N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.

In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.

Barriers and facilitators to implementation and sustainment of guideline-recommended depression screening for patients with breast cancer in medical oncology: a qualitative study.

OBJECTIVES: Implementation of guideline-recommended depression screening in oncology presents numerous challenges. Implementation strategies that are responsive to local context may be critical elements of adoption and sustainment. We evaluated barriers and facilitators to implementation of a depression screening program for breast cancer patients in a community medical oncology setting as part of a cluster randomized controlled trial. METHODS: Guided by the Consolidated Framework for Implementation Research, we employed qualitative methods to evaluate clinician, administrator, and patient perceptions of the program using semi-structured interviews. We used a team-coding approach for the data; thematic development focused on barriers and facilitators to implementation using a grounded theory approach. The codebook was refined through open discussions of subjectivity and unintentional bias, coding, and memo applications (including emergent coding), and the hierarchical structure and relationships of themes. RESULTS: We conducted 20 interviews with 11 clinicians/administrators and 9 patients. Five major themes emerged: (1) gradual acceptance and support of the intervention and workflow; (2) compatibility with system and personal norms and goals; (3) reinforcement of the value of and need for adaptability; (4) self-efficacy within the nursing team; and (5) importance of identifying accountable front-line staff beyond leadership "champions." CONCLUSIONS: Findings suggest a high degree of acceptability and feasibility due to the selection of appropriate implementation strategies, alignment of norms and goals, and a high degree of workflow adaptability. These findings will be uniquely helpful in generating actionable, real-world knowledge to inform the design, implementation, and sustainment of guideline-recommended depression screening programs in oncology. TRIAL REGISTRATION: ClinicalTrials.gov #NCT02941614.

Effect of a Community-Based Medical Oncology Depression Screening Program on Behavioral Health Referrals Among Patients With Breast Cancer: A Randomized Clinical Trial.

Importance: Implementation of guideline-recommended depression screening in medical oncology remains challenging. Evidence suggests that multicomponent care pathways with algorithm-based referral and management are effective, yet implementation of sustainable programs remains limited and implementation-science guided approaches are understudied. Objective: To evaluate the effectiveness of an implementation-strategy guided depression screening program for patients with breast cancer in a community setting. Design, Setting, and Participants: A pragmatic cluster randomized clinical trial conducted within Kaiser Permanente Southern California (KPSC). The trial included 6 medical centers and 1436 patients diagnosed with new primary breast cancer who had a consultation with medical oncology between October 1, 2017, through September 30, 2018. Patients were followed up through study end date of May 31, 2019. Interventions: Six medical centers in Southern California participated and were randomized 1:1 to tailored implementation strategies (intervention, 3 sites, n = 744 patients) or education-only (control, 3 sites, n = 692 patients) groups. The program consisted of screening with the 9-item Patient Health Questionnaire (PHQ-9) and algorithm-based scoring and referral to behavioral health services based on low, moderate, or high score. Clinical teams at tailored intervention sites received program education, audit, and feedback of performance data and implementation facilitation, and clinical workflows were adapted to suit local context. Education-only controls sites received program education. Main Outcomes and Measures: The primary outcome was percent of eligible patients screened and referred (based on PHQ-9 score) at intervention vs control groups measured at the patient level. Secondary outcomes included outpatient health care utilization for behavioral health, primary care, oncology, urgent care, and emergency department. Results: All 1436 eligible patients were randomized at the center level (mean age, 61.5 years; 99% women; 18% Asian, 17% Black, 26% Hispanic, and 37% White) and were followed up to the end of the study, insurance disenrollment, or death. Groups were similar in demographic and tumor characteristics. For the primary outcome, 7.9% (59 of 744) of patients at tailored sites were referred compared with 0.1% (1 of 692) at education-only sites (difference, 7.8%; 95% CI, 5.8%-9.8%). Referrals to a behavioral health clinician were completed by 44 of 59 patients treated at the intervention sites (75%) intervention sites vs 1 of 1 patient at the education-only sites (100%). In adjusted models patients at tailored sites had significantly fewer outpatient visits in medical oncology (rate ratio, 0.86; 95% CI, 0.86-0.89; P = .001), and no significant difference in utilization of primary care, urgent care, and emergency department visits. Conclusions and Relevance: Among patients with breast cancer treated in community-based oncology practices, tailored strategies for implementation of routine depression screening compared with an education-only control group resulted in a greater proportion of referrals to behavioral care. Further research is needed to understand the clinical benefit and cost-effectiveness of this program. Trial Registration: ClinicalTrials.gov Identifier: NCT02941614.

Zika virus seroprevalence in blood donors from the Northeastern region of São Paulo State, Brazil, between 2015 and 2017.

