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Myler, P. J.

Publications and source records attributed to Myler, P. J..

17 recordsLinked to original sources
bioRxiv2026

Relatedness of Gardnerella prophages and their endolysins

The Gardnerella genus includes a complex sub-taxonomy of bacterial species and subgroups which have been implicated in bacterial vaginosis. We analyzed G. leopoldii, G. swidsinski, G. piotii, and G. vaginalis genomes available in public databases and predicted proviral sequences using Cenote-Taker3. We clustered 266 prophage sequences into 140 viral genera level groupings that encode four distinct gene clusters and 19 different lysin proteins. Analysis of the bacterial and prophage sequence phylogenies reveals that while there were no specific prophage sequence lineages that infected each bacterial host, there is clustering of the prophage sequences by bacterial species within the Gardnerella genus.

microbiology↗
bioRxiv2026

Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and crystal structures of TvPPi-PFK are reported. TvPPi-PFK is the first reported eukaryotic PPi-PFK structure. TvPPi-PFK retains the overall PPi-PFK topology observed in bacterial PPi-PFK including conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. In addition to the catalytic PPi-PFK binding sites, TvPPi-PFK has two additional ligand binding sites. The first binds AMP usurped during protein production and helps stabilize the TvPPi-PFK tetramer. A second ligand binding site was observed in proximity to the AMP-binding site and accommodates sugar phosphates soaked into preformed crystals. This sugar phosphates binding site is distinct from the TvPPi-PFK active site that binds fructose-6-phosphate. Future mutagenesis and activity studies are planned to determine the relevance of both sites. SynopsisThe production, crystallization, and crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis (TvPPi-PFK) are reported. TvPPi-PFK has a prototypical PPi-PFK active site as well as unexpected AMP and sugar-phosphate binding sites at the dimer interface.

biochemistry↗
bioRxiv2026

Automated extraction and optimization of protein purification protocols using multi-agent large language models

Recent advances in Large Language Models (LLMs) present new opportunities for automating critical bottlenecks in scientific workflows such as literature reviews or protocol design. One such bottleneck is the purification of recombinant proteins, a vital aspect of biomedical research that frequently fails. To improve success rates, researchers must manually define optimal large-scale purification conditions and establish robust rescue protocols for proteins with low stability or solubility - a time-intensive process. To address this gap, we introduce a multi-agent LLM system that automates the creation and optimization of protein purification protocols to facilitate the production of high-concentration, high-purity protein samples. Our application streamlines the labor-intensive manual process of sequence similarity searches, literature reviews, and protocol comparison. Operating in a tool-like constrained workflow, the system identifies analogous proteins, leverages specialized LLM agents to extract successful purification methodologies from primary source literature, and cross-references them against failed protocols to generate optimization recommendations. Evaluation on a select number of targets demonstrated high accuracy in protocol extraction and the generation of scientifically sound, expert-validated optimization recommendations. While this system reduces complex analysis time from hours to minutes, we identify the lack of programmatic open access to literature, specifically primary citations in the Protein Data Bank, as a fundamental limitation to LLM agent-based scientific workflows. Ultimately, this system demonstrates the feasibility of using LLM agents to streamline wet-lab workflows while preserving methodological transparency and reproducibility.

bioinformatics↗
bioRxiv2026

Structural Basis of Glycolytic Control in Trypanosoma cruzi: Insights from Enolase and PGI

Trypanosoma cruzi, the etiological agent of Chagas disease, depends on glycolysis for ATP production, rendering its glycolytic enzymes attractive targets for therapeutic development. Here, we report the high-resolution crystal structures of two essential glycolytic enzymes, glucose-6-phosphate isomerase (Tc PGI, 1.8 [A]) and enolase (Tc enolase, 2.4 [A]) and provide structural and computational analyses to support structure-based drug design. Tc PGI adopts a dimeric {beta} sandwich fold and features a parasite-specific 53-residue N-terminal extension and a unique C-terminal hook region which both distinguish it from its human ortholog. Tc enolase exhibits the conserved (/{beta}) 8 TIM barrel fold but harbors minor distinct structural deviations, including an extended 17 helix and a structured 1 region, which differentiate it from human isoforms. Both enzymes exhibited high thermal stability, consistent with adaptation to the parasites complex life cycle. Structure-based virtual screening using a scaffold with known multi-target potential identified distinct high-affinity inhibitors for each enzyme. Molecular dynamics simulations further confirmed stable enzyme-inhibitor interactions and favorable binding energetics. Collectively, these findings reveal structural signatures unique to T. cruzi glycolytic enzymes and lay the groundwork for the development of selective antiparasitic therapeutics.

biochemistry↗
bioRxiv2023

Identification of potent and selective N-myristoyltransferase inhibitors of Plasmodium vivax liver stage hypnozoites and schizonts

