Kari B. Basso

Virulent bacteriophages (phages) can kill bacterial prey, potentially reducing burden of infection. In cholera, a high phage to Vibrio cholerae ratio is associated with mild dehydration, yet the relative importance of this ratio in disease severity remains unclear. We used machine learning to rank select host, microbial, and environmental factors as determinants of dehydration severity in over 600 cholera patients from across Bangladesh. We found the phage:pathogen ratio ranked among the top classifiers for mild dehydration, behind age and location. We advocate that phage predation be included as a key factor in cholera characterization for scientific, clinical and epidemiological applications.

The synthesis and use of ionic liquids (ILs) has increased since their inception, driven by their properties and tunability, and it follows that concerns have grown about potential environmental release and persistence. There is thus a motivation to develop and improve methods for the analysis of IL species. However, focus on the detection and characterization of IL species from dilute solutions, rather than the characterization of their bulk properties, is still lacking. The goal of this work is to (1) explore whether DART‐MS might be a useful tool to pursue for rapid detection of known ionic liquid species from solution, (2) introduce class‐based diagnostic product ions for common aromatic N‐heterocyclic ionic liquid cations, and (3) demonstrate the m/z 83 ion as an example of such a diagnostic marker for methyl‐imidazolium‐based IL cations. The results demonstrated that DART‐MS allows for the observation of intact IL cations from dilute solutions of ILs and is thus promising for rapid detection of IL species from solution, although additional steps such as preconcentration may be necessary for many environmentally relevant concentrations. Furthermore, in‐source CID screening for the m/z 83 characteristic product ion for methyl‐imidazolium‐based IL cations was demonstrated in proof‐of‐principle examples, showing promise for future method development. While the initial motivation for this work was the dearth of methods exploration for environmental contamination detection, these results are also broadly applicable to other cases where detection and characterization of IL species are needed and provide initial results upon which further method development can build for specific applications.

Sample normalization is essential for lipid quantitation. While normalization methods involving pre- or postgravimetric measurements, counting, or protein prequantitation exist, a single, common sample normalization method is not widely accepted. Previously, we proposed and evaluated the sulfo-phospho-vanillin assay (SPVA), a total lipid quantitation reaction, for prequantifying total lipids for LC-MS/MS sample normalization purposes. This current investigation furthers our evaluation of the SPVA as a sample normalization method in untargeted lipidomic LC-MS/MS by comparing SPVA total lipid prequantitation to protein prequantitation and gravimetric measurements. This study was applied to a wide selection of matrices, including Escherichia coli, plasma, brain, heart, and kidney. Resulting relative lipid concentrations showed smaller bioreplica variation when a prequantitation method was applied, either measuring total lipid or total protein; however, several relative lipid abundances showed inverse concentration relationships when normalizing with total protein compared to total lipid (from SPVA measurements) or gravimetric sample normalization. Further investigation using lipid extracts linearly spiked with pure, non-native lipid showed that gravimetric and protein normalization nonlinearly overproduced significant lipids, while linear increases in significant lipid features were observed from SPVA normalization. Lipid extracts linearly spiked with pure, non-native protein yielded fewer significant lipid features using SPVA normalization, and this was unchanged as protein was added; however, both gravimetric and protein normalization continued to yield large numbers of significant lipid features. Together, these results suggest neither gravimetric nor protein sample normalization appropriately normalizes quantitative lipidomic experiments and greatly overgenerates statistically significant lipid features for biomarker investigation. SPVA normalization more accurately adjusts for lipid changes in bioreplicate samples, leading to fewer but more biologically relevant statistically significant lipid features.

Cholera is an acute diarrhoeal disease caused by Vibrio cholerae. It remains a major public health challenge worldwide, and particularly in the endemic region around the Bay of Bengal. Over decadal time scales, one lineage typically dominates and spreads in global pandemic waves. However, it remains unclear to what extent diverse lineages co-circulate during a single outbreak. Defining the pool of diversity over finer time-scales is important because the selective pressures that impact V. cholerae, namely antibiotics and phages, are dynamic on these scales. To study the nationwide diversity of V. cholerae, we long-read sequenced 273 V. cholerae genomes from seven hospitals over 1 year (2018) in Bangladesh. Four major V. cholerae lineages were identified: three known lineages, BD-1, BD-2a and BD-2b, and a novel lineage that we call BD-3. In 2022, BD-1 caused a large cholera outbreak in Dhaka, at which point it had replaced BD-2 as the most common lineage in Bangladesh. We show that, in 2018, BD-1 was already predominant in the five northern regions, including Dhaka, consistent with an origin from northern India. By contrast, we observed a higher diversity of lineages in the two southern regions near the coast. The four lineages differed in pangenome content, including integrative and conjugative elements (ICEs) and genes involved in resistance to bacteriophages and antibiotics. Notably, BD-2a lacked an ICE and is predicted to be more sensitive to phages and antibiotics, yet persisted throughout the sampling period. Genes previously associated with antibiotic resistance in V. cholerae isolated from Bangladesh in the prior decade were entirely absent from all lineages in 2018–2019, suggesting shifting costs and benefits of encoding these genes. Our results highlight the diverse nature of the V. cholerae pangenome and geographic structure within a single outbreak season. This diversity provides the raw material for adaptation to antibiotics, phages and other selective pressures.

