The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking.

Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.

Definition of the immune evasion-replication interface of rabies virus P protein.

Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a 'positive patch' of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.

Antagonism of STAT3 signalling by Ebola virus.

Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer.

Safety and Effectiveness of Weekly Carfilzomib, Lenalidomide, Dexamethasone, and Daratumumab Combination Therapy for Patients With Newly Diagnosed Multiple Myeloma: The MANHATTAN Nonrandomized Clinical Trial.

IMPORTANCE: Recently, the benefit of adding daratumumab to the proteasome inhibitor-based, 3-drug combination of bortezomib, lenalidomide, and dexamethasone for patients with newly diagnosed multiple myeloma who underwent high-dose melphalan chemotherapy and autologous hemopoietic cell transplant was assessed. Here, we examine the addition of daratumumab to the second-generation proteasome inhibitor-based, 3-drug combination of carfilzomib, lenalidomide, and dexamethasone. OBJECTIVE: To assess the safety and effectiveness of carfilzomib-lenalidomide-dexamethasone-daratumumab combination therapy for patients with newly diagnosed multiple myeloma, in the absence of high-dose melphalan chemotherapy and autologous hemopoietic cell transplant. DESIGN, SETTING, AND PARTICIPANTS: Clinical and correlative pilot study at the Memorial Sloan Kettering Cancer Center in New York, New York. Patients with newly diagnosed multiple myeloma were enrolled between October 1, 2018, and November 15, 2019. The median follow-up from start of treatment was 20.3 months (95% CI, 19.2-21.9 months). INTERVENTIONS: Eight 28-day cycles with intravenous carfilzomib, 20/56 mg/m2 (days 1, 8, and 15); oral lenalidomide, 25 mg, (days 1-21); dexamethasone, 40 mg weekly, orally or intravenously (cycles 1-4), and 20 mg after cycle 4; and intravenous daratumumab, 16 mg/kg (days 1, 8, 15, and 22 [cycles 1-2]; days 1 and 15 [cycles 3-6]; and day 1 [cycles 7 and 8]). MAIN OUTCOMES AND MEASURES: The primary end point was the minimal residual disease (MRD) rate, in the absence of high-dose melphalan chemotherapy and autologous hemopoietic cell transplant. Secondary end points included determining safety and tolerability, evaluating rates of clinical response per the International Myeloma Working Group, and estimating progression-free survival (PFS) and overall survival (OS) rates. RESULTS: Forty-one evaluable patients were enrolled (median age, 59 years; range, 30-70 years); 25 (61%) were female, and 20 (49%) had high-risk multiple myeloma. The primary end point (MRD negativity in the bone marrow; 10-5 sensitivity) was achieved in 29 of 41 patients (71%; 95% CI, 54%-83%), and therefore the trial was deemed successful. Median time to MRD negativity was 6 cycles (range, 1-8 cycles). Secondary end points of the overall response rate and the very good partial response or complete response rate were 100% (41 of 41 patients) and 95% (39 of 41 patients), respectively. At 11 months of the median follow-up, the 1-year PFS rate and the OS rate were 98% (95% CI, 93%-100%) and 100%, respectively. Most common (≥2 patients) grade 3 or 4 adverse events were neutropenia (12 patients [27%]), rash (4 patients [9%]), lung infection (3 patients [7%]), and increased alanine aminotransferase level (2 patients [4%]). There were no deaths. CONCLUSIONS AND RELEVANCE: In this nonrandomized clinical trial, carfilzomib-lenalidomide-dexamethasone-daratumumab combination therapy was associated with high rates of MRD negativity in patients with newly diagnosed multiple myeloma and high rates of PFS.

Lyssavirus P-protein selectively targets STAT3-STAT1 heterodimers to modulate cytokine signalling.

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.

The Dynamic Interface of Viruses with STATs.