OBJECTIVES: Although primarily transmitted by Aedes aegypti mosquitos, Zika virus (ZIKV) can also be transmitted by blood transfusion, due to the fact that some of the infected donors can establish asymptomatic viremia. ZIKV seroprevalence in Brazilian blood donors is unknown. The main reason for this gap in the knowledge originates from the difficulty in evaluating ZIKV humoral immunity due to antigenic cross-reactivity between the different Brazilian flaviviruses and, in particular, dengue virus (DENV). The objective of this study was to evaluate the anti-ZIKV IgG prevalence in blood donors from the Northeast region of the São Paulo State, Brazil, which experienced a ZIKV outbreak in 2016. METHODS: We evaluated the ZIKV seroprevalence using the NS1 anti-ZIKV IgG test (Euroimmun), followed by confirmation of the positive and borderline results using the Plaque Reduction Neutralization Test (PRNT). ZIKV seroprevalence was estimated by testing plasma samples collected in 2015 (before the ZIKV outbreak), 2016 (during the outbreak) and 2017 (after the outbreak). In order to investigate possible antigenic cross - reactivity between ZIKV and DENV we also included samples that were taken well before the ZIKV outbreak, in years 2010 and 2013. RESULTS: The results obtained by the Euroimmun anti-ZIKV IgG test demonstrated ZIKV seroreactivity in 2015, 2016, and 2017 with prevalences of 5.3%, 12.8% and 13.2%, respectively. The inclusion of blood donor samples from 2010 to 2013, demonstrated anti-ZIKV IgG reactivity only for 2013 (1.7%). The PRNT testing of the ZIKV positive and borderline ELISA reacting samples generated positive results only for the years of 2016 and 2017 (prevalences of 5.6% and 9.1%) which coincided with the introduction of ZIKV in our region. CONCLUSIONS: Our results estimate for the first time the ZIKV seroprevalence among Brazilian blood donors from a region with apparently extensive ZIKV circulation and which, at the same time, is highly endemic for DENV. We detected relatively low ZIKV seroprevalence in blood donors from the studied region probably due to the lower intensity of the outbreak compared to other Brazilian locations. Our study adds to the global understanding of ZIKV circulation and the herd immunity of the exposed population.

A microfluidic approach to study the effect of mechanical stress on erythrocytes in sickle cell disease.

The human red blood cell is a biconcave disc of 6-8 × 2 µm that is highly elastic. This capacity to deform enables it to stretch while circulating through narrow capillaries to ensure its main function of gas exchange. Red cell shape and deformability are altered in membrane disorders because of defects in skeletal or membrane proteins affecting protein-protein interactions. Red cell properties are also altered in other pathologies such as sickle cell disease. Sickle cell disease is a genetic hereditary disorder caused by a single point mutation in the ß-globin gene generating sickle haemoglobin (HbS). Hypoxia drives HbS polymerisation that is responsible for red cell sickling and reduced deformability. The main clinical features of sickle cell disease are vaso-occlusive crises and haemolytic anaemia. Foetal haemoglobin (HbF) inhibits HbS polymerisation and positively impacts red cell survival in the circulation but the mechanism through which it exerts this action is not fully characterized. In this study, we designed a microfluidic biochip mimicking the dimensions of human capillaries to measure the impact of repeated mechanical stress on the survival of red cells at the single cell scale under controlled pressure. We show that mechanical stress is a critical parameter underlying intravascular haemolysis in sickle cell disease and that high intracellular levels of HbF protect against lysis. The biochip is a promising tool to address red cell deformability in pathological situations and to screen for molecules positively impacting this parameter in order to improve red cell survival in the circulation.

Acute pulmonary embolism masquerading as acute myocardial infarction.

Pulmonary embolism can be extremely difficult to diagnose based on clinical presentation. Many studies have demonstrated certain electrocardiographic patterns commonly seen in pulmonary embolism, but few have described changes consistent with ST segment elevation myocardial infarction. In this report, we describe a patient who presented to the emergency department with electrocardiographic findings consistent with an anteroseptal myocardial infarction and his subsequent clinical course.

Tissue-specific inactivation of type 2 deiodinase reveals multilevel control of fatty acid oxidation by thyroid hormone in the mouse.

Type 2 deiodinase (D2) converts the prohormone thyroxine (T4) to the metabolically active molecule 3,5,3'-triiodothyronine (T3), but its global inactivation unexpectedly lowers the respiratory exchange rate (respiratory quotient [RQ]) and decreases food intake. Here we used FloxD2 mice to generate systemically euthyroid fat-specific (FAT), astrocyte-specific (ASTRO), or skeletal-muscle-specific (SKM) D2 knockout (D2KO) mice that were monitored continuously. The ASTRO-D2KO mice also exhibited lower diurnal RQ and greater contribution of fatty acid oxidation to energy expenditure, but no differences in food intake were observed. In contrast, the FAT-D2KO mouse exhibited sustained (24 h) increase in RQ values, increased food intake, tolerance to glucose, and sensitivity to insulin, all supporting greater contribution of carbohydrate oxidation to energy expenditure. Furthermore, FAT-D2KO animals that were kept on a high-fat diet for 8 weeks gained more body weight and fat, indicating impaired brown adipose tissue (BAT) thermogenesis and/or inability to oxidize the fat excess. Acclimatization of FAT-D2KO mice at thermoneutrality dissipated both features of this phenotype. Muscle D2 does not seem to play a significant metabolic role given that SKM-D2KO animals exhibited no phenotype. The present findings are unique in that they were obtained in systemically euthyroid animals, revealing that brain D2 plays a dominant albeit indirect role in fatty acid oxidation via its sympathetic control of BAT activity. D2-generated T3 in BAT accelerates fatty acid oxidation and protects against diet-induced obesity.

