Biology subjects

Morris, J.

Publications and source records attributed to Morris, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Host lipids regulate multicellular behavior of a predator of a human pathogen

As symbionts of animals, microbial eukaryotes benefit and harm their hosts in myriad ways. A model microeukaryote (Capsaspora owczarzaki) is a symbiont of Biomphalaria glabrata snails and may prevent transmission of parasitic schistosomes from snails to humans. However, it is unclear which host factors determine Capsasporas ability to colonize snails. Here, we discovered that Capsaspora forms multicellular aggregates when exposed to snail hemolymph. We identified a molecular cue for aggregation: a hemolymph-derived phosphatidylcholine, which becomes elevated in schistosome-infected snails. Therefore, Capsaspora aggregation may be a response to the physiological state of its host, and it may determine its ability to colonize snails and exclude parasitic schistosomes. Furthermore, Capsaspora is an evolutionary model organism whose aggregation may be ancestral to animals. This discovery, that a prevalent lipid induces Capsaspora multicellularity, suggests that this aggregation phenotype may be ancient. Additionally, the specific lipid will be a useful tool for further aggregation studies.

biochemistry↗

bioRxiv2024

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 value of 0.14 {+/-} 0.04 {micro}M) that was toxic to trophozoites (EC50 value of 0.21 {+/-} 0.02 {micro}M) while the reported CC50 was >300 {micro}M. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.

biochemistry↗

bioRxiv2024

Diverse GABA signaling in the inner retina enables spatiotemporal coding

Summary paragraphGABA ({psi}-aminobutyric acid) is the primary inhibitory neurotransmitter in the mammalian central nervous system (CNS) 1,2. There is a wide range of GABAergic neuronal types, each of which plays an important role in neural processing and the etiology of neurological disorders 3-5. However, there is no comprehensive understanding of this functional diversity, due to the lack of genetic tools to target and study the multitude of cell types. Here we perform two-photon imaging of GABA release in the inner plexiform layer (IPL) of the mouse retina using the newly developed GABA sensor iGABASnFR2. By applying varied light stimuli to isolated retinae, we reveal over 40 different GABA-releasing neurons, including some not previously described. Individual types show unique distributions of synaptic release sites in the sublayers comprising the IPL, allowing layer-specific visual encoding. Synaptic input and output sites are aligned along specific retinal orientations for multiple neuronal types. Furthermore, computational modeling reveals that the combination of cell type-specific spatial structure and unique release kinetics enables inhibitory neurons to suppress and sculpt excitatory signals in response to a wide range of behaviorally relevant motion structures. Our high-throughput approach provides the first comprehensive physiological characterization of inhibitory signaling in the vertebrate CNS. Future applications of this method will enable interrogation of the function and dysfunction of diverse inhibitory circuits in health and disease.

neuroscience↗

bioRxiv2023

Connectivity Regression

SO_SCPLOWUMMARYC_SCPLOWAssessing how brain functional connectivity networks vary across individuals promises to uncover important scientific questions such as patterns of healthy brain aging through the lifespan or dysconnectivity associated with disease. In this article we introduce a general regression framework, Connectivity Regression (ConnReg), for regressing subject-specific functional connectivity networks on covariates while accounting for within-network inter-edge dependence. ConnReg utilizes a multivariate generalization of Fishers transformation to project network objects into an alternative space where Gaussian assumptions are justified and positive semidefinite constraints are automatically satisfied. Penalized multivariate regression is fit in the transformed space to simultaneously induce sparsity in regression coefficients and in covariance elements, which capture within network inter-edge dependence. We use permutation tests to perform multiplicity-adjusted inference to identify covariates associated with connectivity, and stability selection scores to identify network edges that vary with selected covariates. Simulation studies validate the inferential properties of our proposed method and demonstrate how estimating and accounting for within-network inter-edge dependence leads to more efficient estimation, more powerful inference, and more accurate selection of covariate-dependent network edges. We apply ConnReg to the Human Connectome Project Young Adult study, revealing insights into how connectivity varies with language processing covariates and structural brain features.

