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Phillips, M. A.

Publications and source records attributed to Phillips, M. A..

At least 19 recordsLinked to original sources
bioRxiv2026

Signal, noise, and sampling: How pool size and replication shape metabolomic inference

Metabolomics provides a direct readout of physiological state and is increasingly used in evolutionary and systems biology. In small organisms such as Drosophila melanogaster, metabolomic analyses typically require pooling individuals to obtain sufficient material, yet pool sizes vary widely across studies with little justification. How pooling and biological replication influence metabolome characterization and the detection of biological signal remains poorly understood. Here, we evaluate the effects of pool size and biological replication on metabolomic profiles and signal detection using two complementary experimental designs. In the first, we assess how pooling (5, 50, or 100 individuals) influences metabolomic structure and reproducibility in inbred and outbred populations. In the second, we test how pool size interacts with systematic variation in replicate number to affect detection of diet-associated metabolite changes under a high-sugar perturbation. Pool size strongly influenced metabolomic profiles, with samples pooled at five individuals consistently differing from larger pools, while profiles from 50 and 100 individuals were more similar. Larger pools improved reproducibility in a dataset-dependent manner. In the dietary experiment, smaller pool sizes substantially reduced sensitivity, leading to loss of true diet-associated metabolites without increasing false discoveries. Replicate downsampling further revealed that both pool size and biological replication jointly determine signal retention, with smaller pools accelerating the loss of detectable metabolites under reduced replication. Across all analyses, the ability to detect metabolite signals was strongly dependent on effect size and variability. Metabolites with larger and more stable effect estimates were consistently retained, whereas those with smaller or more variable effects were rapidly lost under reduced sampling. Linear mixed-effects modeling confirmed that detection probability is governed by a balance between biological signal strength and measurement variability, with pool size and replication jointly modulating this relationship. More broadly, our results demonstrate that metabolomic inference is governed by the interplay of signal, noise, and sampling design, with pool size and replication jointly shaping the detectability, stability, and interpretation of biological signals.

molecular biology↗
bioRxiv2026

Beyond Fixation: Persistent Genetic Variation Under Intense Selection

Understanding how and why genetic variation is maintained under sustained selection remains a central question in evolutionary genetics. Experimental evolution shows that adaptation in sexually reproducing populations is often highly polygenic, proceeding through coordinated, genome-wide allele frequency shifts from standing variation rather than classic hard sweeps. Recent explanations emphasize highly polygenic architectures, optimizing-selection, and genetic redundancy, which can slow fixation by distributing selection across many loci during adaptation. However, observations from long-term selection experiments reveal a pattern these frameworks do not fully explain: substantial genetic variation persists after hundreds of generations of intense directional selection in constant environments. Here, we use long-term experimental evolution in Drosophila melanogaster to test whether balancing-selection actively maintains genetic variation under strong life-history selection and preserves evolutionary reversibility. Longstanding populations selected for accelerated or delayed reproduction were shifted to the opposing regime, imposing age-structured fitness trade-offs. Notably, selection for early reproduction is associated with substantial loss of genetic variation, providing a stringent test of whether standing variation is truly depleted. Following reciprocal shifts, populations showed rapid phenotypic convergence toward the target regime. At the genomic level, allele-frequency trajectories were strongly antiparallel and highly repeatable across replicates, revealing coordinated polygenic responses. Relaxing long-standing early-life selection produced a pronounced rebound in genome-wide heterozygosity. Deep sequencing uncovered ultra-rare alleles at sites appearing fixed under standard coverage, indicating low-frequency functional variation persists below detection thresholds. These results suggest that substantial genetic variation can persist under intense directional selection and be rapidly redeployed when selection reverses, consistent with widespread balancing-selection.

