Biology subjects

Overmyer, K.

Publications and source records attributed to Overmyer, K..

17 recordsLinked to original sources

bioRxiv2026

Proteotyping reactivant toxoplasmic encephalitis reveals virulence-associated dense granule protein GRA5 polymorphisms

A fatal case of toxoplasmic encephalitis, and others like it, has caused microbiologists and clinicians to question whether different strains of T. gondii have more pathogenic potential than others. This raises significant concern, as T. gondii is a widely spread parasitic organism that is presumed to lie dormant in a third of the worlds general population. In this study, we expand on a previously published proteomicanalysis reactivated toxoplasmic encephalitis and have been able to identify T. gondii-specific peptides in the cerebrospinal fluid (CSF) of this patient and two additional cases of toxoplasmic reactivation. Multilocus PCR-restriction fragment-length polymorphism (PCR-RFLP) was used to genetically identify the T. gondii strain that resulted in this fatal case, which belonged to ToxoDB PCR-RFLP genotype #7. Using other T. gondii strains of the same genotype, we performed bioassays to compare the pathogenicity of this genotype with that of a clinically relevant strain, ME49. Both of the tested genotype #7 strains appear to have a greater pathogenic potential, although through likely different mechanisms. Of the most abundant T. gondii-specific CSF peptides across multiple patients, we identified a polymorphic region of the dense granule protein GRA5 that appears to have strain specificity. This approach could represent a "proteotype" that allows for T. gondii strain risk stratification within clinical samples. Ultimately spinal fluid could be a valuable tool in distinguishing between T. gondii exposed individuals with no cyst forms in the brain versus those exposed individuals that harbor "clinically silent" but viable brain cysts. SIGNIFICANCEA third of the worlds population is exposed to the parasite Toxoplasma gondii, residing in a dormant, encysted stage within neurons. T. gondii is a diverse microorganism with some strains having greater reactivation potential. There is no means of identifying which individuals are at risk of reactivation. Proteomic analysis of cerebrospinal fluid from patients with reactivated toxoplasmosis demonstrated a consistent pattern of T. gondii peptides, including GRA5, a secreted virulence factor. Postmortem analysis identified a ToxoDB PCR-RFLP genotype #7 strain associated with a fatality. Cerebrospinal fluid could provide clues to persistent brain infection and may identify strains able to reactivate.

microbiology↗

bioRxiv2025

Regarding Emitter Positioning for Nanoflow Electrospray Ionization

Nanoflow electrospray ionization is commonly used for proteomics due to its high sensitivity. Signal intensity, however, is dependent on optimal emitter positioning relative to the mass spectrometer inlet. Here, we characterize the effect of varied emitter positions on peptide signal intensity in all three dimensions using emitters and flows consistent with standard proteomic analyses. We observe improved signal robustness to x/y variations at increasing z distances and demonstrate that positioning within 1 to 2 mm of the optimal location will maintain consistent signal. Signal intensity behavior is consistent across the m/z range, suggesting a certain level of analyte-independence for proteomics analyses. These results provide insight for proteomics researchers using nanoflow LC-MS/MS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/695161v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a976ccorg.highwire.dtl.DTLVardef@b6627forg.highwire.dtl.DTLVardef@1fca93eorg.highwire.dtl.DTLVardef@cb61f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Developmentally-specific physiological and metabolic responses support drought resilience in switchgrass and constrains biofuel yield

Switchgrass (Panicum virgatum) is a promising bioenergy crop due in part to its resilience to drought stress. However, the significance of drought timing remains poorly understood, both from a plant biology perspective and its impact on downstream biofuel production. This study determines the developmental stage-specific physiological and metabolic responses of switchgrass to drought stress and its implications for biofuel production using a custom-built programmable irrigation system. Vegetative, flowering, and senescence-stage drought significantly reduced CO2 assimilation, and stomatal conductance without affecting biomass yield. Metabolic profiling revealed significant accumulation of glucose, fructose, quinic acid, shikimate and GABA during vegetative-stage drought, while flowering and senescence stages exhibited limited metabolic changes. Similarly, specialized metabolites also displayed distinct developmental patterns, with vegetative-stage drought driving the most pronounced metabolic alterations. Thermochemically-treated and hydrolyzed switchgrass biomass from vegetative-stage drought showed elevated lignocellulose-derived compounds and saponins with the latter most positively correlating with fermentation lag times. Conversely, senescence-stage drought enhanced ethanol yields while lowering saponin levels in the hydrolysates. While vegetative-stage drought enhanced physiological resilience, it compromises downstream biofuel production by introducing fermentation inhibitors, particularly saponins.