Main conclusion Proteins coded by At1g54570 and At3g25840 previously characterized in the synthesis of phytyl acyl esters also catalyze xanthophyll acyl esterification under tissue-culture-induced stress and when expressed in transgenic tomato. Abstract Fatty acyl esters of xanthophylls accumulate in flower petals and ripening fruit of many plant taxa. The genes involved in xanthophyll esterification are, therefore, of interest as xanthophyll esters are known in determining flower and fruit coloration and the accumulation and stability of nutritionally valuable carotenoids. Arabidopsis thaliana proteins homologous to tomato xanthophyll acyltransferases PYP1 and PYP1-like coded by At1g54570 (AtPES1) and At3g26840 (AtPES2), respectively, were previously implicated in the synthesis of fatty acid phytyl esters and triacylglycerol. Here, we tested the hypothesis that AtPES1 and AtPES2 proteins could have roles in xanthophyll esterification also. Chromatographic analyses quantifying carotenoids from tissue-cultured callus of WT, pes1, pes2, and the double mutant pes1/pes2 revealed that AtPES1 and AtPES2 have overlapping functions in the synthesis of xanthophyll mono-, di-, and tri-esters. The xanthophyll esters from Arabidopsis callus were further confirmed and identified by LC–MS. Given AtPES1 and AtPES2’s homology to PYP1 and PYP1-like in tomato, complementation tests were performed in the tomato mutant, pyp1-1(H7L) impaired for xanthophyll esterification, in which AtPES1 and AtPES2 were overexpressed. Transgenic expression of PES1 restored the dull yellow petal phenotype of pyp1-1(H7L) to bright yellow like that of the wild-type and xanthophyll esters could be identified in their petal extracts. Transgenic expression of AtPES2 had similar, but less effective impact on restoring petal coloration and xanthophyll ester profiles than AtPES1. Together, our results indicate that Arabidopsis AtPES1 and AtPES2 proteins have overlapping functions in the synthesis of xanthophyll acyl esters.

Phage predation is inversely associated with severe cholera yet its importance as a determinant of dehydration severity is unknown relative to other factors. Here we used machine learning to assess and rank potential host, microbial, and environmental factors as determinants of severe dehydration among a cohort of cholera patients enrolled at hospital admission across Bangladesh. We found the phage to pathogen ratio ranked among the top classifying features, placing just behind patient age and admission location. We advocate that phage predation is a key factor to include in the characterization of cholera for scientific, clinical and epidemiological applications.

The nematode Caenorhabditis elegans produces a large family of ascaroside pheromones, which it uses in chemical communication to coordinate the development and behavior of the population. The acyl-CoA oxidase (ACOX) enzymes, which catalyze the first rate-limiting step in peroxisomal β-oxidation, act as gatekeepers for the biosynthesis of ascarosides with specific side-chain lengths. By performing unbiased comparative metabolomics on acox-1.1, -1.2, -1.3, -1.4, and -3 mutant worms and acox-1.1;acox-3 double mutant worms, we provide a comprehensive view of the different roles of these enzymes in ascaroside biosynthesis and implicate them in a number of additional biosynthetic pathways. Our data show that acox-1.1 and acox-3 are required for the biosynthesis of a broad range of medium- and long-chain ascarosides, while acox-1.2, acox-1.3, and acox-1.4 specialize in ascarosides with specific side-chain lengths. Specific acox mutants accumulate a variety of modified ascarosides that are likely shunt products. Furthermore, we show that acox-1.1 and acox-3, but not other acox genes, are required for the biosynthesis of a specific subset of N-acylethanolamines (NAEs), many of which have hydroxyl groups at specific positions in their fatty acyl side chains. Through stable-isotope labeling, feeding experiments, and chemical synthesis, we characterize the structures of these NAEs and show that their fatty acyl groups are derived from both bacteria and nematode sources. One of the most strongly acox-dependent NAEs that has a β-hydroxy fatty acyl group is attractive to C. elegans at attomolar concentrations, whereas a closely related NAE with a γ-hydroxy fatty acyl group is not, indicating that a subset of secreted NAEs may influence worm behavior.

Fibrosis is a dysregulated wound healing response characterized by excessive accumulation of dense scar tissue that inhibits organ function and is estimated to contribute to up to 45% of deaths in the industrialized world. In this work, we sought to uncover new ways to address fibrosis by drawing inspiration from an animal that does not develop fibrosis. The Spiny Mouse (Acomys) has the most extensive regenerative capabilities of any known mammal and can regenerate injuries to the skin, kidney, heart, skeletal muscle, and spine with little to no fibrosis. We hypothesize that the regenerative abilities of Acomys are due, in part, to altered stiffness-mediated fibroblast-to-myofibroblast transition (FMT). In this work, we interrogated stiffness-mediated FMT in Acomys and Mus dermal fibroblasts in vitro by performing RNA Sequencing and found no differential gene expression in Acomys fibroblasts cultured on soft vs stiff substrates. We further investigated the direct impact of stiffness-mediated FMT and species differences on ECM deposition by fabricating cell-derived matrices (CDMs) from Acomys and Mus fibroblasts cultured on varying stiffnesses. After assessing the composition of these CDMs using label-free quantitative proteomics, fibrosis-associated extracellular matrix proteins including fibrillin-1, ADAMTS1, SPARC, and galectin-1 were found to be significantly reduced or absent in Acomys CDMs compared to Mus CDMs. In addition, proteins that have been connected to fibrosis resolution, including Col12a1 and clusterin, were upregulated in Acomys CDMs. When cultured on Acomys CDMs, mouse macrophages downregulated MMP9 mRNA expression and maintained increased expression of iNOS in response to IL-4, a pro-fibrotic cytokine. These results indicate a direct impact of species-specific ECM compositions on macrophage phenotype and suggest that ECM produced by Acomys fibroblasts may impede the development of a pro-fibrotic macrophage phenotype in the presence of pro-fibrotic stimuli.