-99b can aggravate ALI in mice by down-regulating the expression of PRDM1.

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

Postoperative recurrence from microscopic residual disease must be prevented to cure intractable cancers, including pancreatic cancer. Key to this goal is the elimination of cancer stem cells (CSCs) endowed with tumor-initiating capacity and drug resistance. However, current therapeutic strategies capable of accomplishing this are insufficient. Using in vitro models of CSCs and in vivo models of tumor initiation in which CSCs give rise to xenograft tumors, we show that dexamethasone induces expression of MKP-1, a MAPK phosphatase, via glucocorticoid receptor activation, thereby inactivating JNK, which is required for self-renewal and tumor initiation by pancreatic CSCs as well as for their expression of survivin, an anti-apoptotic protein implicated in multidrug resistance. We also demonstrate that systemic administration of clinically relevant doses of dexamethasone together with gemcitabine prevents tumor formation by CSCs in a pancreatic cancer xenograft model. Our study thus provides preclinical evidence for the efficacy of dexamethasone as an adjuvant therapy to prevent postoperative recurrence in patients with pancreatic cancer.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3'-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

When the drug Taxol® was approved by the United States Food and Drug Administration in 1993, it was a game changer for cancer patients. The compound, which arrests cell division by preventing the disassembly of tubulin microfibers, has been used over the past three decades to treat millions of cases of breast, lung, and ovarian cancer as well as Kaposi's sarcoma. In 1990, Bristol Myers Squibb applied to trademark the name Taxol, which was approved in 1992, changing the drug's generic name to paclitaxel.At the time that Taxol was entering clinical trials in the late 1970s, it also proved to be a valuable tool for cytoskeletal research. Tubulin had been discovered in the late 1960s, but it was still unclear how the soluble protein dimer polymerized (Fig. 1) to form the long, complex structures of the cytoskeleton.jbc;295/41/13994/F1F1F1Figure 1.Strands of tubulin, a protein in the cell's skeleton, photographed using a high-resolution microscopy technique. Image made by Pakorn Kanchanawong (National University of Singapore) and Clare Waterman (NHLBI, National Institutes of Health).“Back then, people were just discovering the most basic functions of tubulin and how it polymerized, and then they found a drug that affected this,” said Velia Fowler, a cell biologist at the University of Delaware and former Associate Editor at the Journal of Biological Chemistry.The drug and its cytoskeletal activity intersected in the 1981 JBC paper “Taxol-induced polymerization of purified tubulin” (1), the subject of this JBC Classic. In the single-author paper, Nirbhay Kumar, then a postdoctoral fellow at the National...

Priyanka Tripathi
Dec 30, 2020; 0:jlr.RA120001190v1-jlr.RA120001190
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Stephanie E. Hood
Dec 1, 2020; 61:1720-1732
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Melissa Gómez
Dec 1, 2020; 61:1658-1674
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Martin Prescher
Dec 1, 2020; 61:1605-1616
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Elisa Vidal
Dec 1, 2020; 61:1733-1746
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Montgomery Blencowe
Dec 23, 2020; 0:jlr.RA120000713v1-jlr.RA120000713
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Auguste Dargent
Dec 1, 2020; 61:1776-1783
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Genta Kakiyama
Dec 1, 2020; 61:1629-1644
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Natalie Bruiners
Dec 1, 2020; 61:1617-1628
Research Articles

Michael E. Abrams
Dec 1, 2020; 61:1538-1538
Images in Lipid Research

Julia V Busik
Dec 23, 2020; 0:jlr.TR120000981v1-jlr.TR120000981
Thematic Reviews

Aloïs Dusuel
Dec 9, 2020; 0:jlr.RA120000704v1-jlr.RA120000704
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Anja Jaeschke
Dec 9, 2020; 0:jlr.RA120001141v1-jlr.RA120001141
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Abudukadier Abulizi
Dec 1, 2020; 61:1565-1576
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Stacey N Keenan
Dec 17, 2020; 0:jlr.RA120001126v1-jlr.RA120001126
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Gerhard Liebisch
Dec 1, 2020; 61:1539-1555
Special Report

In SAS Customer Intelligence Studio, you might notice that the user interface becomes unresponsive, as shown below: imgalt="SAS Customer Intelligence Studio UI becomes unresponsive" src="{fusion_66537

In SAS Customer Intelligence Studio, you can choose to create a new calculated variable in the Edit Value dialog box when you populate a treatment custom detail. Following creation of the new calculated

Editing and/or saving an action column can take up to a minute to display a window. There are no workarounds identified at this time.

