NAPRT silencing in FH-deficient renal cell carcinoma confers therapeutic vulnerabilities via NAD+ depletion.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by loss of function mutations in fumarate hydratase (FH) and results in an aggressive subtype of renal cell carcinoma with limited treatment options. Loss of FH leads to accumulation of fumarate, an oncometabolite that disrupts multiple cellular processes and drives tumor progression. High levels of fumarate inhibit alpha ketoglutarate-dependent dioxygenases, including the ten eleven translocation (TET) enzymes and can lead to global DNA hypermethylation. Here, we report patterns of hypermethylation in FH-mutant cell lines and tumor samples are associated with silencing of nicotinate phosphoribosyl transferase (NAPRT), a rate-limiting enzyme in the Preiss-Handler pathway of NAD+ biosynthesis in a subset of HLRCC cases. NAPRT is hypermethylated at a CpG island in the promoter in cell line models and patient samples, resulting in loss of NAPRT expression. We find that FH-deficient RCC models with loss of NAPRT expression, as well as other oncometabolite-producing cancer models that silence NAPRT, are extremely sensitive to nicotinamide phosphoribosyl transferase inhibitors (NAMPTis). NAPRT silencing was also associated with synergistic tumor cell killing with poly(ADP)-ribose polymerase inhibitors (PARPis) and NAMPTis, which was associated with effects on PAR-mediated DNA repair. Overall, our findings indicate that NAPRT-silencing can be targeted in oncometabolite-producing cancers and elucidates how oncometabolite associated hypermethylation can impact diverse cellular processes and leads to therapeutically relevant vulnerabilities in cancer cells. Implications: NAPRT is a novel biomarker for targeting NAD+ metabolism in FH-deficient HLRCCs with NAMPTis alone and targeting DNA repair processes with the combination of NAMPTis and PARPis.

Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system atrophy.

Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded α-synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in α-synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in α-synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo , providing key evidence to support the hypothesis that α-synuclein adopts unique strains in patients. Here, to further interrogate α-synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate α-synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of α-synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro , demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support α-synuclein misfolding. Overall, our studies use a structure-based approach to investigate α-synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

The ER membrane protein complex restricts mitophagy by controlling BNIP3 turnover.

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. This screen revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system and the ubiquitin-proteosome system regulated BNIP3 independently. Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. More broadly, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that tightly regulate endogenous tail-anchored protein localization.

Impact of a high sensitivity troponin accelerated diagnostic protocol on the safety and emergency department length of stay of chest pain in an academic tertiary hospital: a quality improvement study.

PURPOSE: High-sensitivity troponin (hsTn) accelerated diagnostic protocols are highly recommended for evaluating acute coronary syndromes. Our goal was to improve care for chest pain patients through the safe adoption of an accelerated diagnostic protocol in our academic Emergency Department (ED) with an aim to reduce mean ED length of stay for chest pain patients by 1 h over 1.5 years. Pre-accelerated diagnostic protocol, our mean ED length of stay for chest pain patients was 9.0 h. METHODS: Using the Model for Improvement, we implemented a two-hour accelerated diagnostic protocol and conducted two Plan-Do-Study-Act cycles and education efforts to improve accelerated diagnostic protocol compliance and decrease ED length of stay. Using control charts, we measured the mean monthly ED length of stay for chest pain patients to look for special cause evidence of improvement. Process measures measured compliance with the accelerated diagnostic protocol. Balancing measures included the ED length of stay for abdominal pain patients and the number of admissions and deaths at 7 days for chest pain patients. RESULTS: Mean ED length of stay for chest pain patients decreased from 9.0 to 8.2 h post-accelerated diagnostic protocol. The mean time between troponins decreased from 3.9 to 3.0 h, and the percentage of second troponins repeated at < 2.75 h increased from 22.3% to 58.6%. For abdominal pain patients, ED length of stay decreased from 10.8 to 10.5 h. No chest pain patients died within 7 days pre- or post-accelerated diagnostic protocol. Pre-accelerated diagnostic protocol, 0.84% (41/4,905) were admitted within 7 days. Post-accelerated diagnostic protocol and accelerated diagnostic protocol compliant, 0.70% (13/1,844) were admitted. Post-accelerated diagnostic protocol and accelerated diagnostic protocol non-compliant, 1.1% (13/1,183) were admitted. CONCLUSION: We safely introduced a hsTn accelerated diagnostic protocol in an academic ED. ED length of stay decreased for chest pain patients but did not meet our 1-h goal.

