Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

The metabolic milieu of metastases.

To colonize the liver, colon cancer metastases must overcome hypoxia and other metabolic stress. Loo et al. now show that metastatic cells achieve this by decreasing miR-483 and miR-551a expression, which derepresses creatine kinase expression and allows energy to be captured from extracellular ATP through generation and import of phosphocreatine.

Wolbachia endosymbionts manipulate the self-renewal and differentiation of germline stem cells to reinforce fertility of their fruit fly host.

The alphaproteobacterium Wolbachia pipientis infects arthropod and nematode species worldwide, making it a key target for host biological control. Wolbachia-driven host reproductive manipulations, such as cytoplasmic incompatibility (CI), are credited for catapulting these intracellular bacteria to high frequencies in host populations. Positive, perhaps mutualistic, reproductive manipulations also increase infection frequencies, but are not well understood. Here, we identify molecular and cellular mechanisms by which Wolbachia influences the molecularly distinct processes of germline stem cell (GSC) self-renewal and differentiation. We demonstrate that wMel infection rescues the fertility of flies lacking the translational regulator mei-P26 and is sufficient to sustain infertile homozygous mei-P26-knockdown stocks indefinitely. Cytology revealed that wMel mitigates the impact of mei-P26 loss through restoring proper pMad, Bam, Sxl, and Orb expression. In Oregon R files with wild-type fertility, wMel infection elevates lifetime egg hatch rates. Exploring these phenotypes through dual-RNAseq quantification of eukaryotic and bacterial transcripts revealed that wMel infection rescues and offsets many gene expression changes induced by mei-P26 loss at the mRNA level. Overall, we show that wMel infection beneficially reinforces host fertility at mRNA, protein, and phenotypic levels, and these mechanisms may promote the emergence of mutualism and the breakdown of host reproductive manipulations.

BubR1- and Polo-coated DNA tethers facilitate poleward segregation of acentric chromatids.

The mechanisms that safeguard cells against chromosomal instability (CIN) are of great interest, as CIN contributes to tumorigenesis. To gain insight into these mechanisms, we studied the behavior of cells entering mitosis with damaged chromosomes. We used the endonuclease I-CreI to generate acentric chromosomes in Drosophila larvae. While I-CreI expression produces acentric chromosomes in the majority of neuronal stem cells, remarkably, it has no effect on adult survival. Our live studies reveal that acentric chromatids segregate efficiently to opposite poles. The acentric chromatid poleward movement is mediated through DNA tethers decorated with BubR1, Polo, INCENP, and Aurora-B. Reduced BubR1 or Polo function results in abnormal segregation of acentric chromatids, a decrease in acentric chromosome tethering, and a great reduction in adult survival. We propose that BubR1 and Polo facilitate the accurate segregation of acentric chromatids by maintaining the integrity of the tethers that connect acentric chromosomes to their centric partners.

Kinetochore-independent mechanisms of sister chromosome separation.

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

m6A RNA methylation facilitates pre-mRNA 3'-end formation and is essential for viability of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3'-boundary of mRNAs. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3'-end processing of pre-mRNAs. Loss of METTL3, WTAP, or YTH1 led to a defect in transcript 3'-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3'-ends.