You might receive a Segmentation Violation when opening a database table in SAS. The SAS Log contains the error and traceback:

An error might occur when you use the SAS 9 BULKLOAD= and BULKEXTRACT= options load data to or extract data from HP Neoview on the HP Itanium platform. The problem occurs because Hewlett-Packard changed the name of one of

In SAS Retail Space Management, it should be possible to click on any location object, then Show Properties, and change the location fill color. This can be done on the gray-filled objects. However, w

When you use SAS/ACCESS Interface to PostgreSQL to query a Yellowbrick database, the SAS OBS= option is not generating a limit clause on the query that is passed to the database. Click the

In SAS Customer Intelligence Studio,  when you add  Reply- node variable values in the Define Value dialog box, you might notice that two identically labeled data-grid variables are

The SAS 9.4M4 (TS1M4) Hot Fix D7G004 for ODS Templates installs national language support (NLS) content regardless of whether the languages were installed during the initial deployment. Having sparse

Certain tables that are created in SAS Scalable Performance Data (SPD) Server might not be displayed correctly by SAS Federation Server Manager. Tables that have Latin5 characters in column names encounter this

This manuscript has been withdrawn by the Author.

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet–microbiome interactions. Here, we used a click chemistry–based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine (sphinganine alkyne [SAA]) into the murine gut microbial community (Bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet–microbiome interactions.

Since the publication of the Age-Related Eye Disease Study (AREDS2) in 2013, the macular pigment carotenoids lutein and zeaxanthin have become well known to both the eye care community and the public. It is a fascinating aspect of evolution that primates have repurposed photoprotective pigments and binding proteins from plants and insects to protect and enhance visual acuity. Moreover, utilization of these plant-derived nutrients has been widely embraced for preventing vision loss from age-related macular degeneration (AMD). More recently, there has been growing awareness that these nutrients can also play a role in improving visual performance in adults. On the other hand, the potential benefits of lutein and zeaxanthin supplementation at very young ages have been underappreciated. In this review, we examine the biochemical mechanisms and supportive data for lutein and zeaxanthin supplementation throughout the lifespan, with particular emphasis on prenatal supplementation. We propose that prenatal nutritional recommendations may aim at improving maternal and infant carotenoid status. Prenatal supplementation with lutein and zeaxanthin might enhance infant visual development and performance and may even prevent retinopathy of prematurity, possibilities that should be examined in future clinical studies.

The cornea is densely innervated, mainly by sensory nerves of the ophthalmic branch of the trigeminal ganglia (TG). These nerves  are important to maintain corneal homeostasis, and nerve damage can lead to a decrease in wound healing, an increase in corneal ulceration and dry eye disease (DED), and neuropathic pain. Pathologies, such as diabetes, aging, viral and bacterial infection, as well as  prolonged use of contact lenses and surgeries to correct vision can produce nerve damage. There are no effective therapies to alleviate DED (a multifunctional disease) and several clinical trials using -3 supplementation show unclear and sometimes negative results. Using animal models of corneal nerve damage, we show that treating corneas with pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) increases nerve regeneration, wound healing, and tear secretion. The mechanism involves the activation of a calcium-independent phospholipase A2 (iPLA2) that releases the incorporated DHA from phospholipids and enhances the synthesis of docosanoids neuroprotectin D1 (NPD1) and a new resolvin stereoisomer  RvD6i. NPD1 stimulates the synthesis of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and of semaphorin 7A (Sema7A).  RvD6i treatment of injured corneas modulates gene expression in the TG resulting in enhanced neurogenesis; decreased neuropathic pain and increased sensitivity. Taken together, these results represent a promising therapeutic option to re-establish the homeostasis of the cornea.

Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established to participate in numerous processes, such as neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is, therefore, crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) emerges as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine-1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide-1-phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

The LDL receptor-related protein-1 (LRP1) is highly expressed in numerous cell types, and its impairment is associated with obesity, diabetes, and fatty liver disease. However, the mechanisms linking LRP1 to metabolic disease are not completely understood. Here, we compared the metabolic phenotype of C57BL/6J wild type and LRP1 knock-in mice carrying an inactivating mutation in the distal NPxY motif after feeding a low fat (LF) diet or high fat diets with (HFHC) or without (HF) cholesterol supplementation. In response to HF feeding, both groups developed hyperglycemia, hyperinsulinemia, and hyperlipidemia, as well as increased adiposity with adipose tissue inflammation and liver steatosis. However, when animals were fed the HF diet supplemented with cholesterol, the LRP1 NPxY mutation prevents hypercholesterolemia, reduces adipose tissue and brain inflammation, and limits liver progression to steatohepatitis. Nevertheless, insulin signaling is impaired in LRP1 NPxY mutant hepatocytes and this mutation does not protect against HFHC-induced insulin resistance. The selective metabolic improvement observed in HFHC-fed LRP1 NPxY mutant mice is due to an apparent increase of hepatic LDL receptor levels, leading to an elevated rate of plasma lipoprotein clearance and lowering of plasma and hepatic cholesterol levels. The unique metabolic phenotypes displayed by LRP1 NPxY mutant mice in response to HF or HFHC diet feeding indicate an LRP1-cholesterol axis in modulating tissue inflammation. The LRP1 NPxY mutant mouse phenotype differs from phenotypes observed in mice with tissue-specific LRP1 inactivation, thus highlighting the importance of an integrative approach to evaluate how global LRP1 dysfunction contributes to metabolic disease development.