New drugs targeting multiple stages of the malaria-causing parasite, Plasmodium, are needed to reduce and eliminate malaria worldwide. N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme, and a validated chemically tractable drug target for malaria. Previous efforts have failed to target NMT owing to the low selectivity for the Plasmodium enzyme compared with human NMTs. Herein, we applied a structure-guided approach using previously reported NMT inhibitors as scaffolds to develop a new generation of Plasmodium vivax NMT (PvNMT) targeting compounds. We report a series of compounds with IC50 values in the nM range and an order of magnitude improved selectivity to Plasmodium NMT over human NMT (HsNMT). X-ray co-crystallization of PvNMT with a representative lead compound, 12b, supported the prevailing hypothesis that a conformational difference in a key tyrosine residue of PvNMT and HsNMT drives the selectivity between these enzymes. The compounds were triaged based on their selectivity for PvNMT. They significantly decreased P. falciparum blood-stage parasite load, with IC50 values ranging from 0.36 M to 1.25 M. The compounds exhibited a dose-dependent inhibition of P. vivax liver stage schizont and hypnozoite infection, consistently, in three different P. vivax isolates with IC50 values ranging from 2.2 M to 6 M and from 1.2 M to 12 M. Our data provide evidence that NMT inhibitors could be multistage antimalarials, targeting both dormant and developing liver stage parasites, which is essential for malaria elimination. One Sentence SummaryPotent and selective N-myristoyltransferase inhibitors of Plasmodium vivax hypnozoites and schizonts were synthesized and tested.

pharmacology and toxicology↗
bioRxiv2022

A benchmark of Hi-C scaffolders using reference genomes and de novo assemblies

BackgroundStudying a new species using high-throughput sequencing requires a high-quality reference genome. However, assembling chromosome length sequences remains challenging. Recent advances in chromatin conformation capture (Hi-C) have provided a new approach to scaffolding genome assemblies, and the last ten years have seen a proliferation of such methods. However, to our knowledge no comprehensive benchmarking of Hi-C scaffolders has been conducted to date. ResultsThrough a literature review we identified the most popular Hi-C scaffolders - Lachesis, HiRise, 3d-dna, SALSA, and AllHiC. We tested their ability to scaffold four well studied genomes - S. cerevisiae, L. tarentolae, A. thaliana, and H. sapiens. Scaffolders were tasked with both scaffolding fragmented versions of the reference genome as well as de novo assemblies derived from long read datasets. We found that all scaffolders can exceed 80% accuracy under ideal circumstances but that their performance quickly deteriorates under more challenging conditions. Surprisingly, many scaffolders also showed poor performance on the best assemblies, where contigs are near chromosome length. Overall, we found that HiRise and Lachesis offer the best performance on average across all conditions. ConclusionsWe compare the performance of five Hi-C scaffolders using multiple reference species under both ideal and real-life conditions, thereby illuminating their strengths and weaknesses.

genomics↗
bioRxiv2022

Edison: measuring scaffolding accuracy with edit distance

MotivationThe quality of reference genomes critically affects analyses of next generation sequencing experiments. During the construction of the reference genome, contigs are organized into their underlying chromosomes in the scaffolding step. Historically, the quality of scaffolding software has been difficult to evaluate in a systematic and quantitative fashion. To this end, we identified genomic edit distance as a compelling method for evaluating the quality of a scaffold. ResultsWe present Edison, a Python implementation of the Double Cut and Join (DCJ) edit distance algorithm. Edison calculates the overall accuracy of a given scaffold using a reference genome and also provides scores for characterizing different aspects of the scaffolding accuracy, including grouping, ordering, and orientation. All metrics are calculated on a length-weighted basis, which rewards the correct placement of longer contigs over shorter ones. By creating 1000 random assemblies of the S. cerevisiae genome, we show that our scaffolding accuracy provides a more reliable metric than the commonly used metric, N50. Edison can be used to benchmark new scaffolding algorithms, providing insights into the strengths and weaknesses of each approach. Availability and ImplementationEdison is available under an MIT license at https://github.com/Noble-Lab/edison.

genomics↗
bioRxiv2022

Regulation of multiple dimeric states of E-cadherin by adhesion activating antibodies revealed through Cryo-EM and X-ray crystallography

E-cadherin adhesion is regulated at the cell surface, a process that can be replicated by activating antibodies. We use cryo-EM and X-ray crystallography to examine functional states of the cadherin adhesive dimer. This dimer is mediated by N-terminal beta strand-swapping involving Trp2, and forms via a different transient X-dimer intermediate. X-dimers are observed in cryo-EM along with monomers and strand-swap dimers, indicating that X-dimers form stable interactions. A novel EC4-mediated dimer was also observed. Activating Fab binding caused no gross structural changes in E- cadherin monomers but can facilitate strand swapping. Moreover, activating Fab binding is incompatible with the formation of the X-dimer. Both cryo-EM and X-ray crystallography reveal a distinctive twisted strand-swap dimer conformation caused by an outward shift in the N-terminal beta strand that may represent a strengthened state. Thus, regulation of adhesion involves changes in cadherin dimer configurations.

biochemistry↗
bioRxiv2022

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using x-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif - the strand-swap dimer interface. Molecular dynamics simulations and single molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes, one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped {beta}-strand and its complimentary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

biophysics↗
bioRxiv2021

Genome instability drives epistatic adaptation in the human pathogen Leishmania

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches we provide first evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover post-transcriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in snoRNA gene dosage with changes in rRNA 2-O-methylation and pseudouridylation, suggesting translational control is an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression, and genome-independent, compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery, and may be relevant to other fast evolving, eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer. One Sentence SummaryEpistatic interactions harness genome instability for Leishmania fitness gain.

