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Model systems for studying the assembly, trafficking, and secretion of apoB lipoproteins using fluorescent fusion proteins [Research Articles]

apoB exists as apoB100 and apoB48, which are mainly found in hepatic VLDLs and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive the assembly, intracellular trafficking, secretion, and function of Blps remains challenging. Our understanding of the intracellular and intraorganism trafficking of Blps can be greatly enhanced, however, with the availability of fusion proteins that can help visualize Blp transport within cells and between tissues. We designed three plasmids expressing human apoB fluorescent fusion proteins: apoB48-GFP, apoB100-GFP, and apoB48-mCherry. In Cos-7 cells, transiently expressed fluorescent apoB proteins colocalized with calnexin and were only secreted if cells were cotransfected with microsomal triglyceride transfer protein. The secreted apoB-fusion proteins retained the fluorescent protein and were secreted as lipoproteins with flotation densities similar to plasma HDL and LDL. In a rat hepatoma McA-RH7777 cell line, the human apoB100 fusion protein was secreted as VLDL- and LDL-sized particles, and the apoB48 fusion proteins were secreted as LDL- and HDL-sized particles. To monitor lipoprotein trafficking in vivo, the apoB48-mCherry construct was transiently expressed in zebrafish larvae and was detected throughout the liver. These experiments show that the addition of fluorescent proteins to the C terminus of apoB does not disrupt their assembly, localization, secretion, or endocytosis. The availability of fluorescently labeled apoB proteins will facilitate the exploration of the assembly, degradation, and transport of Blps and help to identify novel compounds that interfere with these processes via high-throughput screening.




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A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time [Methods]

The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceride-derived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues. Physiologically active LPL is normally bound to the endothelial cell protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), which transports LPL across endothelial cells, anchors LPL to the vascular wall, and stabilizes LPL activity. Disruption of LPL-GPIHBP1 binding significantly alters triglyceride metabolism and lipid partitioning. In this study, we modified the NanoLuc® Binary Technology split-luciferase system to develop a novel assay that monitors the binding of LPL to GPIHBP1 on endothelial cells in real time. We validated the specificity and sensitivity of the assay using endothelial lipase and a mutant version of LPL and found that this assay reliably and specifically detected the interaction between LPL and GPIHBP1. We then interrogated various endogenous and exogenous inhibitors of LPL-mediated lipolysis for their ability to disrupt the binding of LPL to GPIHBP1. We found that angiopoietin-like (ANGPTL)4 and ANGPTL3-ANGPTL8 complexes disrupted the interactions of LPL and GPIHBP1, whereas the exogenous LPL blockers we tested (tyloxapol, poloxamer-407, and tetrahydrolipstatin) did not. We also found that chylomicrons could dissociate LPL from GPIHBP1 and found evidence that this dissociation was mediated in part by the fatty acids produced by lipolysis. These results demonstrate the ability of this assay to monitor LPL-GPIHBP1 binding and to probe how various agents influence this important complex.




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HDL inhibits endoplasmic reticulum stress-induced apoptosis of pancreatic {beta}-cells in vitro by activation of Smoothened [Research Articles]

Loss of pancreatic β-cell mass and function as a result of sustained ER stress is a core step in the pathogenesis of diabetes mellitus type 2. The complex control of β-cells and insulin production involves hedgehog (Hh) signaling pathways as well as cholesterol-mediated effects. In fact, data from studies in humans and animal models suggest that HDL protects against the development of diabetes through inhibition of ER stress and β-cell apoptosis. We investigated the mechanism by which HDL inhibits ER stress and apoptosis induced by thapsigargin, a sarco/ER Ca2+-ATPase inhibitor, in β-cells of a rat insulinoma cell line, INS1e. We further explored effects on the Hh signaling receptor Smoothened (SMO) with pharmacologic agonists and inhibitors. Interference with sterol synthesis or efflux enhanced β-cell apoptosis and abrogated the anti-apoptotic activity of HDL. During ER stress, HDL facilitated the efflux of specific oxysterols, including 24-hydroxycholesterol (OHC). Supplementation of reconstituted HDL with 24-OHC enhanced and, in cells lacking ABCG1 or the 24-OHC synthesizing enzyme CYP46A1, restored the protective activity of HDL. Inhibition of SMO countered the beneficial effects of HDL and also LDL, and SMO agonists decreased β-cell apoptosis in the absence of ABCG1 or CYP46A1. The translocation of the SMO-activated transcription factor glioma-associated oncogene GLI-1 was inhibited by ER stress but restored by both HDL and 24-OHC. In conclusion, the protective effect of HDL to counter ER stress and β-cell death involves the transport, generation, and mobilization of oxysterols for activation of the Hh signaling receptor SMO




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SSO and other putative inhibitors of FA transport across membranes by CD36 disrupt intracellular metabolism, but do not affect FA translocation [Research Articles]

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.