neuroscience↗

bioRxiv2023

Increasing hub disruption parallels dementia severity in autosomal dominant Alzheimer disease

Hub regions in the brain, recognized for their roles in ensuring efficient information transfer, are vulnerable to pathological alterations in neurodegenerative conditions, including Alzheimer Disease (AD). Given their essential role in neural communication, disruptions to these hubs have profound implications for overall brain network integrity and functionality. Hub disruption, or targeted impairment of functional connectivity at the hubs, is recognized in AD patients. Computational models paired with evidence from animal experiments hint at a mechanistic explanation, suggesting that these hubs may be preferentially targeted in neurodegeneration, due to their high neuronal activity levels--a phenomenon termed "activity-dependent degeneration". Yet, two critical issues were unresolved. First, past research hasnt definitively shown whether hub regions face a higher likelihood of impairment (targeted attack) compared to other regions or if impairment likelihood is uniformly distributed (random attack). Second, human studies offering support for activity-dependent explanations remain scarce. We applied a refined hub disruption index to determine the presence of targeted attacks in AD. Furthermore, we explored potential evidence for activity-dependent degeneration by evaluating if hub vulnerability is better explained by global connectivity or connectivity variations across functional systems, as well as comparing its timing relative to amyloid beta deposition in the brain. Our unique cohort of participants with autosomal dominant Alzheimer Disease (ADAD) allowed us to probe into the preclinical stages of AD to determine the hub disruption timeline in relation to expected symptom emergence. Our findings reveal a hub disruption pattern in ADAD aligned with targeted attacks, detectable even in pre-clinical stages. Notably, the disruptions severity amplified alongside symptomatic progression. Moreover, since excessive local neuronal activity has been shown to increase amyloid deposition and high connectivity regions show high level of neuronal activity, our observation that hub disruption was primarily tied to regional differences in global connectivity and sequentially followed changes observed in A{beta} PET cortical markers is consistent with the activity-dependent degeneration model. Intriguingly, these disruptions were discernible 8 years before the expected age of symptom onset. Taken together, our findings not only align with the targeted attack on hubs model but also suggest that activity-dependent degeneration might be the cause of hub vulnerability. This deepened understanding could be instrumental in refining diagnostic techniques and developing targeted therapeutic strategies for AD in the future.

neuroscience↗

bioRxiv2023

Inhibition of CERS1 in skeletal muscle exacerbates age-related muscle dysfunction

Age-related muscle wasting and dysfunction render the elderly population vulnerable and incapacitated, while underlying mechanisms are poorly understood. Here, we implicate the CERS1 enzyme of the de novo sphingolipid synthesis pathway in the pathogenesis of age-related skeletal muscle impairment. In humans, CERS1 abundance declines with aging in skeletal muscle cells and, correlates with biological pathways involved in muscle function and myogenesis. Furthermore, CERS1 is upregulated during myogenic differentiation. Pharmacological or genetic inhibition of CERS1 in aged mice blunts myogenesis and deteriorates aged skeletal muscle mass and function, which is associated with the occurrence of morphological features typical of inflammation and fibrosis. Ablation of the CERS1 orthologue lagr-1 in C. elegans similarly exacerbates the age-associated decline in muscle function and integrity. We discover genetic variants reducing CERS1 expression in human skeletal muscle and Mendelian randomization analysis in the UK biobank cohort shows that these variants reduce muscle grip strength and overall health. In summary, our findings link age-related impairments in muscle function to a reduction in CERS1, thereby underlining the importance of the sphingolipid biosynthesis pathway in age-related muscle homeostasis.