Viruses commonly antagonize the antiviral type I interferon response by targeting signal transducer and activator of transcription 1 (STAT1) and STAT2, key mediators of interferon signaling. Other STAT family members mediate signaling by diverse cytokines important to infection, but their relationship with viruses is more complex. Importantly, virus-STAT interaction can be antagonistic or stimulatory depending on diverse viral and cellular factors. While STAT antagonism can suppress immune pathways, many viruses promote activation of specific STATs to support viral gene expression and/or produce cellular conditions conducive to infection. It is also becoming increasingly clear that viruses can hijack noncanonical STAT functions to benefit infection. For a number of viruses, STAT function is dynamically modulated through infection as requirements for replication change. Given the critical role of STATs in infection by diverse viruses, the virus-STAT interface is an attractive target for the development of antivirals and live-attenuated viral vaccines. Here, we review current understanding of the complex and dynamic virus-STAT interface and discuss how this relationship might be harnessed for medical applications.

Evaluation of a Peer-Led Implementation of a Suicide Prevention Gatekeeper Training Program for College Students.

Background: Research on models of implementation that enable widespread dissemination of suicide prevention to young adults is needed to address the critical public health issue of suicide among young adults. A peer-to-peer implementation approach may increase widespread dissemination of suicide prevention on college campuses. Aims: The current study involved the evaluation of a peer-led implementation of the evidence-based program Question, Persuade, and Refer Gatekeeper Training for Suicide Prevention (QPR). Method: A total of 161 college students attended one of eight QPR implementations conducted by student peer educators certified as QPR trainers. Questionnaires were administered at pretest and posttest to assess knowledge of suicide, likelihood of intervening with someone suicidal, and self-efficacy to intervene with someone suicidal. Results: Results from a series of paired-samples t tests showed significant increases from pretest to posttest on the three outcomes of interest - knowledge of suicide, self-efficacy to intervene with someone suicidal, and likelihood to intervene with someone suicidal. Limitations: The short time frame of the current study is a limitation. Conclusion: Support for a peer-led model of implementation for college students has critical implications in terms of increasing the capacity for widespread dissemination of suicide prevention efforts on college campuses.

Design, synthesis and biological evaluation of imidazole and oxazole fragments as HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens.

We describe here the synthesis of libraries of novel 1-subtituted-5-aryl-1H-imidazole, 5-aryl-4-tosyl-4,5-dihydro-1,3-oxazole and 5-aryl-1,3-oxazole fragments via microwave (MW)-assisted cycloaddition of para-toluenesulfonylmethyl isocyanide (TosMIC) to imines and aldehydes. The compounds obtained were biologically evaluated in an AlphaScreen HIV-1 IN-LEDGF/p75 inhibition assay with six imidazole-based compounds (16c, 16f, 17c, 17f, 20a and 20d) displaying more than 50% inhibition at 10 µM, with IC50 values ranging from 7.0 to 30.4 µM. Additionally the hypothesis model developed predicts all active scaffolds except 20d to occupy similar areas as the N-heterocyclic (A) moiety and two aromatic rings (B and C) of previously identified inhibitor 5. These results indicate that the identified compounds represent a viable starting point for their use as templates in the design of next generation inhibitors targeting the HIV-1 IN and LEDGF/p75 protein-protein interaction. In addition, the in vitro antimicrobial properties of these fragments were tested by minimum inhibitory concentration (MIC) assays showing that compound 16f exhibited a MIC value of 15.6 µg/ml against S. aureus, while 17f displayed a similar MIC value against B. cereus, suggesting that these compounds could be further developed to specifically target those microbial pathogens.

Effectiveness of a Faculty Development Course on Delivering Learner-Centered Feedback Utilizing the Flipped Training Model.