genomics↗
bioRxiv2021

A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesis

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote development into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastea. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of PKAR3 or PKAC3 null mutants lose their elongated shape and are round but remain flagellated. Truncation of an N-terminal formin homology-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 kinase activity via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARI{beta} and PKARII{beta} binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 does not bind cAMP but nucleoside analogs with a very high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape via PKA activity.

cell biology↗
bioRxiv2020

Naegleria fowleri: protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brains frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogens high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. N. fowleri genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

molecular biology↗
bioRxiv2020

The transcriptome of Balamuthia mandrillaris trophozoites for structure-based drug design

Balamuthia mandrillaris, a pathogenic free-living amoeba (FLA), causes cutaneous skin lesions as well as the brain-eating disease: Balamuthia granulomatous amoebic encephalitis (GAE). These diseases, and diseases caused by other pathogenic FLA, Naegleria fowleri or Acanthamoeba species, are minimally studied from a drug discovery perspective; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Chemotherapies for CNS disease caused by B. mandrillaris require vast improvement. Current therapeutics are limited to a small number of drugs that were previously discovered in the last century through in vitro testing or identified after use in the small pool of surviving reports. Using our recently published methodology to identify potentially useful therapeutics, we screened a collection of 85 compounds that have previously been reported to have antiparasitic activity. We identified 59 compounds that impacted growth at concentrations below 220 M. Since there is no fully annotated genome or proteome, we used RNA-Seq to reconstruct the transcriptome of B. mandrillaris and locate the coding sequences of the specific genes potentially targeted by the compounds identified to inhibit trophozoite growth. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing.

genomics↗
bioRxiv2020

Chromatin-associated protein complexes link DNA base J and transcription termination in Leishmania

Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression; relying instead on post-transcriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base ({beta}-D-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation sites of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP-tagging) and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatic analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein (JBP3), which is also part of another complex containing proteins with domains suggestive of a role in chromatin modification/remodeling. Additionally, JBP3 associates (albeit transiently and/or indirectly) with the trypanosomatid equivalent of the PAF1 complex involved in regulation of transcription in other eukaryotes. Down-regulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional read-through at the 3 end of most PTUs. We propose that JBP3 recruits one or more of these complexes to the J-containing regions at the end of PTUs, where they halt progression of the RNA polymerase. This de-coupling of transcription termination from splicing of individual genes enables the parasites unique reliance on polycistronic transcription and post-transcriptional regulation of gene expression. ImportanceLeishmania parasites cause a variety of serious human diseases, with no effective vaccine and emerging resistance to current drug therapy. We have previously shown that a novel DNA base called J is critical for transcription termination at the ends of the polycistronic gene clusters that are a hallmark of Leishmania and related trypanosomatids. Here, we describe a new J-binding protein (JBP3) associated with three different protein complexes that are reminiscent to those involved in control of transcription in other eukaryotes. However, the parasite complexes have been reprogrammed to regulate transcription and gene expression in trypanosomatids differently than in the mammalian hosts, providing new opportunities to develop novel chemotherapeutic treatments against these important pathogens.

molecular biology↗
bioRxiv2020

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).

bioinformatics↗
bioRxiv2019

The hunger games: sensing host arginine is essential for Leishmania parasite virulence

Arginine homeostasis in lysosomes is critical for growth and metabolism of mammalian cells. They employ a specific sensor (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORC1 activity. Lysosomes of macrophages (phagolysosomes) are the niche where the parasitic protozoan Leishmania resides and causes important human disease. Several years ago, we discovered that upon arginine starvation, cultured Leishmania parasites promptly activate a MAPK2-mediated Arginine Deprivation Response (ADR) pathway, resulting in up-regulation of the Leishmania arginine transporter (AAP3), as well as a small group of other transporters. Significantly, ADR is also activated during macrophage infection, implying that the intracellular parasite actively depletes arginine within the host phagolysosome, likely to prevent mTORC1 activation and enhance intracellular development. We hypothesize that ADR-mediated up-regulation of AAP3 activity is necessary to withstand the resultant arginine starvation. Both copies of the AAP3 genes are located (in tandem) on a tetrasomic chromosome (chr31), but only one (AAP3.2) is responsive to arginine deprivation. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport (mediated by AAP3.1), but lack a functional copy of AAP3.2 and are therefore not responsive to arginine starvation. While these mutants grow normally in culture as promastigotes, they were impaired in their ability to develop inside THP1 macrophages grown under physiological concentrations of arginine (0.1 mM). However, flooding the macrophage growth medium with arginine (1.5 mM) restored parasite infectivity and intracellular growth to that of wild type. The results indicate that inside the host macrophage, Leishmania must overcome the arginine \"Hunger Games\" by up-regulating transport of arginine via the ADR. Furthermore, the AAP3.2 mutants were ~70-80% less virulent in Balb/C mice, showing, for the first time, that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.

cell biology↗