SARS-CoV-2 Omicron BA.1 Variant Infection of Human Colon Epithelial Cells.

The Omicron variant of SARS-CoV-2, characterized by multiple subvariants including BA.1, XBB.1.5, EG.5, and JN.1, became the predominant strain in early 2022. Studies indicate that Omicron replicates less efficiently in lung tissue compared to the ancestral strain. However, the infectivity of Omicron in the gastrointestinal tract is not fully defined, despite the fact that 70% of COVID-19 patients experience digestive disease symptoms. Here, using primary human colonoids, we found that, regardless of individual variability, Omicron infects colon cells similarly or less effectively than the ancestral strain or the Delta variant. The variant induced limited type III interferon expression and showed no significant impact on epithelial integrity. Further experiments revealed inefficient cell-to-cell spread and spike protein cleavage in the Omicron spike protein, possibly contributing to its lower infectious particle levels. The findings highlight the variant-specific replication differences in human colonoids, providing insights into the enteric tropism of Omicron and its relevance to long COVID symptoms.

Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury.

Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase-/-) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation-induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase-/- mice are resistant to cisplatin-induced AKI. nCDase-/- mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase-/- mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.

CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism.

The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.

Dynamics of sphingolipids and the serine palmitoyltransferase complex in rat oligodendrocytes during myelination.

Myelin is a unique lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical myelin components. Yet sphingolipid content and synthesis have not been well characterized in oligodendrocytes, the myelin-producing cells of the CNS. Here, using quantitative real-time PCR, LC-MS/MS-based lipid analysis, and biochemical assays, we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we characterized sphingolipid production in isolated oligodendrocytes. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination, levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of two alternative catalytic subunits, SPTLC2 and -3, in the relative levels of isoforms of the small subunits, ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. Myelination progression was accompanied by distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. We conclude that the distribution of these changes among sphingolipid family members is indicative of a selective channeling of the ceramide backbone toward specific downstream metabolic pathways during myelination. Our findings provide insights into myelin production in oligodendrocytes and suggest how dysregulation of the biosynthesis of this highly specialized membrane could contribute to demyelinating diseases.

Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity.

Serine palmitoyltranferase (SPT) is a pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be studied in both cells and in a cell free system. This assay procedure is applicable to any type of mammalian cell. Here we provide the detailed protocol to measure SPT activity in the presence of either short chain (C8-ceramide) or long chain ceramide (C24-ceramide). One of the greatest advantages of this protocol is the ability to test insoluble long chain ceramides. We accomplished this by generating long chain ceramide through endogenous ceramide synthase by providing exogenous sphingosine and 24:1 acyl CoA in HeLa cell membranes. This SPT assay procedure is simple and easy to perform and does not require sophisticated instruments.

The ORMDL/Orm-serine palmitoyltransferase (SPT) complex is directly regulated by ceramide: Reconstitution of SPT regulation in isolated membranes.

Sphingolipids compose a lipid family critical for membrane structure as well as intra- and intercellular signaling. De novo sphingolipid biosynthesis is initiated by the enzyme serine palmitoyltransferase (SPT), which resides in the endoplasmic reticulum (ER) membrane. In both yeast and mammalian species, SPT activity is homeostatically regulated through small ER membrane proteins, the Orms in yeast and the ORMDLs in mammalian cells. These proteins form stable complexes with SPT. In yeast, the homeostatic regulation of SPT relies, at least in part, on phosphorylation of the Orms. However, this does not appear to be the case for the mammalian ORMDLs. Here, we accomplished a cell-free reconstitution of the sphingolipid regulation of the ORMDL-SPT complex to probe the underlying regulatory mechanism. Sphingolipid and ORMDL-dependent regulation of SPT was demonstrated in isolated membranes, essentially free of cytosol. This suggests that this regulation does not require soluble cytosolic proteins or small molecules such as ATP. We found that this system is particularly responsive to the pro-apoptotic sphingolipid ceramide and that this response is strictly stereospecific, indicating that ceramide regulates the ORMDL-SPT complex via a specific binding interaction. Yeast membranes harboring the Orm-SPT system also directly responded to sphingolipid, suggesting that yeast cells have, in addition to Orm phosphorylation, an additional Orm-dependent SPT regulatory mechanism. Our results indicate that ORMDL/Orm-mediated regulation of SPT involves a direct interaction of sphingolipid with the membrane-bound components of the SPT-regulatory apparatus.