evolutionary biology↗
bioRxiv2026

Derlin-mediated ERAD of lipid regulator ORMDL3 safeguards mitochondrial function

Mammalian Derlin proteins (Derlin-1, Derlin-2, and Derlin-3) are conserved components of the endoplasmic reticulum-associated degradation (ERAD) machinery that mediate the retrotranslocation and proteasomal degradation of misfolded ER-resident proteins. However, their paralog-specific contributions to cellular homeostasis remain poorly understood. Here, we show that Derlin deficiency disrupts mitochondrial architecture and results in mitochondrial fragmentation and tightening of ER-mitochondria contact sites (MERCs) in HEK293 cells. Mechanistically, we identify ORMDL proteins, evolutionarily conserved negative regulators of sphingolipid biosynthesis, as substrates of Derlin-2- and Derlin-3-dependent ERAD. Derlin deficiency leads to selective accumulation of ORMDL3 and its dose-dependent enrichment at MERCs, where it drives mitochondrial dysfunction in respiration and calcium handling. Reducing ORMDL3 levels restores mitochondrial function, establishing ORMDL3 as a key effector downstream of Derlin loss. Our work establishes ERAD as a critical mechanism of protein quantity control that safeguards organelle homeostasis by preventing aberrant accumulation and mislocalization of ER clients at inter-organelle contact sites. Given that ORMDL family members are central regulators of sphingolipid metabolism and are genetically linked to inflammation, cancer, asthma, inflammatory bowel disease, type 1 and type 2 diabetes, multiple sclerosis, obesity, and nonalcoholic fatty liver disease, these findings connect ERAD-dependent spatial control to sphingolipid homeostasis and a broad spectrum of human pathologies.

cell biology↗
bioRxiv2025

Transition of Staphylococcus aureus tetracycline resistance plasmid pT181 from independent multicopy replicon to predominantly integrated chromosomal element over 65 years

Mobile genetic elements (MGEs), including plasmids, phages and genome islands, are major sources of bacterial genetic diversity. The small plasmid pT181 confers tetracycline resistance in bacterial pathogen Staphylococcus aureus via an efflux pump, TetK. pT181 was one of the earliest sequenced S. aureus plasmids and has been isolated in both clinical and livestock-associated strains for decades, both as an independent replicon and integrated in the chromosome as part of staphylococcal cassette chromosome mec (SCCmec). Bacterial genome analysis tools and high-quality sequences with metadata are publicly available, but these resources remain underleveraged for examining historical data, especially when studying the spread of MGEs across a species and over time. Using publicly available reads and metadata, we explored the evolution of pT181 over nearly seven decades of samples to identify temporal trends in sequence evolution, copy number changes, and spread across S. aureus and beyond. pT181 was prevalent across S. aureus (found in 9.5% of 83,366 genomes tested), with a conserved sequence outside of three hypervariable regions. The history of pT181 since 1954 was characterized by spread across strains, significant variation in plasmid copy number of the independent replicon, and increasing frequency of integration of the plasmid into the S. aureus chromosome. We identified multiple chromosomal integration locations of the plasmid, including outside of the previously characterized SCCmec. We found that pT181 was transferred across Staphylococcaceae and into a Gram negative species. The repeated integration of pT181 into the chromosome may indicate coevolution of the plasmid and the host, potentially to facilitate increased antibiotic resistance.

genomics↗
bioRxiv2025

Identification of α-azacyclic acetamide-based inhibitors of P. falciparum Na+ pump (PfATP4) with fast-killing asexual blood-stage antimalarial activity by phenotypic screening

Malaria treatments are compromised by drug resistance, creating an urgent need to discover new drugs. We used a phenotypic high-throughput screening (HTS) platform to identify new antimalarials, uncovering three related pyrrole-, indole-, and indoline-based series with a shared -azacyclic acetamide core. These compounds showed fast-killing activity on asexual blood-stage Plasmodium falciparum parasites, were not cytotoxic, and disrupted parasite intracellular pH and Na+ regulation similarly to cipargamin (KAE609), a clinically advanced inhibitor of the P. falciparum Na+ pump (PfATP4). PfATP4 is localized to the parasite plasma membrane and is essential for maintaining a low cytosolic Na+ concentration. Resistance selections on P. falciparum parasites with two -azacyclic acetamide analogs identified mutations in PfATP4, and cross-resistance was observed across the -azacyclic acetamides and KAE609, confirming PfATP4 as the target. PfATP4 is a well-established antimalarial target, and identification of additional PfATP4 inhibitors provides alternative avenues to disrupt its function.

microbiology↗
bioRxiv2024

A chromosome level reference genome of Diviners sage (Salvia divinorum) provides insight into salvinorin A biosynthesis