plant biology↗

bioRxiv2023

Heme biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue

With age, people tend to accumulate body fat and reduce energy expenditure1. Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles2,3. The activity of brown and beige depots inversely correlates with BMI and age4-11, suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease12-15. Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function16. Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis17. However, BCAAs contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux18. Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.

cell biology↗

bioRxiv2023

SCD1 and monounsaturated lipids are required for autophagy and survival of adipocytes

Exposure of adipocytes to cool temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (SCD1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological SCD1 inhibition, to investigate further the roles of SCD1 in adipocytes. Our study reveals that production of monounsaturated lipids by SCD1 is necessary for fusion of autophagosomes to lysosomes and that with a SCD1-deficiency, autophagosomes accumulate. In addition, SCD1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of SCD1-deficient adipocytes. Taken together, our results demonstrate that in vitro inhibition of SCD1 in adipocytes leads to autophagy-dependent cell death, and in vivo depletion leads to loss of bone marrow adipocytes.

molecular biology↗

bioRxiv2022

Early Endosome Capture Proteomics and its Application to Amyloid Precursor Protein Intramembrane Processing by β and γ-Secretases

Degradation and recycling of plasma membrane-localized proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. The endolysosomal system also participates in intracellular membrane protein trafficking, signaling, and protein degradation via autophagy. Our understanding of the endolysosomal system has been significantly advanced through the development of methods for selective capture of lysosomes (termed Lyso-IP), but methods for analogous isolation of early intermediates in the endolysosomal system are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of early endosomes in action. In addition to resident endosomal components, we identify numerous recycling, regulatory and membrane fusion complexes as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes that we distinguished from lysosomes via comparative proteomics. The approach allows capture of internalized transferrin within minutes of its addition to cells, indicating the utility of the approach for dynamic analysis of early/sorting endosomes. Among the endocytic cargo identified by Endo-IP was the amyloid precursor protein (APP) genetically linked with Alzheimers disease. Processing of APP to amyloidogenic A{beta} peptides by {beta}- and {gamma}-Secretases can occur within the endolysosomal system among other compartments, but methods for spatial quantification of A{beta} products in individual organelles are lacking. We combined Endo- and Lyso-IP with targeted proteomics to provide a spatial digital snapshot of A{beta} products. This analysis revealed that products of A{beta} processing by {beta}- and {gamma}-Secretases, and alterations in the specificity of cleavage by small molecule {gamma}-Secretase modulators, can be quantified in both early/sorting endosomes and lysosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of the many processes that are coordinated on early endosomes.

cell biology↗

bioRxiv2022

BOS1 is a positive regulator of wounding induced cell death and plant susceptibility to Botrytis

Programmed cell death (PCD) is required for many aspects of plant biology, including stress responses, immunity, and plant development including root and flower development. Our understanding of PCD regulation is incomplete, especially concerning regulators involved in multiple divergent processes. The botrytis-suscetible1 (bos1) mutant is one of the genotypes most susceptible to Botrytis cinerea (Botrytis) and has revealed the role of BOS1 in cell death propagation during plant responses to wounding. The bos1-1 allele harbours a T-DNA located in the 5UTR upstream from the start codon that results in elevated BOS1 transcript levels. Here, we resequenced the bos1-1 genome and found a MAS promoter at the ends of the T-DNAs. Expression of the BOS1 gene under control of the MAS promoter conferred the characteristic bos1-1 Botrytis- sensitivity and wounding phenotypes in wildtype plants. We used Crispr-Cas9 to create new bos1 alleles that disrupt exons. These lines lacked the typical bos1-1 wounding and Botrytis phenotypes, but exhibited reduced fertility, as previously observed in other bos1 T-DNA alleles. With multiple overexpression lines of BOS1, we demonstrate that BOS1 is involved in regulation of cell death propagation in a dosage dependent manner. Our data support that bos1-1 is a gain-of-function mutant and that BOS1 acts as a positive regulator of wounding and Botrytis-induced PCD. Taken together these finding suggest that BOS1 function in both fertility and Botrytis response could be unified under cell death control.