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A novel GPER antagonist protects against the formation of estrogen-induced cholesterol gallstones in female mice [Research Articles]

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­




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Vitamin E does not prevent Western diet-induced NASH progression and increases metabolic flux dysregulation in mice [Research Articles]

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by 2H/13C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.




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Commentary on SSO and other putative inhibitors of FA transport across membranes by CD36 disrupt intracellular metabolism, but do not affect fatty acid translocation [Commentaries]




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GPIHBP1, a partner protein for lipoprotein lipase, is expressed only in capillary endothelial cells [Images In Lipid Research]




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Problem Notes for SAS®9 - 65852: The PANEL procedure produces incorrect results for certain models when the NOINT and RANONE options are specified

The estimation results might be incorrect in PROC PANEL when the RANONE and NOINT options are specified in the MODEL statement.




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Problem Notes for SAS®9 - 65940: You might receive "ERROR: PI Point not found" when you query a PI tag name that contains a special character such as an ampersand (&)

When you query a PI tag name or element that contains a special character, such as an ampersand (&), you might receive the following error:



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Problem Notes for SAS®9 - 65939: "ERROR: Unable to transcode data to/from UCS-2 encoding" occurs when you run an SQL query using SAS/ACCESS Interface to ODBC on SAS 9.4M5 with UTF-8

When you run an SQL query using SAS/ACCESS Interface to ODBC under the following conditions, you might receive an error: You run SAS 9.4M5 (TS1M5) or SAS 9.4M6 (TS1M6)  i




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Problem Notes for SAS®9 - 65938: Incorrect values might be written to Hadoop for columns defined with the BIGINT data type

Large numeric values consisting of 16 digits in SAS might be incorrect when written to Hadoop for columns defined with the BIGINT data type.  This problem was introduced in SAS 9




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Problem Notes for SAS®9 - 65900: Registering an Oracle table to the metadata might fail and generate an error

When you register an Oracle table to the metadata, it might fail and generate an error similar to the following: "ERROR: An exception has been encountered...ERROR: Read Access Violation METALIB..."




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Problem Notes for SAS®9 - 35066: When a bulk-loading process fails with "SQL*Loader 2026" error, error message appears as a warning in the SAS log

If a bulk-loading process fails when you use SAS with SAS/ACCESS Interface to Oracle, you will receive the warning: "WARNING: All or some rows were rejected/discarded.: The actual error is "SQL*Loader-2026: The load was aborted because SQL




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Problem Notes for SAS®9 - 65834: PROC METADATA returns various errors when the input contains certain multi-byte characters

The METADATA procedure might return an error similar to one of the following:

  • ERROR: Missing root element definition.
  • Full Article


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    Problem Notes for SAS®9 - 65899: "ERROR: ORACLE disconnect error: ORA-03135" occurs when SAS disconnects from the Oracle database server

    When you run 32-bit SAS on Windows and disconnect from the Oracle database server, you might see the error: "ERROR: ORACLE disconnect error: ORA-03135: connection lost contact."




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    Problem Notes for SAS®9 - 60332: A SAS 9.4 installation in Update mode notifies you about unwritable files in the "SASHome\SASWebApplicationServer" directory

    When you run SAS Deployment Wizard to install or update SAS 9.4 software, the file system is examined. If any files that the wizard needs to delete are found to be locked, they are reported as unwritable f




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    Problem Notes for SAS®9 - 65918: SAS Workflow Services fails to respond after a com.sas.workflow.engine.policy.PolicyExecutionException error occurs for a workflow instance

    When the problem occurs, you are unable to perform any workflow actions in a SAS solution that uses SAS Workflow Services.




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    Problem Notes for SAS®9 - 65929: A grid-enabled sign-on to SAS 9.4M6 (TS1M6) fails with errors, including "Remote signon … canceled"

    A sign-on to a grid-enabled environment fails while it is trying to communicate with the client host. The following errors then appear in the SAS log:


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    Problem Notes for SAS®9 - 64285: The SCD Type 2 Loader transformation in SAS Data Integration Studio generates "ERROR 22-322: Syntax error, expecting one of the following:..."