Genome-wide localization of histone variants in Toxoplasma gondii implicates variant exchange in stage-specific gene expression.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that differentiates from acute tachyzoite stages to latent bradyzoite forms in response to environmental cues that modify the epigenome. We studied the distribution of the histone variants CenH3, H3.3, H2A.X, H2A.Z and H2B.Z, by genome-wide chromatin immunoprecipitation to understand the role of variant histones in developmental transitions of T. gondii parasites. RESULTS: H3.3 and H2A.X were detected in telomere and telomere associated sequences, whereas H3.3, H2A.X and CenH3 were enriched in centromeres. Histones H2A.Z and H2B.Z colocalize with the transcriptional activation mark H3K4me3 in promoter regions surrounding the nucleosome-free region upstream of the transcription start site. The H2B.Z/H2A.Z histone pair also localizes to the gene bodies of genes that are silent but poised for activation, including bradyzoite stage-specific genes. The majority of H2A.X and H2A.Z/H2B.Z loci do not overlap, consistent with variant histones demarcating specific functional regions of chromatin. The extent of enrichment of H2A.Z/H2B.Z (and H3.3 and H2A.X) within the entire gene (5'UTR and gene body) reflects the timing of gene expression during the cell cycle, suggesting that dynamic turnover of H2B.Z/H2A.Z occurs during the tachyzoite cell cycle. Thus, the distribution of the variant histone H2A.Z/H2B.Z dimer defines active and developmentally silenced regions of the T. gondii epigenome including genes that are poised for expression. CONCLUSIONS: Histone variants mark functional regions of parasite genomes with the dynamic placement of the H2A.Z/H2B.Z dimer implicated as an evolutionarily conserved regulator of parasite and eukaryotic differentiation.

Host sensing and signal transduction during Toxoplasma stage conversion.

The intracellular parasite Toxoplasma gondii infects nucleated cells in virtually all warm-blooded vertebrates, including one-third of the human population. While immunocompetent hosts do not typically show symptoms of acute infection, parasites are retained in latent tissue cysts that can be reactivated upon immune suppression, potentially damaging key organ systems. Toxoplasma has a multistage life cycle that is intimately linked to environmental stresses and host signals. As this protozoan pathogen is transmitted between multiple hosts and tissues, it evaluates these external signals to appropriately differentiate into distinct life cycle stages, such as the transition from its replicative stage (tachyzoite) to the latent stage (bradyzoite) that persists as tissue cysts. Additionally, in the gut of its definitive host, felines, Toxoplasma converts into gametocytes that produce infectious oocysts (sporozoites) that are expelled into the environment. In this review, we highlight recent advances that have illuminated the interfaces between Toxoplasma and host and how these interactions control parasite stage conversion. Mechanisms underlying these stage transitions are important targets for therapeutic intervention aimed at thwarting parasite transmission and pathogenesis.

The endosymbiont Wolbachia rebounds following antibiotic treatment.

Antibiotic treatment has emerged as a promising strategy to sterilize and kill filarial nematodes due to their dependence on their endosymbiotic bacteria, Wolbachia. Several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity. However, it remains unclear if antibiotics can permanently deplete Wolbachia and cause sterility for the lifespan of the adult worms. Concerns about resistance arising from mass drug administration necessitate a careful exploration of potential Wolbachia recrudescence. In the present study, we investigated the long-term effects of the FDA-approved antibiotic, rifampicin, in the Brugia pahangi jird model of infection. Initially, rifampicin treatment depleted Wolbachia in adult worms and simultaneously impaired female worm fecundity. However, during an 8-month washout period, Wolbachia titers rebounded and embryogenesis returned to normal. Genome sequence analyses of Wolbachia revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of densely packed Wolbachia within the worm's ovarian tissues were observed by confocal microscopy and remained in worms treated with rifampicin, suggesting that they may serve as privileged sites that allow Wolbachia to persist in worms while treated with antibiotic. To our knowledge, these clusters have not been previously described and may be the source of the Wolbachia rebound.

SPINE20 recommendations 2022: spine care-working together to recover stronger.