BackgroundDiviners sage (Salvia divinorum; Lamiaceae) is the source of the powerful hallucinogen salvinorin A (SalA). This neoclerodane diterpenoid is an agonist of the human K opioid receptor with potential medical applications in the treatment of chronic pain, addiction, and post-traumatic stress disorder. Only two steps of the approximately twelve step biosynthetic sequence leading to SalA have been resolved to date. ResultsTo facilitate pathway elucidation in this ethnomedicinal plant species, here we report a chromosome level genome assembly. A high-quality genome sequence was assembled with an N50 value of 41.4 Mb and a BUSCO completeness score of 98.4%. The diploid (2n = 22) genome of [~]541 Mb is comparable in size and ploidy to most other members of this genus. Two diterpene biosynthetic gene clusters were identified and are highly enriched in previously unidentified cytochrome P450s as well as crotonolide G synthase, which forms the dihydrofuran ring early in the SalA pathway. Coding sequences for other enzyme classes with likely involvement in downstream steps of the SalA pathway (BAHD acyl transferases, alcohol dehydrogenases, and O-methyl transferases) were scattered throughout the genome with no clear indication of clustering. Differential gene expression analysis suggests that most of these genes are not inducible by methyl jasmonate treatment. ConclusionsThis genome sequence and associated gene annotation are among the highest resolution in Salvia, a genus well known for the medicinal properties of its members. Here we have identified the cohort of genes responsible for the remaining steps in the SalA pathway. This genome sequence and associated candidate genes will facilitate the elucidation of SalA biosynthesis and enable an exploration of its full clinical potential.

genomics↗
bioRxiv2024

Selection for early reproduction leads to accelerated aging and extensive metabolic remodeling in Drosophila melanogaster populations

Experimental evolution studies that feature selection on life-history characters are a proven approach for studying the evolution of aging and variation in rates of senescence. Recently, the incorporation of genomic and transcriptomic approaches into this framework has led to the identification of hundreds of genes associated with different aging patterns. However, our understanding of the specific molecular mechanisms underlying these aging patterns remains limited. Here, we incorporated extensive metabolomic profiling into this framework to generate mechanistic insights into aging patterns in Drosophila melanogaster. Specifically, we characterized metabolomic change over adult lifespan in populations of D. melanogaster where selection for early reproduction has led to an accelerated aging phenotype relative to their controls. Using these data we: i) evaluated evolutionary repeatability across the metabolome; ii) assessed the value of the metabolome as a predictor of "biological age" in this system; and iii) identified specific metabolites associated with accelerated aging. Generally, our findings suggest that selection for early reproduction resulted in highly repeatable alterations to the metabolome and the metabolome itself is a reliable predictor of "biological age". Specifically, we find clusters of metabolites that are associated with the different rates of senescence observed between our accelerated aging population and their controls, adding new insights into the metabolites that may be driving the accelerated aging phenotype. SignificanceWhile experimental evolution studies featuring Drosophila melanogaster have generated significant insights into the forces that shape aging and life history patterns, more recent efforts incorporating genomic and transcriptomic data have had comparatively little success identifying the molecular mechanisms underlying these patterns. Here we work to incorporate molecular phenotyping into this general framework as a way forward. Specifically, we characterize how the metabolome changes with age in populations of D. melanogaster where hundreds of generations of selection for early reproduction have led to enrichment for an accelerated aging phenotype. By comparing to control populations, we show that the metabolome does appear to capture true signals of "biological age" and provides a new avenue for understanding the factors that underlie complex trait variation in real populations.

