Biology subjects

Smith, E. W.

Publications and source records attributed to Smith, E. W..

5 recordsLinked to original sources

bioRxiv2026

Glucosylceramide synthase is required for C6-ceramide nanoliposome-induced organelle stress and cell death

Sphingolipids are bioactive lipids that regulate key signaling pathways both directly as ligands and through membrane re-organization. Ceramide sits at the center of this network and is considered pro-death in many contexts, making ceramide accumulation an attractive therapeutic strategy. Given the wide-ranging regulation of cellular responses this network exerts, better understanding ceramide metabolism may promote therapeutic efficacy of sphingolipid-based therapeutics. Ceramide glycosylation, catalyzed by glucosylceramide synthase (GCS; UGCG), is in turn widely regarded as a detoxification route thus limiting efficacy of ceramide- based therapeutics. Here we show the opposite. Delivery of short-chain C6-ceramide via a ceramide nanoliposome (CNL) induced organelle stress and cell death in chronic lymphocytic leukemia (CLL) via the accumulation of glycosphingolipids (GSLs), rather than through ceramide itself. Pharmacologic and genetic blockade of GCS protected B-cell leukemia, breast carcinoma, glioblastoma, lung adenocarcinoma, and non- malignant embryonic kidney cells from CNL-induced death. Conversely, exogenous C8-glucosylceramide was sufficient to kill cells that cannot degrade it. We show that GSL accumulation drives an ordered organelle response beginning with lysosomal deacidification, endoplasmic reticulum stress, followed by mitochondrial respiratory capacity decline, each attenuated by inhibition of GSL synthesis. These effects were accompanied by MLKL phosphorylation, increased activity of the stress sensor JNK and CHOP induction, with JNK inhibition partially protecting from death. These findings invert the prevailing view of ceramide glycosylation as a resistance mechanism and identify glycosphingolipid flux as a required effector arm of ceramide-directed therapy.

cancer biology↗

bioRxiv2026

CD8scape: an accessible, command-line tool for predicting viral escape from the CD8+ T cell response

The CD8+ T cell response is a critical component of antiviral immunity, particularly in hosts who are immunocompromised or undergoing B cell-depleting therapy, such as rituximab. As viral evolution can lead to escape from CD8+ T cell recognition, tools that predict such escape are increasingly relevant. Here, we present CD8scape, an accessible command-line tool designed to predict viral escape from the CD8+ T cell response based on within-host sequence variation and HLA class I genotype. CD8scape is primarily a Julia wrapper for NetMHCpan v4.2, a neural network-based predictor trained on mass spectrometry-derived peptide presentation data. CD8scape integrates variant data and viral reading frames to identify all overlapping 8-11mer peptides at variant sites in both ancestral and derived states. These peptides are evaluated using NetMHCpan, which outputs eluted ligand (EL) scores as allele-specific percentile ranks to account for differences in MHC binding fastidiousness, and these are passed back to CD8scape itself. For each variant, the best-ranking peptide across all alleles is identified, and a harmonic mean is used to summarize presentation likelihood across the hosts HLA genotype. A fold-change between ancestral and derived harmonic means quantifies the likelihood of immune escape, with values >1 indicating reduced predicted presentation, and therefore a potential escape from the CD8+ T cell response. This is converted to a log2 value of this fold-change so that the metric is symmetric around 0, with positive values representing predicted escape. CD8scape can operate with known HLA genotypes or a representative HLA supertype panel for generalizable predictions. We demonstrate our method by application to within-host SARS-CoV-2 evolution in a rituximab-treated patient and discuss its implications for population-level CD8+ T cell escape.

bioinformatics↗

bioRxiv2025

Splitting Hairs: Optimized Sample Preparation Strategy for Mass Spectrometry Imaging of Nematode Caenorhabditis elegans

Caenorhabditis elegans is a powerful model organism for studying development, neurobiology, aging, and toxicology, yet most investigations rely on genetic and phenotypic measurements that do not directly capture their biochemical landscape. Mass spectrometry (MS)-based metabolomics has begun to address this gap by enabling detection of small-molecule metabolites and lipids. However, conventional liquid or gas chromatography-MS approaches require homogenization of whole worms, eliminating spatial information. Mass spectrometry imaging (MSI) offers an avenue for visualizing metabolite distributions in situ, but its application to C. elegans is hindered by the nematodes small size and thick cuticle which make it challenging for structure preservation during cryo-sectioning and ion image interpretation. We develop an integrated sample preparation workflow to overcome these challenges and enable high-resolution MSI of C. elegans adult hermaphrodites. To facilitate anatomical and ion images interpretation, we employed a dual-fluorescent reporter strain expressing GFP in intestinal cells and RFP in neuronal nuclei. We optimized embedding media compositions and freezing strategies to preserve tissue architecture and genetically encoded reporter fluorescence during cryo-sectioning. Additionally, we implemented a simple "sandwiching" method that reproducibly orients worms, allowing consistent longitudinal sectioning. Using this optimized workflow, we achieved matrix-assisted laser desorption/ionization (MALDI) MSI of 10-m spatial resolution and demonstrated tissue-specific distributions of lipids and metabolites in adult hermaphrodites. With structural preservation, consistent orientation, and fluorescence-guided spatial annotation, our approach provides a foundation for investigating metabolic heterogeneity in C. elegans and can be adapted to other microscale biological systems for MSI applications.

biochemistry↗

bioRxiv2022

Structural and molecular biology of Acheta domesticus segmented densovirus, the first parvovirus to harbor a bipartite genome

Parvoviruses (family Parvoviridae) are defined by their linear monopartite ssDNA genome, T=1 icosahedral capsid, and distinct structural (VP) and non-structural (NS) protein expression cassettes within their genome. Here, we report the first parvovirus with a segmented genome, Acheta domesticus segmented densovirus (AdSDV), a house cricket (Acheta domesticus) pathogen. The AdSDV harbors its NS and VP cassettes on two separate segmented genomes. Its VP segment acquired a phospholipase A2-encoding gene via inter-subfamily recombination, which is absent from its capsid. The AdSDV evolved a transcription profile in response to its multipartite replication strategy that has diverged from its Brevihamaparvovirus ancestors. Furthermore, AdSDV assembles three capsid populations, which package one genome segment per particle. The cryoEM structures of these three capsids (2.3 to 3.3 [A] resolution) reveal a genome packaging mechanism, which differs from other parvoviruses. This study provides a new perspective on ssDNA genome segmentation and on the plasticity of parvovirus biology.

microbiology↗

bioRxiv2020

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

BackgroundSARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. ResultsWe used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. ConclusionsWe present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

genomics↗