physiology↗

bioRxiv2023

Extensive variation in germline de novo mutations in Poecilia reticulata

The rate of germline mutation is fundamental to evolutionary processes, as it generates the variation upon which selection acts. The guppy, Poecilia reticulata, is a model of rapid adaptation, however the relative contribution of standing genetic variation versus de novo mutation to evolution in this species remains unclear. Here, we use pedigree-based approaches to quantify and characterize de novo mutations (DNMs) in three large guppy families. Our results suggest germline mutation rate in the guppy varies substantially across individuals and families. Most DNMs are shared across multiple siblings, suggesting they arose during early zygotic development. DNMs are randomly distributed throughout the genome, and male-biased mutation rate is low, as would be expected from the short guppy generation time. Overall, our study demonstrates remarkable variation in germline mutation rate and provides insights into rapid evolution of guppies.

evolutionary biology↗

bioRxiv2023

Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (under chow and high fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

cell biology↗

bioRxiv2022

LARP1 regulates metabolism and mTORC1 activity in cancer

The protein mammalian target of rapamycin (mTOR) is a master regulator of cell homeostasis. Although mTOR is aberrantly overactivated in 70% ovarian cancers, mTOR cascade inhibitors (such as those blocking the kinase activity of mTOR itself or upstream kinases PI3K/AKT) have demonstrated disappointing activity in ovarian cancer clinical trials. These findings indicate that, despite its pivotal role in normal cells, hyperactivated mTOR does not act as a master regulator of metabolism in this cancer context. Surprisingly, we have identified that the RNA binding protein LARP1, a known phospho-target of mTORC1 and activator of ribosomal biogenesis, is responsible for metabolic reprogramming in mTOR-dysregulated cancers. LARP1 post-transcriptionally regulates the expression of several hundred rate-limiting enzymes involved in multiple aspects of metabolism, including glycolysis and oxidative phosphorylation. Through this mechanism LARP1 sustains ATP production and mTORC1 localisation on the lysosome, thereby activating cell proliferation despite the scarcity of extracellular nutrients. Our findings show that, by sustaining global cellular metabolism in response to growth factor signalling, LARP1 has a central post-transcriptional role in controlling mTORC1 localisation and driving cancer progression, a key cancer hallmark.

cancer biology↗

bioRxiv2022

Multimodal brain age estimates relate to Alzheimer disease biomarkers and cognition in early stages: a cross-sectional observational study

BackgroundEstimates of "brain-predicted age" quantify apparent brain age compared to normative trajectories of neuroimaging features. The brain age gap (BAG) between predicted and chronological age is elevated in symptomatic Alzheimer disease (AD), but has not been well explored in preclinical AD. Prior studies have typically modeled BAG with structural magnetic resonance imaging (MRI), but more recently other modalities, including functional connectivity (FC) and multimodal MRI, have been explored. MethodsWe trained three models to predict age from FC, volumetric (Vol), or multimodal MRI (Vol+FC) in 390 control participants (18-89 years old). In independent samples of 144 older adult controls, 154 preclinical AD participants, and 154 cognitively impaired (CI; CDR > 0) participants, we tested relationships between BAG and AD biomarkers of amyloid, tau, and neurodegeneration, as well as a global cognitive composite. ResultsAll models predicted age in the control training set, with the multimodal model outperforming the unimodal models. All three BAG estimates were significantly elevated in CI compared to controls. FC-BAG and Vol+FC-BAG were marginally reduced in preclinical AD participants compared to controls. In CI participants only, elevated Vol-BAG and Vol+FC-BAG were associated with more advanced AD pathology and lower cognitive performance. ConclusionsBoth FC-BAG and Vol-BAG are elevated in CI participants. However, FC and volumetric MRI also capture complementary signals. Specifically, FC-BAG may capture a unique biphasic response to preclinical AD pathology, while Vol-BAG may capture pathological progression and cognitive decline in the symptomatic stage. A multimodal age-prediction model captures these modality-specific patterns, and further, improves sensitivity to healthy age differences. FundingThis work was supported by the National Institutes of Health (P01-AG026276, P01-AG03991, P30-AG066444, 5-R01-AG052550, 5-R01-AG057680, 1-R01-AG067505, 1S10RR022984-01A1, U19-AG032438), the BrightFocus Foundation (A2022014F), and the Alzheimers Association (SG-20-690363-DIAN).