evolutionary biology↗
bioRxiv2024

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes

Background & AimsAging is associated with a significant decline in mitochondrial function in the liver, leading to an increased risk of liver disease. This study examines age-related changes in the mitochondrial structure of human and murine livers using a combination of Serial Block-Face Scanning Electron Microscopy (SBF-SEM) and mass spectrometry approaches. MethodsThis study integrates mitochondrial structure analysis in a murine model with an analysis of liver architecture, lipogenesis, and genetically regulated gene expression in human cohorts. We explored the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex using SBF-SEM, three-dimensional reconstruction with Amira software, and mass spectrometry techniques. ResultsAging leads to a reduction in mitochondrial size and complexity, resulting in changes in the metabolomic and lipidomic profiles of murine liver cells that are comparable to those observed in aged human samples. We find that genetically modeled expression of MICOS complex genes OPA1 and CHCHD3 is associated with chronic liver disease phenotypes within a large biobank population. Furthermore, we observed dysregulated mitochondrial calcium handling and increased oxidative stress due to the disruption of the MICOS complex. ConclusionOur study highlights the age-associated decline in mitochondrial complexity and metabolic regulation within the aging murine liver and the human population. We have identified that these changes are partially attributable to the age-related loss of the MICOS complex. Impact and implicationsThis study offers new insights into the changes to mitochondrial ultrastructure that occur during aging. Using SBF-SEM, the quantification of young and aged murine mitochondrial structure was performed, which had previously been an underexplored avenue for measuring mitochondrial changes. The discovery of mitochondrial ultrastructural changes, in conjunction with measurements of age-associated metabolic alterations and gene association data, provides a model for how changes in MICOS expression may modulate age-related impairment of hepatic mitochondria. These results provide a new model by which changes in MICOS protein expression may both cause and be a potential therapeutic target for age-related impairment in hepatic function. HighlightsDecreased modeled expression of CHCHD3 in individuals of European genetic ancestry is linked to liver transplant and cirrhosis, while decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic liver disease and cirrhosis. Aging alters liver lipid accumulation, MICOS mRNA levels, and disease markers. Aging reduces the volume and complexity of murine liver ultrastructure. Aging and diet significantly alter the MICOS complex in mice. Knockdown of Mic60 and Chchd6 lowers Ca2+ uptake, retention, and induces oxidative stress in HepG2 cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/599846v3_ufig1.gif" ALT="Figure 1000"> View larger version (47K): org.highwire.dtl.DTLVardef@1cdd61corg.highwire.dtl.DTLVardef@a3fb74org.highwire.dtl.DTLVardef@1d1ad36org.highwire.dtl.DTLVardef@c2e55f_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver aging causes metabolic, lipidomic, and mitochondrial structural alterations, reflecting age-dependent losses in the MICOS complex. Key components of the MICOS complex (MIC60, CHCHD3 and CHCHD6) are illustrated.

physiology↗
bioRxiv2024

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

Due to aging, the efficiency of kidney function begins to decrease. Dysfunction in mitochondria and their cristae is a hallmark of aging. Therefore, age-related decline in kidney function could be attributed to changes in mitochondrial ultrastructure, increased reactive oxygen species, and alterations in metabolism and lipid composition. We sought to understand how mitochondrial ultrastructure is altered over time in tubular kidney cells. A serial block facing-scanning electron microscope and manual segmentation using the Amira software were employed to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we demonstrate that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney during aging. Disruption of the MICOS complex resulted in altered mitochondrial metabolic function and increased ROS levels. We found significant, detrimental structural changes in the mitochondria of aged kidney tubules, suggesting a potential mechanism underlying the increased frequency of kidney disease with aging. We hypothesize that disruption of the MICOS complex exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, which impacts kidney health. Impact and ImplicationsDue to aging, the efficiency of kidney function begins to decrease, and the risk of kidney diseases may increase; however, the specific regulators of mitochondrial age-related changes are poorly understood. This study demonstrates that the MICOS complex may be a target for mitigating age-related mitochondrial changes. The MICOS complex is associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies. HighlightsO_LIAging alters the MICOS mRNA levels and disease markers. C_LIO_LIAging reduces cristae architecture, mitochondrial volume and complexity in murine kidney ultrastructure C_LIO_LIReducing MIC60 and CHCHD6 lowers Ca2+ uptake and retention and induces oxidative stress in HEK cells. C_LIO_LIMetabolomic Profiling revealed that NAD+ and amino acid metabolism were altered in aged kidneys. C_LIO_LIMICOS deficiency alters the reduced basal, ATP-linked, maximal capacity and spare capacity. C_LIO_LIDecreased modeled expression of CHCHD6 in individuals of European genetic ancestry is linked to chronic kidney disease, whereas decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic kidney disease. C_LI Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/598108v3_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a868f0org.highwire.dtl.DTLVardef@1817bfborg.highwire.dtl.DTLVardef@1f2a1a3org.highwire.dtl.DTLVardef@520692_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗
bioRxiv2024