plant biology↗

bioRxiv2021

Convergent evolution of mevalonate pathway in Inonotus obliquus and Betula pendula.

Inonotus obliquus, Chaga mushroom, is a fungal species from Hymenochaetaceae family (Basidiomycota) which has been widely used for traditional medicine in Europe and Asia. Here, chaga genome was sequenced using Pacbio sequencing into a 50.7Mbp assembly consisting of 301 primary contigs with an N50 value of 375 kbp. Genome evolution analyses revealed a lineage-specific whole genome duplication event and an expansion of Cytochrome P450 superfamily. Fungal biosynthetic clusters were enriched for tandemly duplicated genes, suggesting that biosynthetic pathway evolution has proceeded through small-scale duplications. Metabolomic fingerprinting confirmed a highly complex terpene biosynthesis chemistry when compared against related fungal species lacking the genome duplication event.

evolutionary biology↗

bioRxiv2021

A Taphrina strain infecting the model plant Arabidopsis thaliana.

Yeasts are important plant-associated organisms that can modulate host immunity to either promote or prevent disease. Mechanisms of plant-yeast interactions, specifically of yeast perception by the plant innate immune system, remain unknown. Progress has been hindered by the scarcity of yeast species associated with the model plant Arabidopsis thaliana (Arabidopsis). We have previously isolated Taphrina strain M11 from wild Arabidopsis in the field. Taphrina are poorly studied dimorphic yeast-like fungi that are plant pathogens, often producing plant hormones and causing tumour-like and leaf deformation symptoms on their hosts. Here we characterize the interaction of M11 with Arabidopsis. Infection of Arabidopsis with the birch pathogen T. betulina, used as a non-host control, shows early HR, enhanced ROS accumulation, and limitation of growth, demonstrating that Arabidopsis had immunity against non-adapted yeasts. M11 triggered limited cell death, an attenuated ROS response, and grew in planta, as well as subtle but clear leaf deformation symptoms, demonstrating it is pathogenic. Hormone responsive promoter-reporter analysis demonstrated activation of cytokinin signalling during infection. Mutant infection assays indicate jasmonate and ethylene were required for immunity against M11. Analysis of the Taphrina M11 genome was used to mine evidence for yeast specific PAMPs which may underlie host immune responses against yeast-like fungi.

plant biology↗

bioRxiv2021

The isolation and characterization of Taphrina betulina and other yeasts residing in the Betula pendula phylloplane.

The phylloplane is an important microbial habitat and a reservoir of organisms that affect plant health, both positively and negatively. Taphrina betulina is the causative agent of birch witches broom disease. Taphrina are dimorphic, invading theirs hosts in a filamentous form and residing in the host phyllosphere in their non-infectious yeast form. As such, they are widely accepted to be found a resident yeasts on their hosts, even on healthy tissues; however, there is little experimental data to support this. With the aim of exploring the local infection ecology of T. betulina, we had isolated yeasts from the phylloplane of birch, using three classes of samples; from infected symptom bearing leaves inside brooms, healthy leaves from branches away from brooms on broom bearing trees, and symptom-free leaves from symptom-free trees. Isolations yielded 224 yeast strains, representing 11 taxa, including T. betulina, which was the most common isolate and was found in all sample classes, including asymptomatic leaves. Genotyping with two genetic markers revealed genetic diversity among these T. betulina isolates, with seven distinct genotype differentiated by the markers used. Of the 57 T. betulina strains, 22 representative strains were selected for further studies and preliminarily characterized, revealing differences in size and the ability to produced compounds with activity to activate the signalling pathway for the plant hormone auxin.

