High throughput screening identifies dasatinib as synergistic with trametinib in low grade serous ovarian carcinoma.

BACKGROUND: Low grade serous ovarian carcinoma (LGSOC) is a distinct histotype of ovarian cancer characterised high levels of intrinsic chemoresistance, highlighting the urgent need for new treatments. High throughput screening in clinically-informative cell-based models represents an attractive strategy for identifying candidate treatment options for prioritisation in clinical studies. METHODS: We performed a high throughput drug screen of 1610 agents across a panel of 6 LGSOC cell lines (3 RAS/RAF-mutant, 3 RAS/RAF-wildtype) to identify novel candidate therapeutic approaches. Validation comprised dose-response analysis across 9 LGSOC models and 5 high grade serous comparator lines. RESULTS: 16 hits of 1610 screened compounds were prioritised for validation based on >50% reduction in nuclei counts in over half of screened cell lines at 1000 nM concentration. 11 compounds passed validation, and the four agents of greatest interest (dasatinib, tyrosine kinase inhibitor; disulfiram, aldehyde dehydrogenase inhibitor; carfilzomib, proteasome inhibitor; romidepsin, histone deacetylase inhibitor) underwent synergy profiling with the recently approved MEK inhibitor trametinib. Disulfiram demonstrated excellent selectivity for LGSOC versus high grade serous ovarian carcinoma comparator lines (P = 0.003 for IC50 comparison), while the tyrosine kinase inhibitor dasatinib demonstrated favourable synergy with trametinib across multiple LGSOC models (maximum zero interaction potency synergy score 46.9). The novel, highly selective Src family kinase (SFK) inhibitor NXP900 demonstrated a similar trametinib synergy profile to dasatinib, suggesting that SFK inhibition is the likely driver of synergy. CONCLUSION: Dasatinib and other SFK inhibitors represent novel candidate treatments for LGSOC and demonstrate synergy with trametinib. Disulfiram represents an additional treatment strategy worthy of investigation.

Negative pressure suction test: An intraoperative airway maneuver to assess effectiveness of surgical correction of tracheobronchomalacia.

BACKGROUND: Surgical correction of tracheobronchomalacia (TBM) has evolved greatly over the past decade, with select pediatric institutions establishing dedicated surgery and anesthesia teams to navigate the complexities and challenges of surgical airway repairs. Although anesthetic techniques have evolved internally over many years to improve patient safety and outcomes, many of these methods remain undescribed in literature. TECHNIQUE: In this article, we describe the intraoperative negative pressure suction test. This simulates the negative pressure seen in awake and spontaneously breathing patients, including the higher pressures seen during coughing which induce airway collapse in patients with TBM. Also known as the Munoz maneuver in surgical literature, this test has been performed on over 300 patients since 2015. DISCUSSION: The negative pressure suction test allows for controlled intraoperative assessment of surgical airway repairs, replaces the need for risky intraoperative wake-up tests, increases the chances of a successful surgical repair, and improves anesthetic management for emergence and extubation. We provide a guide on how to perform the test and videos demonstrating its efficacy in intraoperative airway evaluation. CONCLUSIONS: As surgeries to repair TBM become more prevalent in other pediatric institutions, we believe that pediatric patients and anesthesia providers will benefit from the insights and methods described here.

Impact of population based indoor residual spraying with and without mass drug administration with dihydroartemisinin-piperaquine on malaria prevalence in a high transmission setting: a quasi-experimental controlled before-and-after trial in northeastern Uganda.

BACKGROUND: Declines in malaria burden in Uganda have slowed. Modelling predicts that indoor residual spraying (IRS) and mass drug administration (MDA), when co-timed, have synergistic impact. This study investigated additional protective impact of population-based MDA on malaria prevalence, if any, when added to IRS, as compared with IRS alone and with standard of care (SOC). METHODS: The 32-month quasi-experimental controlled before-and-after trial enrolled an open cohort of residents (46,765 individuals, 1st enumeration and 52,133, 4th enumeration) of Katakwi District in northeastern Uganda. Consented participants were assigned to three arms based on residential subcounty at study start: MDA+IRS, IRS, SOC. IRS with pirimiphos methyl and MDA with dihydroartemisinin- piperaquine were delivered in 4 co-timed campaign-style rounds 8 months apart. The primary endpoint was population prevalence of malaria, estimated by 6 cross-sectional surveys, starting at baseline and preceding each subsequent round. RESULTS: Comparing malaria prevalence in MDA+IRS and IRS only arms over all 6 surveys (intention-to-treat analysis), roughly every 6 months post-interventions, a geostatistical model found a significant additional 15.5% (95% confidence interval (CI): [13.7%, 17.5%], Z = 9.6, p = 5e-20) decrease in the adjusted odds ratio (aOR) due to MDA for all ages, a 13.3% reduction in under 5's (95% CI: [10.5%, 16.8%], Z = 4.02, p = 5e-5), and a 10.1% reduction in children 5-15 (95% CI: [8.5%, 11.8%], Z = 4.7, p = 2e-5). All ages residents of the MDA + IRS arm enjoyed an overall 80.1% reduction (95% CI: [80.0%, 83.0%], p = 0.0001) in odds of qPCR confirmed malaria compared with SOC residents. Secondary difference-in-difference analyses comparing surveys at different timepoints to baseline showed aOR (MDA + IRS vs IRS) of qPCR positivity between 0.28 and 0.66 (p < 0.001). Of three serious adverse events, one (nonfatal) was considered related to study medications. Limitations include the initial non-random assignment of study arms, the single large cluster per arm, and the lack of an MDA-only arm, considered to violate equipoise. CONCLUSIONS: Despite being assessed at long time points 5-7 months post-round, MDA plus IRS provided significant additional protection from malaria infection over IRS alone. Randomized trials of MDA in large areas undergoing IRS recommended as well as cohort studies of impact on incidence. TRIAL REGISTRATION: This trial was retrospectively registered 11/07/2018 with the Pan African Clinical Trials Registry (PACTR201807166695568).

