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Malaria is a highly oxidative parasitic disease in which anemia is the most common clinical symptom. A major contributor to malarial anemia pathogenesis is the destruction of bystander, uninfected red blood cells. Metabolic fluctuations are known to occur in the plasma of individuals with acute malaria, emphasizing the role of metabolic changes in disease progression and severity. Here, we report that conditioned media fromPlasmodium falciparumculture induces oxidative stress in healthy uninfected RBCs. Additionally, we show the benefit of amino acid pre-exposure for RBCs and how this pre-treatment intrinsically prepares RBCs to mitigate oxidative stress.Key pointsIntracellular ROS is acquired in red blood cells incubated withPlasmodium falciparumconditioned mediaGlutamine, cysteine, and glycine amino acid supplementation increased glutathione biosynthesis and reduced ROS levels in stressed RBCs

Alpha/beta hydrolase domain-containing protein 4 (ABHD4) catalyzes the deacylation ofN-acyl phosphatidyl-ethanolamine (NAPE) and Lyso-NAPE to produce Glycerophospho-N-acyl ethanolamine (GP-NAE). Through a variety of metabolic enzymes, NAPE, Lyso-NAPE, and GP-NAE are ultimately converted into NAE, a group of bioactive lipids that control many physiological processes including inflammation, cognition, and food intake (i.e., oleoylethanolamide or OEA). In a diet-induced obese mouse model, adipose ABHD4 gene expression positively correlated with adiposity. However, it is unknown whether ABHD4 is a causal or a reactive gene to obesity. To fill this knowledge gap, we generated an ABHD4 knockout (KO) 3T3-L1 pre-adipocyte. During adipogenic stimulation, ABHD4 KO pre-adipocytes had increased adipogenesis and lipid accumulation, suggesting ABHD4 is responding to (a reactive gene), not contributing to (not a causal gene), adiposity and may serve as a mechanism for protecting against obesity. However, we did not observe any differences in adiposity and metabolic outcomes between adipocyte specific ABHD4 KO mice or whole body ABHD4 KO mice and their littermate control mice on chow or a high fat diet. This might be because we found that deletion of ABHD4 did not affect NAE such as OEA production, even though ABHD4 was highly expressed in adipose tissue and correlated with fasting adipose OEA levels and lipolysis. These data suggest that ABHD4 plays a role in adipocyte differentiation in vitro but not in adipose tissue lipid metabolism in mice despite nutrient overload, possibly due to compensation from other NAPE and NAE metabolic enzymes.

Despite the rapid development of virtual medical Spanish educational materials, online resources lack transparency and a peer-review process. The purpose of this interdisciplinary study was to provide a critical inventory of virtual resources for medical Spanish education, thereby providing a panorama of the current state of online medical Spanish.Research team members conducted iterative searches to identify medical Spanish online resources, which were then screened for predetermined inclusion/exclusion criteria. Between June and August 2020, a panel of medical and language experts then adapted and applied a previously published evaluation tool to determine whether resources that met study criteria would help learners achieve medical Spanish core competencies and to what extent each resource incorporated communicative language activities. Consensus meetings were conducted to resolve disagreements and identify gaps in online education.Out of 465 resources, 127 were further screened, and eight were selected for evaluation. Medical and language specialists independently scored each resource and, following discussions, achieved consensus. Overall, no resource met suitability criteria for all five medical Spanish learner competencies or cultural elements, and only one was suitable for achieving the self-assessment competency.Interdisciplinary consensus meetings provide an important avenue for resolving differences of opinion and for integrating both language and medical perspectives into the evaluation process. Existing online resources should be used in conjunction with other materials to ensure that all core competencies for medical Spanish education are addressed. This study revealed important gaps in online resources, including a need to target advanced Spanish learners, apply authentic communicative activities, include assessment opportunities, and integrate culture in the learning program. Based on the current state of online medical Spanish, we offer recommendations for future resources.

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells., Changes in intracellular GTP levels are not considered as a regulatory event in RAC1 activation in live cells since total GTP levels are substantially higher than the RAC1 GTP dissociation constant determined in vitro. Here, the authors demonstrate that the availability of free GTP in live cells controls the activity of RAC1 and cell invasion.

The goal of this study is to identify pharmacological inhibitors that target a recently identified novel mediator of breast cancer brain metastasis (BCBM), truncated glioma-associated oncogene homolog 1 (tGLI1). Inhibitors of tGLI1 are not yet available. To identify compounds that selectively kill tGLI1-expressing breast cancer, we screened 1527 compounds using two sets of isogenic breast cancer and brain-tropic breast cancer cell lines engineered to stably express the control, GLI1, or tGLI1 vector, and identified the FDA-approved antifungal ketoconazole (KCZ) to selectively target tGLI1-positive breast cancer cells and breast cancer stem cells, but not tGLI1-negative breast cancer and normal cells. KCZ’s effects are dependent on tGLI1. Two experimental mouse metastasis studies have demonstrated that systemic KCZ administration prevented the preferential brain metastasis of tGLI1-positive breast cancer and suppressed the progression of established tGLI1-positive BCBM without liver toxicities. We further developed six KCZ derivatives, two of which (KCZ-5 and KCZ-7) retained tGLI1-selectivity in vitro. KCZ-7 exhibited higher blood–brain barrier penetration than KCZ/KCZ-5 and more effectively reduced the BCBM frequency. In contrast, itraconazole, another FDA-approved antifungal, failed to suppress BCBM. The mechanistic studies suggest that KCZ and KCZ-7 inhibit tGLI1’s ability to bind to DNA, activate its target stemness genes Nanog and OCT4, and promote tumor proliferation and angiogenesis. Our study establishes the rationale for using KCZ and KCZ-7 for treating and preventing BCBM and identifies their mechanism of action.