Rubisco supplies pyruvate for the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) produces pyruvate in the chloroplast through beta-elimination of the aci-carbanion intermediate. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid, and branched chain amino acid biosynthesis in photosynthetically active tissue. 13C labeling studies of whole Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate (PEP) to serve as precursor. Low oxygen stimulates rubisco carboxylase activity and increased pyruvate production and flux through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis. Metabolome analysis of mutants defective in PEP or pyruvate import further supported rubisco as the main source of pyruvate in chloroplasts. Rubisco beta-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin-Benson-Bassham cycle (CBC). These insights solve the so-called pyruvate paradox, improve the fit of metabolic models for central metabolism, and connect the MEP pathway directly to carbon assimilation.

plant biology↗
bioRxiv2023

Human Heart Failure Alters Mitochondria and Fiber 3D Structure Triggering Metabolic Shifts

This study, utilizing SBF-SEM, reveals structural alterations in mitochondria and myofibrils in human heart failure (HF). Mitochondria in HF show changes in structure, while myofibrils exhibit increased cross-sectional area and branching. Metabolomic and lipidomic analyses indicate concomitant dysregulation in key pathways. The findings underscore the need for personalized treatments considering individualized structural changes in HF.

physiology↗
bioRxiv2023

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria we less spherical and more complex, indicating age-related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusin 2. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/566502v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1d2be0eorg.highwire.dtl.DTLVardef@bba6caorg.highwire.dtl.DTLVardef@1c335e5org.highwire.dtl.DTLVardef@10789c9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Age-related skeletal muscle atrophy shows morphological alterations in mitochondrial structure associated with declining function. Our findings propose that exercise intervention may counteract these structural declines by reinstating levels of mitofusin 2, thus highlighting a potential mechanism by which exercise attenuates age-induced mitochondrial dysfunction. C_FIG

biophysics↗
bioRxiv2022

Consequences of blindness for neural dynamics in mouse visual cortex

The role of experience in the development and maintenance of emergent network properties such as cortical oscillations and states is poorly understood. To define how early-life experience affects cortical dynamics in adult visual cortex, we examined the effects of two forms of blindness, bilateral loss of retinal input (enucleation) and degradation of visual input (eyelid-suture), on spontaneous activity in awake head-fixed mice of both sexes. Neither form of deprivation fundamentally altered the state-dependent regulation of firing-rates or local field potentials. However, each form of deprivation did cause a unique set of changes in network behavior. Laminar analysis revealed two different generative mechanisms for low-frequency synchronization, one prevalent during movement, the other during quiet-wakefulness. The former was absent in enucleated mice, suggesting a mouse homolog of human alpha oscillations. In addition, neurons in enucleated animals were less correlated and fired more regularly, but showed no change in mean firing-rate. Chronic lid-suture decreased firing rates during quiet-wakefulness, but not during movement, with no effect on neural correlations or regularity. Sutured animals showed a broadband increase in dEEG power and an increased occurrence, but reduced central frequency, of narrowband gamma oscillations. The complementary--rather than additive--effects of lid-suture and enucleation suggest that the development of these emergent network properties does not require vision but is plastic to modified input. Our results suggest a complex interaction of internal set-points and experience determines the expression of mature cortical activity, with low-frequency synchronization being particularly susceptible to early deprivation. SIGNIFICANCE STATEMENTThe developmental rules that guide how cortex balances internal homeostatic set points with external inputs to establish the emergent network level dynamics critical to its function are unclear. Using multiple methods of early deprivation, we show that the development of dynamics in mouse visual cortex is not dependent on the type of input. Rather, specific neural rhythms, firing-rate set points, and neural correlations are differentially modified by experience. Our deprivations identify one specific rhythm as a likely homolog to human alpha and suggest a mechanism for its loss in blindness. Our results advance our understanding of the regulatory mechanism leading to normal cortical processing, which is altered in blindness and multiple neural disorders.

neuroscience↗
bioRxiv2022

A fast-killing tyrosine amide ((S)-SW228703) with blood and liver-stage antimalarial activity associated with the Cyclic Amine Resistance Locus (PfCARL)

Current malaria treatments are threatened by drug resistance and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations in Plasmodium falciparum cyclic amine resistance locus (PfCARL) and P. falciparum acetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their target(s) are unknown. The tyramine of (S)-SW703 is shared with some reported PfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for activity of (S)-SW703, suggesting differences in mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity on multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.