microbiology↗

bioRxiv2021

Dissecting the subtropical adaptation traits and cuticle synthesis pathways via the genome of the subtropical blueberry Vaccinium darrowii

Vaccinium darrowii is a subtropical wild blueberry species, which was used to breed economically important southern highbush cultivars. The adaptation traits of V. darrowii to subtropical climate would provide valuable information for breeding blueberry and perhaps other plants, especially against the background of global warming. Here, we assembled the V. darrowii genome into 12 pseudochoromosomes using Oxford Nanopore long reads complemented with Hi-C scaffolding technologies, and predicted 41 815 genes using RNAseq evidence. Syntenic analysis across three Vaccinium species revealed a highly conserved genome structure, with the highest collinearity between V. darrowii and V. corymbosum. This conserved genome structure may explain the high fertilization during crossbreeding between V. darrowii and other blueberry cultivars. Gene expansion and tandem duplication analysis indicated possible roles of defense and flowering associated genes in adaptation of V. darrowii to the subtropics. The possible SOC1 genes in V. darrowii were identified with phylogeny and expression analysis. Blueberries are covered in a thick cuticle layer and contain anthocyanins, which confer their powdery blue color. Using RNA-sequencing, the cuticle biosynthesis pathways of Vaccinium species were delineated here in V. darrowii. This result could serve as a reference for breeding berries with customer-desired colors. The V. darrowii reference genome, together with the unique traits of this species, including diploid genome, short vegetative phase, and high compatibility in hybridization with other blueberries, make V. darrowii a potential research model for blueberry species.

plant biology↗

bioRxiv2021

Case study of a rhizosphere microbiome assay on a bamboo rhizome with excessive shoots

Young Moso bamboo shoots are a very popular seasonal food. Bamboo is an important source of income for farmers and the value for cultivation has recently been estimated to $30,000 per hectare. A rare and valuable phenomenon has recently appeared where dozens of adjacent buds within a single Moso bamboo rhizome have grown into shoots. Due to its rarity, this phenomenon, which is of practical importance for the production of edible shoots, has not been scientifically studied. We report the occurrence of a rhizome with 18 shoots, of which the microbiome were analyzed, using rhizomes having one or no shoots as controls. The community of prokaryotes, but not fungi, correlated with the shoot numbers. Burkholderia was the most abundant genus, which negatively correlated with rhizome shoot number, while Clostridia and Ktedonobacteria positively correlated with many shoots. Two Burkholderia strains were isolated and their plant-growth promoting activity was tested. The isolated Burkholderia strains attenuated the growth of bamboo seedlings. Analysis of collected events of enhanced shoot production in China showed no evidence that enhanced shoot development was heritable. Overall, our data provides a firsthand study on excessive shoot development of bamboo.

plant biology↗

bioRxiv2020

The molecular and metabolic program for adaptation of white adipocytesto cool physiologic temperatures

Although visceral adipocytes located within the bodys central core are maintained at ~37{degrees}C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell, and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well-studied; however, comparatively little is known about mechanisms by white adipocytes adapt to temperatures below 37{degrees}C. Here we report that adaptation of cultured adipocytes to 31{degrees}C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, induces extensive changes in gene expression, increased anabolic and catabolic lipid metabolism, and elevated oxygen consumption with reduced reliance on glucose and preferential use of pyruvate, glutamine and fatty acids as energy sources. Cool temperatures up-regulate stearoyl-CoA desaturase-1 expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues, and stearoyl-CoA desaturase-1 activity is required for acquisition of maximal oxygen consumption at 31{degrees}C.

physiology↗

bioRxiv2020

Nano-Emulsion Adjuvant Enhances Cross-presentation by Lipid Body Formation Independent of Glycolysis

Here, we report that a carbomer-based adjuvant, Adjuplex(R) (ADJ), stimulated robust CD8 T-cell responses to subunit antigens by modulating multiple steps in the cytosolic pathway of cross-presentation, and afforded effective immunity against virus and intracellular bacteria. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs and NLRP3-driven caspase 1 activity. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, induction of ROS and lipid bodies (LBs) and alterations in LB composition mediated by ADJ were critical for DC cross-presentation. These findings challenge the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have implications in the rational development of novel adjuvants.

immunology↗

bioRxiv2019

A novel Arabidopsis phyllosphere resident Protomyces sp. and a re-examination of genus Protomyces based on genome sequence data.

