Primitive sterol evolution plays an important role in fossil record interpretation and offers potential therapeutic avenues for human disease resulting from nematode infections. Recognizing that C4-methyl stenol products [8(14)-lophenol] can be synthesized in bacteria while C4-methyl stanol products (dinosterol) can be synthesized in dinoflagellates and preserved as sterane biomarkers in ancient sedimentary rock is key to eukaryotic sterol evolution. In this regard, nematodes have been proposed to convert dietary cholesterol to 8(14)-lophenol by a secondary metabolism pathway that could involve sterol C4 methylation analogous to the C2 methylation of hopanoids (radicle-type mechanism) or C24 methylation of sterols (carbocation-type mechanism). Here, we characterized dichotomous cholesterol metabolic pathways in Caenorhabditis elegans that generate 3-oxo sterol intermediates in separate paths to lophanol (4-methyl stanol) and 8(14)-lophenol (4-methyl stenol). We uncovered alternate C3-sterol oxidation and ⁷ desaturation steps that regulate sterol flux from which branching metabolite networks arise, while lophanol/8(14)-lophenol formation is shown to be dependent on a sterol C4α-methyltransferse (4-SMT) that requires 3-oxo sterol substrates and catalyzes a newly discovered 3-keto-enol tautomerism mechanism linked to S-adenosyl-l-methionine-dependent methylation. Alignment-specific substrate-binding domains similarly conserved in 4-SMT and 24-SMT enzymes, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of methyl sterols. The combination of these results provides evolutionary leads to sterol diversity and points to cryptic C4-methyl steroidogenic pathways of targeted convergence that mediate lineage-specific adaptations.­­

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.

Steroids that contain a 3-hydroxyl group (3-OH steroids) are widely distributed in nature. During analysis with ESI-MS, they easily become dehydrated while in the protonated form, resulting in the production of several precursor ions and leading to low sensitivity of detection. To address this analytical challenge, here, we developed a method for the quantitation of 3-OH steroids by LC-MS/MS coupled with post-column addition of lithium (Li) ions to the mobile phase. The Li ion has a high affinity for the keto group of steroids, stabilizing their structures during ionization and permitting detection of analytes exclusively as the lithiated form. This not only improved the intensities of the precursor ions, but also promoted the formation of typical lithiated fragment ions. This improvement made the quantitation by multiple reaction monitoring more sensitive and reliable, as evidenced by 1.53–188 times enhanced detection sensitivity of 13 steroids that contained at least one keto and two hydroxyl groups or one keto and one 5-olefinic double bond, among 16 different 3-OH steroids. We deployed our newly developed method for profiling steroids in mouse brain tissue and identified six steroids in one tissue sample. Among these, 16-hydroxyestrone, tetrahydrocorticosterone, and 17α-hydroxypregnenolone were detected for the first time in the mouse brain. In summary, the method described here enables the detection of lithiated steroids by LC-MS/MS, including three 3-OH steroids not previously reported in the mouse brain. We anticipate that this new method may allow the determination of 3-OH steroids in different brain regions.

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

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

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

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

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..."

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

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