RéSUMé : OBJECTIF : Les protocoles de diagnostic accélérés à haute sensibilité de la troponine (hsTn) sont fortement recommandés pour évaluer les syndromes coronariens aigus. Notre objectif était d'améliorer les soins pour les patients souffrant de douleurs thoraciques grâce à l'adoption en toute sécurité d'un protocole de diagnostic accéléré dans notre service d'urgence universitaire (ED) dans le but de réduire la durée moyenne de séjour des patients souffrant de douleurs thoraciques d'une heure sur 1,5 an. Protocole de diagnostic pré-accéléré, notre durée moyenne de séjour aux urgences pour les patients souffrant de douleurs thoraciques était de 9 heures. MéTHODES: À l'aide du Modèle d'amélioration, nous avons mis en œuvre un protocole de diagnostic accéléré de deux heures et mené deux cycles Plan-Do-Study-Act et des efforts d'éducation pour améliorer la conformité du protocole de diagnostic accéléré et réduire la durée du séjour aux urgences. À l'aide de tableaux de contrôle, nous avons mesuré la durée moyenne mensuelle du séjour aux urgences pour les patients souffrant de douleurs thoraciques afin de rechercher des preuves d'amélioration de cause spéciale. Le processus mesure la conformité au protocole de diagnostic accéléré. Les mesures d'équilibrage comprenaient la durée du séjour aux urgences pour les patients souffrant de douleurs abdominales et le nombre d'admissions et de décès à sept jours pour les patients souffrant de douleurs thoraciques. RéSULTATS: La durée moyenne du séjour aux urgences chez les patients souffrant de douleurs thoraciques a diminué de 9,0 à 8,2 heures après le protocole de diagnostic accéléré. Le temps moyen entre les troponines a diminué de 3,9 à 3,0 heures, et le pourcentage de deuxième troponines répétées à moins de 2,75 heures a augmenté de 22,3 % à 58,6 %. Pour les patients souffrant de douleurs abdominales, la durée du séjour aux urgences a diminué de 10,8 à 10,5 heures. Aucun patient souffrant de douleurs thoraciques n'est décédé dans les sept jours précédant ou suivant le protocole de diagnostic accéléré. Protocole de diagnostic pré-accéléré, 0,84 % (41/4905) ont été admis dans les sept jours. Protocole de diagnostic post-accéléré et protocole de diagnostic accéléré conforme, 0,70% (13/1844) ont été admis. Le protocole diagnostique post-accéléré et le protocole diagnostique accéléré non conforme, 1,1% (13/1,183) ont été admis. CONCLUSION: Nous avons introduit en toute sécurité un protocole de diagnostic accéléré hsTn dans un ED académique. La durée de séjour des patients souffrant de douleurs thoraciques a diminué, mais n'a pas atteint notre objectif d'une heure.

In vitro flow cytometry assay to assess primary human and mouse macrophage phagocytosis of live cells.

Although tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we present a protocol for assessing macrophage engulfment of tumor cells in vitro using flow cytometry. We describe steps for cell preparation, reseeding macrophages, and setting up phagocytosis. We then detail procedures for collecting samples, staining macrophages, and flow cytometry. The protocol is applicable to both mouse bone-marrow-derived macrophages and human monocyte-derived macrophages. For complete details on the use and execution of this protocol, please refer to Roehle et al. (2021).1.

The ER membrane protein complex governs lysosomal turnover of a mitochondrial tail-anchored protein, BNIP3, to restrict mitophagy.

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, violate this assumption. Rather, BNIP3 and NIX are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all of its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. By this approach, we revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system regulates BNIP3 alongside, but independent of, the ubiquitin-proteosome system (UPS). Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. In short, while BNIP3 can be cleared by parallel and partially compensatory quality control pathways, non-autophagic lysosomal degradation of BNIP3 is a strong post-translational modifier of BNIP3 function. More broadly, these data reveal an unanticipated connection between mitophagy and TA protein quality control, wherein the endolysosomal system provides a critical axis for regulating cellular metabolism. Moreover, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that ensure tight regulation of endogenous TA protein localization.

An Analysis of SNAP Online Purchasing Behavior in California: A Review of the First 7 Months of Program Implementation and Lessons Learned.

PURPOSE: The Supplemental Nutrition Assistance Program (SNAP) Online Purchasing Pilot (OPP) allows for the online purchase of groceries using SNAP benefits. First implemented in California in late April 2020, little is known about program usage. This study assessed initial implementation of SNAP Online in California using SNAP OPP transaction data from April - October 2020. Insights can identify usage differences by demographics, store availability, and rurality to help inform future pilot programs and nutrition initiatives. DESIGN: Using generalized estimating equations, we modeled county-level associations between transactions and county-level demographics, rurality, and retailer availability. SETTING: Transaction data from California's Department of Social Services (CDSS) was linked with publicly-available, county-level demographics. SUBJECTS: Anonymized county-level data on SNAP Online transactions and CalFresh households. MEASURES: The primary outcome was successful SNAP Online food transactions per county. ANALYSIS: Generalized estimating equation models with clustering by county was used. RESULTS: During the first 7 months, median SNAP Online transactions per county per month was 665; 2.7% of total SNAP redemptions were from SNAP Online. Counties with more female-led, disabled, Latino, or Asian CalFresh households had fewer Amazon transactions. Each additional Walmart per county corresponded to 260.7 more Walmart transactions (P < .001). Each percent increase in county zip codes covered by Amazon Fresh corresponded to 45.4 fewer Walmart transactions (P < .05) and 37.3 more Amazon transactions (P < .001). CONCLUSION: Number of stores per county was associated with greater online grocery transactions, whereas rurality was not. County-level SNAP demographics correlated with transactions at particular retailers.