Protomyces is a genus of yeast-like fungi that is currently defined as plant pathogens of only the Umbelliferae (Apiaceae) and Compositae (Asteraceae) family plants. Many Protomyces species have been proposed; however, there is a lack of molecular data and available specimens for Protomyces spp., just over ten species are officially accepted and only six species are preserved and available in public culture collections for examination. Phylogenetic relationships and species boundaries within this genus remain largely controversial. Recently, we isolated Protomyces strains from wild Arabidopsis thaliana (Arabidopsis), a Brassicaceae family plant only distantly related to the accepted Protomyces hosts. We have previously sequenced the genomes of all the currently public available Protomyces species, together with a strain (SC29) we isolated from the Arabidopsis phyllosphere. Genome-wide phylogenetic analysis suggests that SC29 occupies a unique phylogenetic position in the genus Protomyces. The SC29 genome has low average nucleotide identity values in comparison with other species genomes. As physiological evidence, SC29 has morphological characteristics and carbon assimilation patterns that distinguish it from the other six Protomyces species. Analysis with several nuclear gene phylogenetic markers further confirms SC29 as a novel Protomyces species and suggests the act1 gene DNA sequences can be used together with ITS sequences for the rapid identification of Protomyces species. In our previous study, SC29 could persist on the Arabidopsis phyllosphere in both short term laboratory and overwinter outdoor garden experiments and Protomyces spp. (or OTUs) were found in the Arabidopsis phyllosphere at multiple sites in both Finland and Germany. We conclude that SC29 is a novel Protomyces species isolated from Arabidopsis and propose the name of Protomyces arabidopsidicola sp. nov. Additionally, the genus Protomyces may not be strictly associated with only Compositae or Umbelliferae family host plants, as evidenced by SC29 and Protomyces strains previously isolated from plants in other families. The merging of two Protomyces species found to have high genomic similarity (P. inouyei and P. lactucaedebilis) is also proposed.

microbiology↗

bioRxiv2019

A novel phyllosphere resident Protomyces species that interacts with the Arabidopsis immune system.

We describe the genome contents of six Protomyces spp. that are pathogenic within the typical host range of the genus and a novel Protomyces strain (SC29) that was previously isolated from the phylloplane of wild Arabidopsis thaliana (Arabidopsis), an atypical or possible alternate host. Genome-wide phylogenetic analysis defined SC29 as a distinct Protomyces sp. Analysis of gene family expansions, gene retention, and gene loss patterns among these Protomyces spp. lead us to hypothesize that SC29 may have undergone a host jump. The role of phyllosphere residency in the lifecycle of Protomyces spp. was previously unknown. Genomic changes in SC29 and all other Protomyces spp. were consistent with adaptations to the plant phylloplane. As predicted by our analysis of its mating locus, SC29 did not cause disease on Arabidopsis as a single strain, but could persist in its phylloplane, while the closely related P. inouyei does not. SC29 treated Arabidopsis exhibited enhanced immunity against Botrytis cinerea infection, associated with activation of MAPK3/6, camalexin, and SA-signalling pathways. We conclude that SC29 is a novel Protomyces sp. able to survive in the Arabidopsis phylloplane and that phylloplane residency is an important element in the lifecycle of Protomyces spp.

microbiology↗

bioRxiv2018

Methyl viologen can affect mitochondrial function in Arabidopsis.

Reactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. The chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in Arabidopsis thaliana, we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies implicate mitochondria in the response to ROS induced by MV.

plant biology↗