Trauma Center Outcomes After Transition From Level 2 to Level 1: A National Trauma Data Bank Analysis.

BACKGROUND: Previous US-based studies have shown that a trauma center designation of level 1 is associated with improved patient outcomes. However, most studies are cross-sectional, focus on volume-related issues and are direct comparisons between levels. This study investigates the change in patient characteristics when individual trauma centers transition from level 2 to level 1 and whether the patients have similar outcomes during the initial period of the transition. STUDY DESIGN: We performed a retrospective cohort study that analyzed hospital and patient records included in the National Trauma Data Bank from 2007 to 2016. Patient characteristics were compared before and after their hospitals transitioned their trauma level. Mortality; complications including acute kidney injury, acute respiratory distress syndrome, cardiac arrest with CPR, deep surgical site infection, deep vein thrombosis, extremity compartment syndrome, surgical site infection, osteomyelitis, pulmonary embolism, and so on; ICU admission; ventilation use; unplanned returns to the OR; unplanned ICU transfers; unplanned intubations; and lengths of stay were obtained following propensity score matching, comparing posttransition years with the last pretransition year. RESULTS: Sixteen trauma centers transitioned from level 2 to level 1 between 2007 and 2016. One was excluded due to missing data. After transition, patient characteristics showed differences in the distribution of race, comorbidities, insurance status, injury severity scores, injury mechanisms, and injury type. After propensity score matching, patients treated in a trauma center after transition from level 2 to 1 required significantly fewer ICU admissions and had lower complication rates. However, significantly more unplanned intubations, unplanned returns to the OR, unplanned ICU transfers, ventilation use, surgical site infections, pneumonia, and urinary tract infections and higher mortality were reported after the transition. CONCLUSIONS: Trauma centers that transitioned from level 2 to level 1 had lower overall complications, with fewer patients requiring ICU admission. However, higher mortality and more surgical site infections, pneumonia, urinary tract infections, unplanned intubations, and unplanned ICU transfers were reported after the transition. These findings may have significant implications in the planning of trauma systems for administrators and healthcare leaders.

Volatile anesthetic agents for life-threatening pediatric asthma: A multicenter retrospective cohort study and narrative review.

BACKGROUND: Volatile anesthetic agents are described as rescue therapy for children invasively ventilated for critical asthma. Yet, data are currently limited to case series. AIMS: Using the Virtual Pediatric Systems database, we assessed children admitted to a pediatric intensive care unit invasively ventilated for life-threatening asthma and hypothesized ventilation duration and mortality rates would be lower for subjects exposed to volatile anesthetics compared with those without exposure. METHODS: We performed a multicenter retrospective cohort study among nine institutions including children 5-17 years of age invasively ventilated for asthma from 2013 to 2019 with and without exposure to volatile anesthetics. Primary outcomes were ventilation duration and mortality. Secondary outcomes included patient characteristics, length of stay, and anesthetic-related adverse events. A subgroup analysis was performed evaluating children intubated ≥2 days. RESULTS: Of 203 children included in study, there were 29 (14.3%) with and 174 (85.7%) without exposure to volatiles. No differences in odds of mortality (1.1, 95% CI: 0.3-3.9, p > .999) were observed. Subjects receiving volatiles experienced greater median difference in length of stay (4.8, 95% CI: 1.9-7.8 days, p < .001), ventilation duration (2.3, 95% CI: 1-3.3 days, p < .001), and odds of extracorporeal life support (9.1, 95% CI: 1.9-43.2, p = .009) than those without volatile exposure. For those ventilated ≥2 days, no differences were detected in mortality, ventilation duration, length of stay, arrhythmias, or acute renal failure. However, the odds of extracorporeal life support remained greater for those receiving volatiles (7.6, 95% CI: 1.3-44.5, p = .027). No children experienced malignant hyperthermia or hepatic failure after volatile exposure. CONCLUSIONS: For intubated children for asthma, no differences in mechanical ventilation duration or mortality between those with and without volatile anesthetic exposure were observed. Although volatiles may represent a viable rescue therapy for severe cases of asthma, definitive, and prospective trials are still needed.

M Segment-Based Minigenomes and Virus-Like Particle Assays as an Approach To Assess the Potential of Tick-Borne Phlebovirus Genome Reassortment.