PURPOSE: Globally, spine disorders are the leading cause of disability, affecting more than half a billion individuals. However, less than 50% of G20 countries specifically identify spine health within their public policy priorities. Therefore, it is crucial to raise awareness among policy makers of the disabling effect of spine disorders and their impact on the economic welfare of G20 nations. In 2019, SPINE20 was established as the leading advocacy group to bring global attention to spine disorders. METHODS: Recommendations were developed through two Delphi methods with international and multi-professional panels. RESULTS: In 2022, seven recommendations were delivered to the leaders of G20 countries, urging them to: Develop action plans to provide universal access to evidence-based spine care that incorporates the needs of minorities and vulnerable populations. Invest in the development of sustainable human resource capacity, through multisectoral and inter-professional competency-based education and training to promote evidence-based approaches to spine care, and to build an appropriate healthcare working environment that optimizes the delivery of safe health services. Develop policies using the best available evidence to properly manage spine disorders and to prolong functional healthy life expectancy in the era of an aging population. Create a competent workforce and improve the healthcare infrastructure/facilities including equipment to provide evidence-based inter-professional rehabilitation services to patients with spinal cord injury throughout their continuum of care. Build collaborative and innovative translational research capacity within national, regional, and global healthcare systems for state-of-the-art and cost-effective spine care across the healthcare continuum ensuring equality, diversity, and inclusion of all stakeholders. Develop international consensus statements on patient outcomes and how they can be used to define and develop pathways for value-based care. Recognize that intervening on determinants of health including physical activity, nutrition, physical and psychosocial workplace environment, and smoking-free lifestyle can reduce the burden of spine disabilities and improve the health status and wellness of the population. At the third SPINE20 summit 2022 which took place in Bali, Indonesia, in August 2022, 17 associations endorsed its recommendations. CONCLUSION: SPINE20 advocacy efforts focus on developing public policy recommendations to improve the health, welfare, and wellness of all who suffer from spinal pain and disability. We propose that focusing on facilitating access to systems that prioritize value-based care delivered by a competent healthcare workforce will reduce disability and improve the productivity of the G20 nations.

SPINE20 recommendations 2021: spine care for people's health and prosperity.

PURPOSE: The focus of SPINE20 is to develop evidence-based policy recommendations for the G20 countries to work with governments to reduce the burden of spine disease, and disability. METHODS: On September 17-18, 2021, SPINE20 held its annual meeting in Rome, Italy. Prior to the meeting, the SPINE20 created six proposed recommendations. These recommendations were uploaded to the SPINE20 website 10 days before the meeting and opened to the public for comments. The recommendations were discussed at the meeting allowing the participants to object and provide comments. RESULTS: In total, 27 societies endorsed the following recommendations. SPINE20 calls upon the G20 countries: (1) to expand telehealth for the access to spine care, especially in light of the current situation with COVID-19. (2) To adopt value-based interprofessional spine care as an approach to improve patient outcomes and reduce disability. (3) To facilitate access and invest in the development of a competent rehabilitation workforce to reduce the burden of disability related to spine disorders. (4) To adopt a strategy to promote daily physical activity and exercises among the elderly population to maintain an active and independent life with a healthy spine, particularly after COVID-19 pandemic. (5) To engage in capacity building with emerging countries and underserved communities for the benefit of spine patients. (6) To promote strategies to transfer evidence-based advances into patient benefit through effective implementation processes. CONCLUSIONS: SPINE20's initiatives will make governments and decision makers aware of efforts to reduce needless suffering from disabling spine pain through education that can be instituted across the globe.

Green spaces, especially nearby forest, may reduce the SARS-CoV-2 infection rate: A nationwide study in the United States.

The coronavirus pandemic is an ongoing global crisis that has profoundly harmed public health. Although studies found exposure to green spaces can provide multiple health benefits, the relationship between exposure to green spaces and the SARS-CoV-2 infection rate is unclear. This is a critical knowledge gap for research and practice. In this study, we examined the relationship between total green space, seven types of green space, and a year of SARS-CoV-2 infection data across 3,108 counties in the contiguous United States, after controlling for spatial autocorrelation and multiple types of covariates. First, we examined the association between total green space and SARS-CoV-2 infection rate. Next, we examined the association between different types of green space and SARS-CoV-2 infection rate. Then, we examined forest-infection rate association across five time periods and five urbanicity levels. Lastly, we examined the association between infection rate and population-weighted exposure to forest at varying buffer distances (100 m to 4 km). We found that total green space was negative associated with the SARS-CoV-2 infection rate. Furthermore, two forest variables (forest outside park and forest inside park) had the strongest negative association with the infection rate, while open space variables had mixed associations with the infection rate. Forest outside park was more effective than forest inside park. The optimal buffer distances associated with lowest infection rate are within 1,200 m for forest outside park and within 600 m for forest inside park. Altogether, the findings suggest that green spaces, especially nearby forest, may significantly mitigate risk of SARS-CoV-2 infection.