Disparities in SNAP online grocery delivery and implementation: Lessons learned from California during the 2020-21 COVID pandemic.

During the COVID-19 pandemic in 2020, the Supplemental Nutrition Assistance Program (SNAP) Online Purchasing Pilot (OPP) was rapidly expanded across the US. This program, enabling direct-to-home grocery delivery, could be a transformative step towards improving fresh-food access. However, lack of information on which areas are serviced by SNAP OPP hinders the identification of potential demographic and regional disparities in access. Lessons from the initial implementation period are critical for understanding continuing inequities and informing the implementation of future programs. In California, SNAP OPP expanded food access for 85.9% of the state's SNAP households in 2020-21. Coverage was significantly greater in urban areas, covering 87.2% of CalFresh households in urban limited food access areas as compared with 29.9% of CalFresh households in rural limited food access areas. County-level COVID-19 rates did not have a meaningful association with SNAP OPP coverage.

Discerning Fact From Fiction: An Assessment of Coronavirus-19 Misinformation Among Patients in Rural Michigan.

Coronavirus-19 misinformation poses a unique challenge for public health communication efforts. In rural communities, COVID-19 misinformation is not well studied. We investigate patients' ability to discriminate COVID-19 fact from fiction from their news sources, as well as general COVID-19 knowledge, perceptions, public health practices, and their primary news sources in 258 adult patients at a primary health clinic in rural Michigan. Most of the population surveyed was able to correctly differentiate reliable COVID-19 public health information from fabricated information. However, only 55.4% of participants reported that they would be somewhat or extremely likely to get a COVID-19 vaccine. The most reported news source was mainstream broadcast television channels such as CBS and ABC. Our data support those older participants are better informed and more likely to practice safe public health practices than younger participants. Based on our data, we offer strategies for public health campaigns in rural communities, such as targeted interventions towards younger people and utilizing local television stations and community institutions to disseminate public health communications and health promotions. Public health interventions beyond education should be considered to mitigate the gap between COVID-19 knowledge and prevention behaviors. Future studies should investigate the role of health care providers in COVID-19 communication with patients, understanding hesitations toward COVID-19 vaccination, and communication strategies to best increase COVID-19 vaccine uptake in rural communities.

Consequences of variability in α-synuclein fibril structure on strain biology.

Synucleinopathies are a group of clinically and neuropathologically distinct protein misfolding diseases caused by unique α-synuclein conformations, or strains. While multiple atomic resolution cryo-electron microscopy structures of α-synuclein fibrils are now deposited in Protein Data Bank, significant gaps in the biological consequences arising from each conformation have yet to be unraveled. Mutations in the α-synuclein gene (SNCA), cofactors, and the solvation environment contribute to the formation and maintenance of each disease-causing strain. This review highlights the impact of each of these factors on α-synuclein misfolding and discusses the implications of the resulting structural variability on therapeutic development.

Inter- and intra-mouse variability in odor preferences revealed in an olfactory multiple-choice test.

Animals choose between sensory stimuli, a highly complex behavior which includes detection, discrimination, preference, and memory processes. Rodents are reported to display robust preferences for some odors, for instance, in the context of choosing among possible mates or food items. In contrast to the apparent robustness of responses toward these and other "ethologically relevant" odors, little is known about the robustness of behaviors toward odors which have no overt role in the rodent ecological niche, so-called "nonethologically relevant" odors. We developed an apparatus for monitoring the nose-poking behavior of mice and used this apparatus to explore the prevalence and stability of choices among different odors both across mice, and within mice over successive days. Mice were tested with a panel of either ethologically relevant or nonethologically relevant odors in an olfactory multiple-choice test. Significant preferences to nonethologically relevant odors were observed across the population of mice, with longer investigation durations to some odors more than to others. However, we found substantial inter-mouse variability in these responses, and that responses to these odors even varied within mice across days of testing. Tests with ethologically relevant odors revealed that responses toward these odors were also variable across mice, but within individual mice, responses were somewhat stable. This work establishes an olfactory multiple-choice test for monitoring odor investigation, choice, and preference behaviors and the application of this apparatus to assess across- and within-mouse odor-preference choice stability. These results highlight that odor preferences, as assayed by measuring choice behaviors, are variable. (PsycINFO Database Record