Capillary electrophoresis methods for determining the IVT mRNA critical quality attributes of size and purity.

One result of the Covid-19 pandemic has been an increased awareness of IVT mRNA vaccines and the speed at which they can be produced for disease outbreaks. Currently the only approved IVT mRNA therapeutics are the Covid-19 vaccines, however IVT mRNA is being investigated for other non-Covid prophylactic vaccines, therapeutics, and therapeutic vaccines. IVT mRNAs can range from less than 100 nt in length to longer than 9,000 nt.

Heteroduplex cleavage assay for screening of probable zygosities resulting from CRISPR mutations in diploid single cell lines.

The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events.

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity.

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point.

Plant Sphingolipid Metabolism and Function.

Sphingolipids, a once overlooked class of lipids in plants, are now recognized as abundant and essential components of plasma membrane and other endomembranes of plant cells. In addition to providing structural integrity to plant membranes, sphingolipids contribute to Golgi trafficking and protein organizational domains in the plasma membrane. Sphingolipid metabolites have also been linked to the regulation of cellular processes, including programmed cell death.

Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays.

Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis.

Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis.

Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth, Sphingolipid Metabolism, Programmed Cell Death, and Mycotoxin Resistance.

Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B1 (FB1). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism.

Sphingolipid metabolism is strikingly different between pollen and leaf in Arabidopsis as revealed by compositional and gene expression profiling.

Although sphingolipids are essential for male gametophytic development in Arabidopsis thaliana, sphingolipid composition and biosynthetic gene expression have not been previously examined in pollen. In this report, electrospray ionization (ESI)-MS/MS was applied to characterization of sphingolipid compositional profiles in pollen isolated from wild type Arabidopsis Col-0 and a long-chain base (LCB) Δ4 desaturase mutant. Pollen fractions were highly enriched in glucosylceramides (GlcCer) relative to levels previously reported in leaves.

A mass spectrometry-based method for the assay of ceramide synthase substrate specificity.

The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.