microbiology↗
bioRxiv2022

Building bridges from genome to physiology using machine learning and Drosophila experimental evolution

Drosophila experimental evolution, with its well-defined selection protocols, has long supplied useful genetic material for the analysis of functional physiology. While there is a long tradition of interpreting the effects of large-effect mutants physiologically, in the genomic era identifying and interpreting gene-to-phenotype relationships has been challenging, with many labs not resolving how physiological traits are affected by multiple genes throughout the genome. Drosophila experimental evolution has demonstrated that multiple phenotypes change due to the evolution of many loci across the genome, creating the scientific challenge of sifting out differentiated but noncausal loci for individual characters. The fused lasso additive model method (FLAM) allows us to infer some of the differentiated loci that have relatively greater causal effects on the differentiation of specific phenotypes. The experimental material used in the present study comes from 50 populations that have been selected for different life-histories and levels of stress resistance. Differentiation of cardiac robustness, starvation resistance, desiccation resistance, lipid content, glycogen content, water content, and body masses was assayed among 40 to 50 of these experimentally-evolved populations. Through FLAM, we combined physiological analysis from eight parameters with whole-body pooled-seq genomic data to identify potentially causally linked genomic regions. We have identified approximately 1,900 significantly differentiated 50 kb genomic windows among our 50 populations, with 161 of those identified genomic regions highly likely to have a causal effect connecting specific genome sites to specific physiological characters.

physiology↗
bioRxiv2022

Strength of selection potentiates distinct adaptive responses in an evolution experiment with outcrossing yeast

Experimental evolution studies with sexually-reproducing populations consistently find that adaptation is highly polygenic and fueled by standing genetic variation. However, studies vary substantially with respect to other evolutionary dynamics. Resolving these discrepancies is a crucial next step as we move toward extrapolating findings from laboratory systems to natural populations. Differences in experimental parameters between studies can perhaps answer these questions, and here we assess how one such parameter - selection intensity - influences outcomes. We subject populations of outcrossing Saccharomyces cerevisiae to zero, moderate, and high ethanol stress for [~]200 generations and ask: 1) does stronger selection lead to greater changes in allele frequencies at adaptive sites; and 2) do targets of selection vary with intensity? With respects to sites with large effects, we find some evidence for positive correlations between selection intensity and allele frequency change. While we observe shared genomic responses across treatments, we also identify treatment-specific responses. Combined with evidence of phenotypic trade-offs between treatments, our findings support the hypothesis that selection intensity influences evolutionary outcomes due to pleiotropic and epistatic interactions. We conclude that it should be a major consideration when attempting to generalize inferences across studies; in other words, we argue that different intensities of selection effectively create distinct environments and genotype-by-environment interactions. Lastly, our results demonstrate the importance of clearly-defined controls in experimental evolution. Despite working with a presumably lab-adapted model system, without this element we would not have been able to distinguish genomic responses to ethanol stress from those associated with laboratory conditions.

evolutionary biology↗
bioRxiv2021

A gene coevolution network provides insight into eukaryotic cellular and genomic structure and function

Orthologous gene coevolution--which refers to gene pairs whose evolutionary rates covary across speciation events--is often observed among functionally related genes. We present a comprehensive gene coevolution network inferred from the examination of nearly three million orthologous gene pairs from 332 budding yeast species spanning [~]400 million years of eukaryotic evolution. Modules within the network provide insight into cellular and genomic structure and function, such as genes functioning in distinct cellular compartments and DNA replication. Examination of the phenotypic impact of network perturbation across 14 environmental conditions using deletion mutant data from the bakers yeast Saccharomyces cerevisiae suggests that fitness in diverse environments is impacted by orthologous gene neighborhood and connectivity. By mapping the network onto the chromosomes of S. cerevisiae and the opportunistic human pathogen Candida albicans, which diverged [~]235 million years ago, we discovered that coevolving orthologous genes are not clustered in either species; rather, they are most often located on different chromosomes or far apart on the same chromosome. The budding yeast coevolution network captures the hierarchy of eukaryotic cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as an extensively linked ensemble of genes.

evolutionary biology↗