Oxygen Consumption and Blood Pressure Are Not Influenced by Use of a Backpack Hip Strap.

INTRODUCTION: Several studies have explored the effect of backpack carriage on physiologic responses while walking, but few have focused specifically on the influence of the use of a hip strap on these responses. The aim of this study was to investigate the effect of a backpack hip strap on physiologic responses when walking at a moderate intensity while carrying a backpack with a standardized relative load of 30% of the wearer's body mass. METHODS: Twenty-three healthy, active participants carrying backpacks walked on a treadmill at a speed and grade that elicited 40-50% of their heart rate reserve. Participants completed 2 counterbalanced 30-min trials, one with the hip strap in the strapped condition and one with the hip strap unfastened. Metabolic, heart rate, blood pressure, and muscle oxygen saturation (SmO2) responses were recorded during both trials. For each variable, 5-min intervals were averaged at baseline, 5, 10, 15, 20, 25, and 30 min. A repeated measures ANOVA test was used to evaluate the differences between the conditions at each time point. Data reported are the values from the final 5-min interval (30 min) and are reported as mean±SD. RESULTS: No differences were found between strapped and unstrapped trials for oxygen consumption (strapped 21.9±4.2 mL·kg-1·min-1; unstrapped 22.0±4.4 mL·kg-1·min-1, P=0.842), Δmean arterial pressure (strapped +5±17 Δmm Hg; unstrapped +12±14 Δmm Hg, P=0.128) or muscle oxygen saturation of the quadriceps (strapped 86±15%; unstrapped 90±12%, P=0.359) and calf (strapped 73±19%; unstrapped 81±12%, P=0.888). CONCLUSIONS: These results suggest that wearing a hip strap does not influence physiologic responses up to 30 min of moderate intensity walking while carrying 30% of the wearer's mass.

Correction: Wolbachia and host germline components compete for kinesin-mediated transport to the posterior pole of the Drosophila oocyte.

[This corrects the article DOI: 10.1371/journal.ppat.1007216.].

Regulation of arginine transport by GCN2 eIF2 kinase is important for replication of the intracellular parasite Toxoplasma gondii.

Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma. Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma-infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication.

Mechanisms driving acentric chromosome transmission.

The kinetochore-microtubule association is a core, conserved event that drives chromosome transmission during mitosis. Failure to establish this association on even a single chromosome results in aneuploidy leading to cell death or the development of cancer. However, although many chromosomes lacking centromeres, termed acentrics, fail to segregate, studies in a number of systems reveal robust alternative mechanisms that can drive segregation and successful poleward transport of acentrics. In contrast to the canonical mechanism that relies on end-on microtubule attachments to kinetochores, mechanisms of acentric transmission largely fall into three categories: direct attachments to other chromosomes, kinetochore-independent lateral attachments to microtubules, and long-range tether-based attachments. Here, we review these "non-canonical" methods of acentric chromosome transmission. Just as the discovery and exploration of cell cycle checkpoints provided insight into both the origins of cancer and new therapies, identifying mechanisms and structures specifically involved in acentric segregation may have a significant impact on basic and applied cancer research.

SPINE20 A global advocacy group promoting evidence-based spine care of value.