The p110delta isoform of PI3K differentially regulates beta1 and beta2 integrin-mediated monocyte adhesion and spreading and modulates diapedesis.

OBJECTIVE: Leukocyte diapedesis is misregulated in inflammatory disease and depends on the binding of monocytic LFA-1 and VLA-4 to endothelial ICAM-1 and VCAM-1, respectively. The authors hypothesized that these different molecular interactions elicit specific signaling cascades within monocytes regulating specific steps in adhesion, motility, and diapedesis. METHODS: The authors employed the PI3K p110delta catalytic subunit specific inhibitor IC87114 (2 microM) and the broad-spectrum PI3K inhibitory agents LY294002 (50 microM) and wortmannin (100 nM), to examine the role of PI3Kdelta in monocyte diapedesis through endothelial monolayers and its role in monocyte adhesion and spreading upon carpets of ICAM-1 or VCAM-1. They further explored the effects of PI3Kdelta inhibition on the activation state of beta1 and beta2 integrins with immunocytochemistry and flow cytometry. RESULTS: In human peripheral blood monocytes IC87114 was as effective as wortmannin and LY294002 at inhibiting diapedesis, however, in THP-1 cells LY294002 and wortmannin caused a 5-fold reduction in diapedesis, while IC87114 only decreased diapedesis 2-fold. PI3Kdelta activity was specifically required for THP-1 cell adhesion and spreading on VCAM-1, but not on ICAM-1 protein substrates. Flow cytometric analysis demonstrated that PI3Kdelta inhibition decreased the amount of conformationally active beta 1-integrins, while having no effect on the prevalence of conformationally active beta 2-integrins expressed on the cell surface. In addition, PI3Kdelta inhibition resulted in a 4-fold decrease in the activation state of Rac-1 and Cdc42. CONCLUSIONS: These results demonstrate the specific necessity of PI3Kdelta in regulating monocytic integrin activation and the general role of PI3K signaling during diapedesis, implicating PI3K as a target for therapeutic intervention.

Beta3 integrins facilitate matrix interactions during transendothelial migration of PC3 prostate tumor cells.

BACKGROUND: beta3 integrins play a role in metastatic progression of prostate cancer by mediating adhesion of cancer cells to endothelium and migration through extracellular matrix (ECM). However, the role of beta3 integrins during transendothelial migration (TEM) of prostate tumor cells is poorly understood. We examined the role of beta3 integrins in TEM of PC3 human prostate cancer cells through a monolayer of human lung microvascular endothelial cells (HLMVECs). METHODS: PC3 cells were challenged with beta3 integrin antibodies or antisense nucleotides and their efficiency to migrate through monolayers of endothelial cells (ECs) was assessed using confocal microscopy. RESULTS: beta3 integrins in PC3 cells are not localized in focal contacts and their blockade significantly inhibited TEM by over 50% preferentially during late stages of migration. Formation of PC3 cell pseudopodia on matrigel was significantly reduced by beta3 integrin antisense oligonucleotides. CONCLUSIONS: beta3 integrins play important roles during TEM of PC3 cells while interacting with the matrix underneath the endothelium. These interactions are independent of the ability to cluster beta3 integrins into focal adhesions.

Interleukin-1beta reduces transcellular monocyte diapedesis and compromises endothelial adherens junction integrity.

OBJECTIVE: Diapedesis occurs through endothelial cell-cell junctions (paracellular) or through individual endothelial cells without disrupting junctions (transcellular). While in vitro studies have provided considerable insight into mechanisms controlling paracellular diapedesis, little is known about what regulates transcellular diapedesis. The authors investigated whether transcellular diapedesis is susceptible to IL-1beta exposure of the endothelium. METHODS: Laser scanning confocal microscopy and biochemical analysis were used to determine the effect of IL-1beta pretreatment of the endothelium on adherens junctional morphology and monocyte transcellular diapedesis in cocultures of human peripheral blood monocytes and coronary artery endothelial cells. RESULTS: IL-1beta pretreatment caused a 40% decrease in the number of migrating monocytes that used a transcellular route of diapedesis, and resulted in elongate endothelial cell morphology, a reorganization of the F-actin cytoskeleton, and a significant decrease in transendothelial electrical resistance. In IL-1beta treated monolayers, VE-cadherin and its associated catenins were distributed in a punctate pattern in comparison to the lacy pattern seen in control monolayers. Coimmunoprecipitation of VE-cadherin molecular assemblies revealed that IL-1beta-mediated changes in distribution were associated with a decrease in the presence of cadherin/catenin complexes in the detergent insoluble fraction. CONCLUSIONS: IL-1beta-induced rearrangement of interendothelial adherens junctions facilitates paracellular diapedesis at the expense of transcellular diapedesis.