    If your business key column is a name literal, like " business key "n, a syntax error occurs when that variable name does not follow standard SAS naming conventions.




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    Problem Notes for SAS®9 - 64459: A SAS Data Integration Studio job receives an error that states "The name '; index_name '; has the wrong number of qualifiers"

    An error occurs because of an incorrectly generated CREATE INDEX clause in an SQL query that is sent to DB2 when the DB2 schema value is SESSION . The error message says "The name '; index_name '; has the wrong number of qualifie




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    Problem Notes for SAS®9 - 65908: The IMPORT procedure contains a stack-corruption vulnerability

    Severity: Medium Description: PROC IMPORT contains a stack-corruption vulnerability. Potential Impact: Under certain circumstances (with use of the DBM




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    Problem Notes for SAS®9 - 65906: The EXPORT procedure contains a stack-corruption vulnerability

    Severity: Medium Description: PROC EXPORT contains a stack-corruption vulnerability. Potential Impact: Under certain circumstances, the use of PROC EXP




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    Problem Notes for SAS®9 - 65922: Trying to read a Google BigQuery table that contains a variable defined as an array might result in a panic error and SAS shutting down

    Trying to read a Google BigQuery table that contains a variable that is defined as an array of records might result in an error and cause SAS to shut down. This issue occurs when one of the variables contained in




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    Problem Notes for SAS®9 - 65295: The order of columns is not maintained when you select columns for output in the Business Rules transformation

    In SAS Data Integration Studio, the columns that you select to include in the target table in a Business Rules transformation appear in the Selected columns area in a random order. Th




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    Problem Notes for SAS®9 - 65916: Accessing a Google BigQuery table without including the SCHEMA= option in the LIBNAME statement might result in an error

    When you issue a LIBNAME statement for a Google BigQuery database without including the SCHEMA= option, all tables in the project are shown when the libref is expanded. However, if you try to acces




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    Problem Notes for SAS®9 - 65935: The UNICODE function does not support Numeric Character Representation (NCR) for a surrogate pair

    Using the NCR form of a surrogate pair as an input string to the UNICODE function does not convert the string to the appropriate display character.




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    Problem Notes for SAS®9 - 65031: Grid options set mappings are returned based on which option set is the first in the metadata search chain

    When users are defined in multiple groups associated with grid options set mappings, the first grid option set that is returned by the metadata search chain takes precedence. Only one grid options set mapping is used




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    Problem Notes for SAS®9 - 34294: A missing discrete dependent variable in the selection model together with a OUTPUT statement might cause an Access Violation error

    If the following conditions are met in PROC QLIM: the SELECT option and DISCRETE option are specified in the same MODEL statement or ENDOGENOUS statement the same dependent variable with S




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    Problem Notes for SAS®9 - 65927: The Copy Files task in SAS Enterprise Guide 8.2 fails with the message "ERROR: Target folder does not exist or cannot be accessed"

    When you run the Copy Files task in SAS Enterprise Guide and there is no connection to a SAS server, it fails with the following error: "ERROR: Target folder does not exist or cannot be accessed."




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    Problem Notes for SAS®9 - 65903: You see a "java.lang.IllegalArgumentException" error in the log file when you use the IFRS9_Cycle workflow template in SAS Solution for IFRS 9

    The problem occurs on a content release on the SAS Risk Governance Framework.




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    Problem Notes for SAS®9 - 65872: You see a "java.lang.IllegalArgumentException" error in the log file when you use the CECL_Cycle workflow template in SAS Solution for CECL

    The problem occurs on a content release on the SAS Risk Governance Framework.




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    Problem Notes for SAS®9 - 65904: SAS Federation Server stops responding when you run queries against X_OBJECT_PRIVILEGES in SYSCAT and the queries run for hours

    The select * from "SYSCAT"."SYSCAT"."X_EFFECTIVE_OBJECT_PRIVILEGES" query runs for hours. In this scenario, SAS Federation Server stops responding, making it unavailable for use. Restarting SAS Federation Server solves t




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    Problem Notes for SAS®9 - 65682: Running FedSQL with an Oracle table is slow, even when you use a LIMIT clause

    When you query an Oracle table and use the LIMIT clause using either SAS  Federation Server or FedSQL, a row limit is not passed to the database. In this scenario, a Full Article



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    Problem Notes for SAS®9 - 65914: You see the error "Driver does not support this optional feature" after trying to insert or append data to a Databricks table

    You can create create a Databricks table by using PROC SQL, but you cannot insert data into the table. PROC APPEND cannot create a new table or append data to an existing table.