PURPOSE: The Global Burden of Diseases (GBD) Studies have estimated that low back pain is one of the costliest ailments worldwide. Subsequent to GBD publications, leadership of the four largest global spine societies agreed to form SPINE20. This article introduces the concept of SPINE20, the recommendations, and the future of this global advocacy group linked to G20 annual summits. METHODS: The founders of SPINE20 advocacy group coordinated with G20 Saudi Arabia to conduct the SPINE20 summit in 2020. The summit was intended to promote evidence-based recommendations to use the most reliable information from high-level research. Eight areas of importance to mitigate spine disorders were identified through a voting process of the participating societies. Twelve recommendations were discussed and vetted. RESULTS: The areas of immediate concern were "Aging spine," "Future of spine care," "Spinal cord injuries," "Children and adolescent spine," "Spine-related disability," "Spine Educational Standards," "Patient safety," and "Burden on economy." Twelve recommendations were created and endorsed by 31/33 spine societies and 2 journals globally during a vetted process through the SPINE20.org website and during the virtual inaugural meeting November 10-11, 2020 held from the G20 platform. CONCLUSIONS: This is the first time that international spine societies have joined to support actions to mitigate the burden of spine disorders across the globe. SPINE20 seeks to change awareness and treatment of spine pain by supporting local projects that implement value-based practices with healthcare policies that are culturally sensitive based on scientific evidence.

A Multicenter Curricular Intervention to Address Resident Knowledge and Perceptions of Personal Finance.

OBJECTIVES: We evaluated internal medicine residents' confidence and knowledge of personal finance, perceptions of burnout, and relations between these issues before and after an educational intervention. METHODS: We surveyed internal medicine residents at two university-based training programs in 2018. We developed and implemented a curriculum at both sites, covering topics of budgeting, saving for retirement, investment options, and the costs of investing. Each site used the same content but different strategies for dissemination. One used a condensed-form lecture series (two 1-hour sessions) and the other used a microlecture series (four 30-minute sessions) series. Residents were resurveyed following the intervention for comparison. RESULTS: The preintervention survey response rate was 41.2% (122/296) and the postintervention response rate was 44.3% (120/271). Postintervention mean scores for personal finance knowledge improved for basic concepts (52.6% vs 39.4%, P < 0.001), mutual fund elements (30.8% vs 19.7%, P < 0.001), investment plans (68.5% vs. 49.2%, P < 0.001), and overall knowledge (50.1% vs 36.1%, P < 0.001). A significantly smaller proportion of residents reported feelings of burnout following the intervention (23.3% vs 36.9%, P = 0.022). CONCLUSIONS: Our findings show that residents want to learn about finances. Our brief educational intervention is a practical way to improve overall knowledge. Our intervention suggests that improving knowledge of finance may be associated with decreased feelings of burnout.

TgDrpC, an atypical dynamin-related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division.

Dynamin-related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP-2) complex subunit alpha-1 and a homolog of the ezrin-radixin-moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re-distribution of TgDrpC to the IMC during division is dependent on post-Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.

Efficacy of Guanabenz Combination Therapy against Chronic Toxoplasmosis across Multiple Mouse Strains.

Toxoplasma gondii, an obligate intracellular parasite that can cause life-threatening acute disease, differentiates into a quiescent cyst stage to establish lifelong chronic infections in animal hosts, including humans. This tissue cyst reservoir, which can reactivate into an acute infection, is currently refractory to clinically available therapeutics. Recently, we and others have discovered drugs capable of significantly reducing the brain cyst burden in latently infected mice, but not to undetectable levels. In this study, we examined the use of novel combination therapies possessing multiple mechanisms of action in mouse models of latent toxoplasmosis. Our drug regimens included combinations of pyrimethamine, clindamycin, guanabenz, and endochin-like quinolones (ELQs) and were administered to two different mouse strains in an attempt to eradicate brain tissue cysts. We observed mouse strain-dependent effects with these drug treatments: pyrimethamine-guanabenz showed synergistic efficacy in C57BL/6 mice yet did not improve upon guanabenz monotherapy in BALB/c mice. Contrary to promising in vitro results demonstrating toxicity to bradyzoites, we observed an antagonistic effect between guanabenz and ELQ-334 in vivo While we were unable to completely eliminate the brain cyst burden, we found that a combination treatment with ELQ-334 and pyrimethamine impressively reduced the brain cyst burden by 95% in C57BL/6 mice, which approached the limit of detection. These analyses highlight the importance of evaluating anti-infective drugs in multiple mouse strains and will help inform further preclinical studies of cocktail therapies designed to treat chronic toxoplasmosis.