Biology subjects

Sylvester, K.

Publications and source records attributed to Sylvester, K..

7 recordsLinked to original sources

bioRxiv2024

Label free metabolic imaging to enhance the efficacy of Chimeric Antigen Receptor T cell therapy

Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains challenging due to the complex manufacturing process and the immunosuppressive tumor microenvironment. The manufacturing condition directly impacts CAR T cell yield, phenotype, and metabolism, which correlate with in vivo potency and persistence. Optical metabolic imaging (OMI) is a non-invasive, label-free method to evaluate single cell metabolism based on autofluorescent metabolic coenzymes NAD(P)H and FAD. Using OMI, we identified the dominating impacts of media composition over the selection of antibody stimulation and/or cytokines on anti-GD2 CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrated that OMI parameters were indicative of cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. Notably, OMI accurately predicted oxidative metabolic phenotype of virus-free CRISPR-edited anti-GD2 CAR T cells that correlated to higher in vivo potency against neuroblastoma. Our data supports OMIs potential as a robust, sensitive analytical tool that enables dynamic and optimal manufacturing conditions for increased CAR T cell yield and metabolic fitness. One sentence summaryAutofluorescence imaging informs manufacturing conditions that enhance yield and metabolic fitness of CAR T cells for neuroblastoma.

immunology↗

bioRxiv2021

Substrate, temperature, and geographical patterns among nearly 2,000 natural yeast isolates

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here we used environmental sampling and isolation to assemble a dataset of 1,962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the US, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon - substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa. Take AwayO_LIAnalysis of environmental metadata of nearly 2,000 yeast isolates. C_LIO_LIIndividual yeast taxa associate with specific substrates and plant genera. C_LIO_LIOptimal yeast isolation temperature differs depending on taxonomic rank. C_LIO_LISubstrate type and isolation temperatures affect isolated yeast diversity. C_LI

ecology↗

bioRxiv2020

The tubovesicular network in Plasmodium vivax liver-stage hypnozoites and schizonts associates with host aquaporin 3

The apicomplexan Plasmodium parasites replicate in the liver before causing malaria. P. vivax can also persist in the liver as dormant hypnozoites and cause relapses upon activation. The host water and solute channel aquaporin-3 (AQP3) has been shown to localize to the parasitophorous vacuole membrane (PVM) of P. vivax hypnozoites and liver schizonts, along with other Plasmodium species and stages. In this study, we use high-resolution microscopy to characterize temporal changes of the tubovesicular network (TVN), a PVM-derived network within the host cytosol, during P. vivax liver-stage infection. We demonstrate an unexpected role for the TVN in hypnozoites and reveal AQP3 associates with TVN-derived vesicles and extended membrane features. We further show AQP3 recruitment to Toxoplasma gondii. Our results highlight dynamic host-parasite interactions that occur in both dormant and replicating liver-stage P. vivax forms and implicate AQP3 function during this time. Together, these findings enhance our understanding of AQP3 in apicomplexan infection.

microbiology↗

bioRxiv2020

A Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity

Advances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito microbiome studies and highlights an understudied source of bioactive small molecules.

microbiology↗

bioRxiv2019

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood when compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive RNA-seq analysis throughout the Plasmodium berghei LS, covering as early as 2 hours post infection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi, and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with co-expression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding presents potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasites LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites. IMPORTANCEThe LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasites biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provides insights into these early parasite fluxes that may facilitate establishment of infection, transformation and replication in the liver.

microbiology↗

bioRxiv2019

Genomic diversity and global distribution of Saccharomyces eubayanus, the wild ancestor of hybrid lager-brewing yeasts

S. eubayanus, the wild, cold-tolerant parent of hybrid lager-brewing yeasts, has a complex and understudied natural history. The exploration of this diversity can be used both to develop new brewing applications and to enlighten our understanding of the dynamics of yeast evolution in the wild. Here, we integrate whole genome sequence and phenotypic data of 200 S. eubayanus strains, the largest collection to date. S. eubayanus has a multilayered population structure, consisting of two major populations that are further structured into six subpopulations. Four of these subpopulations are found exclusively in the Patagonian region of South America; one is found predominantly in Patagonia and sparsely in Oceania and North America; and one is specific to the Holarctic ecozone. S. eubayanus is most abundant and genetically diverse in Patagonia, where some locations harbor more genetic diversity than is found outside of South America. All but one subpopulation shows isolation-by-distance, and gene flow between subpopulations is low. However, there are strong signals of ancient and recent outcrossing, including two admixed lineages, one that is sympatric with and one that is mostly isolated from its parental populations. Despite S. eubayanus extensive genetic diversity, it has relatively little phenotypic diversity, and all subpopulations performed similarly under most conditions tested. Using our extensive biogeographical data, we constructed a robust model that predicted all known and a handful of additional regions of the globe that are climatically suitable for S. eubayanus, including Europe. We conclude that this industrially relevant species has rich wild diversity with many factors contributing to its complex distribution and biology.

genomics↗

bioRxiv2018

Pathogenic Budding Yeasts Isolated outside of Clinical Settings

Budding yeasts are distributed across a wide range of habitats, including as human commensals. However, under some conditions, these commensals can cause superficial, invasive, and even lethal infections. Despite their importance to human health, little is known about the ecology of these opportunistic pathogens, aside from their associations with mammals and clinical environments. During a survey of approximately 1000 non-clinical samples across the United States of America, we isolated 54 strains of budding yeast species considered opportunistic pathogens, including Candida albicans and Candida (Nakaseomyces) glabrata. We found that, as a group, pathogenic yeasts were positively associated with fruits and soil environments, while the species Pichia kudriavzevii (syn. Candida krusei syn. Issatchenkia orientalis) had a significant association with plants. These results suggest that pathogenic yeast ecology is more complex and diverse than is currently appreciated and raises the possibility that these additional environments could be a point of contact for human infections.\n\nImportanceWe isolated several opportunistic pathogenic species of yeasts from the subphylum Saccharomycotina from multiple non-clinical environments across the United States of America. Among those strains isolated, over 50% represent the most common opportunistic pathogens. These species, C. albicans, Candida tropicalis, Candida parapsilosis, and C. glabrata, have been rarely isolated from non-clinical settings and have been usually interpreted as contamination. Our extensive isolations from natural settings challenge this assumption, suggesting that opportunistic pathogens can persist in alternative niches and that their ecology may be more complicated than is currently assumed. Non-clinical environments could be a short-term habitat as these yeasts are passed between their predominant hosts, endothermic animals.

microbiology↗