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    Problem Notes for SAS®9 - 65574: Decimal values are rounded after they are inserted into a new Databricks table via SAS/ACCESS Interface to JDBC

    A DATA step and PROC SQL can round numeric values while creating and loading data into a new Databricks table via JDBC.




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    Problem Notes for SAS®9 - 65886: Trying to bulk load data into a Google BigQuery database might result in an error

    When you bulk load data into a Google BigQuery database, you might encounter this error: "Error while reading data, error message: CSV table encountered too many errors, giving up...Error detected while parsing row starting at position: 0...Data




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    Problem Notes for SAS®9 - 65898: A misleading SASTRACE message appears in the log when you insert a row into an Oracle table using SAS/ACCESS Interface to Oracle with DBIDIRECTEXEC

    When you add one row to an Oracle table using DBIDIRECTEXEC, you see the following misleading trace message: "ORACLE: 4294967296 rows inserted/updated/deleted." You should see something similar to the following: "ORACLE: 1 rows inserte




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    Problem Notes for SAS®9 - 65909: SAS Visual Analytics Designer 7.5 responds slowly when you edit large or complex reports

    If your SAS Visual Analytics report contains many sections and objects, you might encounter performance problems when you are editing the report.   A hot fix is planned for this issue.




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    Problem Notes for SAS®9 - 64980: The PRINT procedure contains a buffer-overrun vulnerability

    Severity: Medium Description: PROC PRINT might fail with a buffer overrun when you submit it in conjunction with certain malformed SAS statements.



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    Problem Notes for SAS®9 - 64550: SAS Enterprise Case Management contains a cross-site scripting vulnerability in the CASE_ID parameter

    Severity: Medium Description: SAS Enterprise Case Management contains a cross-site scripting vulnerability in the CASE_ID parameter. Potential Impact:




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    Problem Notes for SAS®9 - 65893: Custom sorts are sorted incorrectly when they are used in a hierarchy in SAS Visual Analytics Designer

    A custom sort might be sorted incorrectly when the data item is used in a custom category, which is then used in a hierarchy. The issue can occur in the following scenario:




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    Problem Notes for SAS®9 - 65844: STRESS task fails with "Fatal error in PMPI_Bcast: Other MPI error, error stack: PMPI_Bcast(1478)"

    In SAS  High-Performance Risk, a STRESS task might fail with a message like the following in the SAS log while the compute server is sending the ScenarioCF/Value data to the HPRisk Engine:



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    Problem Notes for SAS®9 - 65835: A series of PROC SQL queries might not generate a distinct set of rows

    A set of PROC SQL queries that create a view, contain a constant column, contain a computed column, and a create a table do not generate a unique set of rows in the table that is created.




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    Problem Notes for SAS®9 - 65597: An SQL procedure query with a WHERE clause that contains multiple subselects might return incorrect results

    An issue occurs when code contains a complex SQL procedure query with a WHERE clause that contains multiple subselects. Incorrect results might be returned. Click the Hot Fix tab in this note to




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    Problem Notes for SAS®9 - 65572: The length of a string variable might be longer than specified with the MAX_CHAR_LEN= option

    When you read in a BigQuery table, the length of string variables might be longer than the length specified with the MAX_CHAR_LEN= option when running your SAS software   with UTF-8. By




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    Problem Notes for SAS®9 - 65883: SAS Workflow Studio returns a "cannot load" error when you try to open the CECL_Cycle_AFS workflow template for SAS Solution for CECL

    You might see the following error in SAS Workflow Studio when you try to open the CECL_Cycle_AFS workflow template that is shipped with SAS Solution for CECL:



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    Problem Notes for SAS®9 - 65856: The process of updating a lookup table in SAS Business Rules Manager (running in UNIX operating environments) does not work properly

    Under UNIX, the process of updating a lookup table in SAS Business Rules Manager does not work properly. The problem occurs when you perform these steps:  Open a lookup table. Cl




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    Problem Notes for SAS®9 - 65869: SAS Visual Data Builder does not enable you to schedule with multiple time-event triggers

    SAS Visual Data Builder might not enable you to create multiple time-event triggers. The + button to add another trigger is not available to select, as shown in the following display: imgalt="" src="{fusion_658




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    Problem Notes for SAS®9 - 65868: Saving a report distribution in SAS Visual Analytics Designer fails with "The name is invalid"

    When you attempt to save a report distribution in SAS Visual Analytics Designer, you might see the error shown in the following display:  imgalt="" src="{fusion_65868_1_distributionerror.png}" />