Transmembrane topology of mammalian ORMDL proteins in the endoplasmic reticulum as revealed by the substituted cysteine accessibility method (SCAM™).

Sphingolipids are diverse lipids with essential, and occasionally opposing, functions in the cell and therefore tight control over biosynthesis is vital. Mechanisms governing this regulation are not understood. Initial steps in sphingolipid biosynthesis take place on the cytosolic face of the endoplasmic reticulum (ER). Serine palmitoyltransferase (SPT) is an ER-resident enzyme catalyzing the first-committed step in sphingolipid biosynthesis. Not surprisingly, SPT activity is tightly regulated. ORMDLs are ER-resident proteins recently identified as regulators of SPT activity. ORMDL proteins interact directly with SPT but the nature of this interaction is unknown. ORMDL protein sequences contain hydrophobic regions, yet algorithm-based predictions of transmembrane segments are highly ambiguous, making topology of this key regulator unclear. Here we report use of substituted cysteine accessibility to analyze topology of mammalian ORMDLs. We constructed multiple mutant ORMDLs, each containing a single cysteine strategically placed along the protein length. Combined use of selective membrane permeabilization with an impermeant cysteine modification reagent allowed us to assign transmembrane and cytosolic segments of ORMDL. We confirmed that mammalian ORMDL proteins transit the membrane four times, with amino- and carboxy termini facing the cytosol along with a large cytosolic loop. This model will allow us to determine details of the ORMDL-SPT interaction and identify regions acting as the "lipid sensor" to detect changes in cellular sphingolipid levels. We also observe that SPT and ORMDL are substantially resistant to extraction from membranes with non-ionic detergent, indirectly suggesting that both proteins reside in a specialized subdomain of the ER.

Orm/ORMDL proteins: Gate guardians and master regulators.

Sphingolipids comprise a diverse family of lipids that perform multiple functions in both structure of cellular membranes and intra- and inter-cellular signaling. The diversity of this family is generated by an array of enzymes that produce individual classes and molecular species of family members and enzymes which catabolize those lipids for recycling pathways. However, all of these lipids begin their lives with a single step, the condensation of an amino acid, almost always serine, and a fatty acyl-CoA, almost always the 16-carbon, saturated fatty acid, palmitate. The enzyme complex that accomplishes this condensation is serine palmitoyltransferase (SPT), a membrane-bound component of the endoplasmic reticulum. This places SPT in the unique position of regulating the production of the entire sphingolipid pool. Understanding how SPT activity is regulated is currently a central focus in the field of sphingolipid biology. In this review we examine the regulation of SPT activity by a set of small, membrane-bound proteins of the endoplasmic reticulum, the Orms (in yeast) and ORMDLs (in vertebrates). We discuss what is known about how these proteins act as homeostatic regulators by monitoring cellular levels of sphingolipid, but also how the Orms/ORMDLs regulate SPT in response to other stimuli. Finally, we discuss the intriguing connection between one of the mammalian ORMDL isoforms, ORMDL3, and the pervasive pulmonary disease, asthma, in humans.

Complexity overlooked: enhancing cultural competency in the white lesbian counseling trainee through education and supervision.

Self-awareness is often associated with enhanced multicultural competency. Training programs must work to facilitate self-awareness in counseling trainees who hold both privileged and oppressed identities. In this article, I highlight a gap in the literature regarding how best to supervise white lesbian counseling trainees. Facilitating self-awareness through supervision will be explored as a tool for enhancing multicultural competency in the white lesbian counseling trainee. An exploration of understanding white privilege as well as the impact of oppression on lesbian counseling trainees, will be used to draw conclusions regarding effective supervision for this population. Additionally, suggestions for future research will be proposed.

Factors influencing the genesis of neurosurgical technology.

For any new technology to gain acceptance, it must not only adequately fill a true need, but must also function optimally within the confines of coexisting technology and concurrently available support systems. As an example, over the first decades of the 20th century, a number of drill designs used to perform cranial bone cuts appeared, fell out of favor, and later reappeared as certain supportive technologies emerged. Ultimately, it was the power source that caused one device to prevail. In contrast, a brilliant imaging device, designed to demonstrate an axial view of the lumbar spine, was never allowed to gain acceptance because it was immediately superseded by another device of no greater innovation, but one that performed optimally with popular support technology. The authors discuss the factors that have bearing on the evolution of neurosurgical technology.