neuroscience↗

bioRxiv2022

PERK-ATAD3A interaction protects mitochondrial proteins synthesis during ER stress

Widespread repression of protein synthesis rates is a key feature of Endoplasmic Reticulum (ER) stress, mediated by the ER sensor kinase PERK. While select transcripts escape this repression, global translational down-regulation impacts crucial protein levels in all cellular compartments, beyond the ER. How the cell manages this paradox is unclear. PERK has a unique cytoplasmic loop within its kinase domain that binds PERKs target, eIF2. We identified the mitochondrial protein, ATAD3A, as a new interactor of the loop, binding to a highly conserved region within it. During ER stress, increased interaction between ATAD3A and PERK attenuates PERK signalling to eIF2, removing the translational block on several mitochondrial proteins. This occurs at novel context-dependent, mitochondria-ER contact sites. The interaction provides a previously unknown mechanism for fine-tuning translational repression at a local level, mitigating the impact of ER stress on mitochondria. Further, it represents a new target for selective modulation of PERK-eIF2 signalling in diseases from cancer to neurodegeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/501280v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@f74252org.highwire.dtl.DTLVardef@1234633org.highwire.dtl.DTLVardef@d5dc9aorg.highwire.dtl.DTLVardef@8606fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Heavy metal pollution impacts soil bacterial community structure and antimicrobial resistance at the Birmingham 35th Avenue Superfund Site

Heavy metals (HMs) are known to modify bacterial communities both in the laboratory and in situ. As a consequence, soils in HM contaminated sites like the U.S. Environmental Protection Agency (EPA) Superfund sites are predicted to have altered ecosystem functioning, with potential ramifications for the health of organisms, including humans, that live nearby. Further, several studies have shown that metal tolerant bacteria are often also resistant to antimicrobial agents (AMR), and therefore HM contaminated soils could potentially act as reservoirs that could disseminate AMR genes into human-associated pathogenic bacteria. To explore this possibility soil samples were collected from six public locations in the zip code 35207 (the home of the North Birmingham 35th Avenue Superfund site) and in six public areas in a neighboring reference zip code (35214). Sequencing of the V4 region of the bacterial 16S rRNA gene revealed that elevated concentrations of HMs As, Mn, Pb, and Zn reduced microbial diversity and altered community structure within each zip code. While there was no difference between zip codes in the proportion of total culturable microbes that survived antimicrobial or metal exposure, bacterial isolates with HMR almost always also exhibited AMR. Metagenomes inferred using PICRUST2 also predicted significantly higher mean relative frequencies in 35207 for several AMR genes related to both specific and broad-spectrum AMR phenotypes. Together, these results support the hypothesis that chronic HM pollution alters soil bacterial community structure in ecologically meaningful ways and may also select for bacteria with increased potential to contribute to AMR in human bacterial disease.

microbiology↗

bioRxiv2022

Pan-cancer proteomic map of 949 human cell lines reveals principles of cancer vulnerabilities

The proteome provides unique insights into biology and disease beyond the genome and transcriptome. Lack of large proteomic datasets has restricted identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types were analyzed by mass spectrometry. Deploying a clinically-relevant workflow to quantify 8,498 proteins, these data capture evidence of cell type and post-transcriptional modifications. Integrating multi-omics, drug response and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline revealed thousands of protein-specific biomarkers of cancer vulnerabilities. Proteomic data had greater power to predict drug response than the equivalent portion of the transcriptome. Further, random downsampling to only 1,500 proteins had limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger), available at https://cellmodelpassports.sanger.ac.uk, is a comprehensive resource revealing principles of protein regulation with important implications for future clinical studies.