CONTEXT: Effective feedback is an important step in the acquisition of residents' clinical skills and a key component of most adult learning strategies. Faculty-resident feedback discussions can facilitate resident self-assessment and reflection on their performance and motivate them to study and ask questions in areas where their knowledge may be evaluated as deficient. The flipped training model approach, a type of blended learning that reverses the traditional learning environment by delivering instructional content outside of the classroom, has garnered increased support within both graduate medical education (GME) and other healthcare disciplines. METHODS: The overall purpose of this exploratory pilot project was to examine the pre-post impact of a faculty feedback flipped training model course provided to a convenience sample of community-based faculty learners. After receiving campus IRB approval, the authors developed a set of five primary course goals and objectives. A convenience sample of n = 17 community-based faculty who had completed the entire course were administered a pair of pre and post-course surveys regarding their overall feedback satisfaction and comfort levels for supervising residents. RESULTS: In summary, five of the 13 total survey items increased at statistically significant levels from pre-course levels. The majority of qualitative faculty comments also positively evaluated the flipped training model approach. CONCLUSIONS: These promising pilot findings suggest that a flipped GME faculty feedback skills training model can help improve faculty learners' satisfaction and confidence as they supervise residents and/or medical students. The impact of these types of flipped training models for GME faculty needs to be more rigorously examined in project settings with larger samples to identify what specific types of curricular activities might prove to be most effective for diverse faculty learners in GME programs across the nation.

Delivering the AAMC "Teaching for Quality" Program through a Community-Based GME Collaborative: Lessons Learned to Date.

CONTEXT: To address scholarly activity (SA) accreditation standards, the Michigan State University's College of Osteopathic Medicine Statewide Campus System has offered the Association of American Medical Colleges' (AAMC) Teaching for Quality Program for two cohorts of community-based faculty. The purpose of this paper was to describe the design and delivery of the customized program, the authors' initial lessons learned, and their plans for further evaluation and dissemination. METHODS: The authors customized the program to overcome the barriers typically faced by community-based program faculty learners through a graduate medical education (GME) consortium model. This was the first time this program was delivered in this manner. RESULTS: The authors' initial cohort of 19 learners successfully developed 15 projects, with two pairs of learners collaborating on projects. The second cohort of 15 learners developed 11 projects, with one pair of learners collaborating. The authors present a series of principles for community-based GME leaders striving to develop SA projects in their respective GME environments. CONCLUSIONS: The "consortium advantage" derived from entities such as the SCS may prove integral to efficiently coordinating SA project resources and knowledge across diverse GME systems.

The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 µm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 µm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

Chronically ill Canadians' experiences of being unattached to a family doctor: a qualitative study of marginalized patients in British Columbia.

BACKGROUND: Unattached patients do not have a regular primary care provider. Initiatives are being developed to increase attachment rates across Canada. Most existing attention paid to patient unattachment has focused on quantifying the problem and health system costs. Our purpose is to qualitatively identify the implications of chronically ill patients' experiences of unattachment for health policy and planning to provide policy-relevant insights for Canadian attachment initiatives. METHODS: Three focus groups were conducted with marginalized chronically ill individuals residing in a mid-sized city in British Columbia who are unattached to a family doctor. We use the term marginalized as a descriptor to acknowledge that by virtue of their low socio-economic status and lack of attachment the participants are marginalized in Canada's health care system Focus groups were structured as an open conversation organized around a series of probing questions. They were digitally recorded and transcribed verbatim. Thematic analysis was employed. RESULTS: Twenty-six individuals participated in the focus groups. The most common chronic illnesses reported were active drug addiction or recovery (and their associated symptoms), depression, arthritis, and hepatitis C. Participants identified life transitions as being the root cause for not having a family doctor. There was a strong sense that unsuccessful attempts to get a family doctor reflected that they were undesirable patients. Participants wanted to experience having a trusting relationship with a regular family doctor as they believed it would encourage greater honesty and transparency. One of the main health concerns regarding lack of access to a regular family doctor is that participants lacked access to preventative care. Participants were also concerned about having a discontinuous medical record due to unattachment. CONCLUSIONS: Participants perceived that there are many benefits to be had by having attachment to a regular family doctor and that experiencing unattachment challenged their health and access to health care. We encourage more research to be done on the lived experience of unattachment in order to provide on-the-ground insights that policy-makers require in order to develop responsive, patient-centred supports and programs.