systems biology↗

bioRxiv2021

IDENTIFYING PLANT GENES SHAPING MICROBIOTA COMPOSITION IN THE BARLEY RHIZOSPHERE

A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here we use metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identify a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS, emerges as a major determinant of microbiota composition. We subject soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allows us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide insights into the footprint of crop improvement on the plants capacity of shaping rhizosphere microbes.

plant biology↗

bioRxiv2021

Programmable Cleavage of Double-stranded DNA by Combined Action of Argonaute CbAgo from Clostridium butyricum and Nuclease Deficient RecBC Helicase from E.coli

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Discovery of mesophilic Argonautes active at physiological temperature places pAgos closer to their possible application for genome editing as a simpler alternative to CRISPR/Cas nucleases. Currently, the use of mesophilic pAgos as programmable DNA endonucleases is hampered by their poor action on double-stranded DNA (dsDNA), mainly due to their inability to invade the DNA duplex. The present study demonstrates that efficient in vitro cleavage of double-stranded DNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of complementary DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.3-25 kb DNA substrates. When combined with RecBexo-C helicase, CbAgo was capable of cleaving target sequences located 11-12.5 kb from the ends of linear dsDNA at 37{o}C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate dsDNA fragments flanked with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. At present, the combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of dsDNA at any desired location.

molecular biology↗

bioRxiv2021

Microgravity effect on murine T cells exposed to suborbital flight aboard Blue Origins New Shepard vehicle

Numerous scientific experiments have been conducted in space. However, the precise mechanisms mediating successful human body adaption to the hostile space environment are still not delineated. The cost and logistic challenges of sending biological payloads to International Space Station are forcing scientists to find alternative research platforms. In this study, we investigated whether brief exposure to microgravity during the suborbital flight aboard Blue Origins New Shepard rocket modulated the behavior of the gravity-sensitive murine T cells. We assessed the effect of suborbital environment on different T cell subsets, activation markers, functionality, and cytokine secretion capabilities. Thus, to optimize the potential response of T cells, we cultured them in interleukin IL-2 alone or combined with IL-12. We found that exposure to microgravity decreased the expression of T cells with CD4+ cells being more sensitive to suborbital flight as compared to CD8+ cells. Our data indicate that the functional capabilities of flown T cells were reduced. Also, our findings suggest that supplementing cells with IL-2 and IL-12 cytokines may restore microgravity-mediated cellular alterations. Finally, our study provides insights on the microgravity effect on the murine T cells by utilizing a novel suborbital research platform.

cell biology↗

bioRxiv2020

The indole-alkaloid gramine shapes the bacterial communities thriving at the barley root-soil interface

Microbial communities proliferating at the root-soil interface, collectively referred to as the rhizosphere microbiota, represent an untapped beneficial resource for plant growth, development and health. Integral to a rational manipulation of the microbiota for sustainable agriculture is the identification of the molecular determinants of these communities. In plants, biosynthesis of allelochemicals is centre stage in defining inter-organismal relationships in the environment. Intriguingly, this process has been moulded by domestication and breeding selection. The indole-alkaloid gramine, whose occurrence in barley (Hordeum vulgare L.) is widespread among wild genotypes but has been counter selected in several modern varieties, is a paradigmatic example of this phenomenon. This prompted us to investigate how exogenous applications of gramine impacted on the rhizosphere microbiota of two, gramine-free, elite barley varieties grown in a reference agricultural soil. High throughput 16S rRNA gene amplicon sequencing revealed that applications of gramine interfere with the proliferation of a subset of soil microbes with a relatively broad phylogenetic assignment. Strikingly, growth of these bacteria appeared to be rescued by barley plants in a genotype- and dosage-independent manner. In parallel, we discovered that host recruitment cues can interfere with the impact of gramine application in a host genotype-dependent manner. Interestingly, this latter effect displayed a bias for members of the phyla Proteobacteria. These initial observations indicate that gramine can act as a determinant of the prokaryotic communities inhabiting the root-soil interface.

plant 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↗