Bunyaviruses have a tripartite negative-sense RNA genome. Due to the segmented nature of these viruses, if two closely related viruses coinfect the same host or vector cell, it is possible that RNA segments from either of the two parental viruses will be incorporated into progeny virions to give reassortant viruses. Little is known about the ability of tick-borne phleboviruses to reassort. The present study describes the development of minigenome assays for the tick-borne viruses Uukuniemi phlebovirus (UUKV) and Heartland phlebovirus (HRTV). We used these minigenome assays in conjunction with the existing minigenome system of severe fever with thrombocytopenia syndrome (SFTS) phlebovirus (SFTSV) to assess the abilities of viral N and L proteins to recognize, transcribe, and replicate the M segment-based minigenome of a heterologous virus. The highest minigenome activity was detected with the M segment-based minigenomes of cognate viruses. However, our findings indicate that several combinations utilizing N and L proteins of heterologous viruses resulted in M segment minigenome activity. This suggests that the M segment untranslated regions (UTRs) are recognized as functional promoters of transcription and replication by the N and L proteins of related viruses. Further, virus-like particle assays demonstrated that HRTV glycoproteins can package UUKV and SFTSV S and L segment-based minigenomes. Taken together, these results suggest that coinfection with these viruses could lead to the generation of viable reassortant progeny. Thus, the tools developed in this study could aid in understanding the role of genome reassortment in the evolution of these emerging pathogens in an experimental setting.IMPORTANCE In recent years, there has been a large expansion in the number of emerging tick-borne viruses that are assigned to the Phlebovirus genus. Bunyaviruses have a tripartite segmented genome, and infection of the same host cell by two closely related bunyaviruses can, in theory, result in eight progeny viruses with different genome segment combinations. We used genome analogues expressing reporter genes to assess the abilities of Phlebovirus nucleocapsid protein and RNA-dependent RNA polymerase to recognize the untranslated region of a genome segment of a related phlebovirus, and we used virus-like particle assays to assess whether viral glycoproteins can package genome analogues of related phleboviruses. Our results provide strong evidence that these emerging pathogens could reassort their genomes if they were to meet in nature in an infected host or vector. This reassortment process could result in viruses with new pathogenic properties.

Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity.

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Interferon-Stimulated Gene (ISG)-Expression Screening Reveals the Specific Antibunyaviral Activity of ISG20.

Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an "antiviral state." Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ∼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

Synergy and timing: a concurrent mass medical campaign predicted to augment indoor residual spraying for malaria.

BACKGROUND: Control programmes for high burden countries are tasked with charting effective multi-year strategies for malaria control within significant resource constraints. Synergies between different control tools, in which more than additive benefit accrues from interventions used together, are of interest because they may be used to obtain savings or to maximize health impact per expenditure. One commonly used intervention in sub-Saharan Africa is indoor residual spraying (IRS), typically deployed through a mass campaign. While possible synergies between IRS and long-lasting insecticide-treated nets (LLINs) have been investigated in multiple transmission settings, coordinated synergy between IRS and other mass medical distribution campaigns have not attracted much attention. Recently, a strong timing-dependent synergy between an IRS campaign and a mass drug administration (MDA) was theoretically quantified. These synergistic benefits likely differ across settings depending on transmission intensity and its overall seasonal pattern. METHODS: High coverage interventions are modelled in different transmission environments using two methods: a Ross-Macdonald model variant and openmalaria simulations. The impact of each intervention strategy was measured through its ability to prevent host infections over time, and the effects were compared to the baseline case of deploying interventions in isolation. RESULTS: By modelling IRS and MDA together and varying their deployment times, a strong synergy was found when the administered interventions overlapped. The added benefit of co-timed interventions was robust to differences in the models. In the Ross-Macdonald model, the impact compared was roughly double the sequential interventions in most transmission settings. Openmalaria simulations of this medical control augmentation of an IRS campaign show an even stronger response with the same timing relationship. CONCLUSIONS: The strong synergies found for these control tools between the complementary interventions demonstrate a general feature of effective concurrent campaign-style vector and medical interventions. A mass treatment campaign is normally short-lived, especially in higher transmission settings. When co-timed, the rapid clearing of the host parasite reservoir via chemotherapy is protected from resurgence by the longer duration of the vector control. An effective synchronous treatment campaign has the potential to greatly augment the impact of indoor residual spraying. Mass screening and treatment (MSAT) with highly sensitive rapid diagnostic tests may demonstrate a comparable trend while mass LLIN campaigns may similarly coordinate with MDA/MSAT.

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase.

The M genome segment of Bunyamwera virus (BUNV)-the prototype of both the Bunyaviridae family and the Orthobunyavirus genus-encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SP(NSm) and NSm domain V as SP(Gc) Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SP(Gc)) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17-312 or nearby residues; NSm, 332-477; and Gc, 478-1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies.

Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion.

An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus-host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens.

Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer.

OBJECTIVE: Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited. DESIGN: To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets. RESULTS: By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer. CONCLUSIONS: BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453). TRIAL REGISTRATION NUMBER: NCT02884453; Pre-results.

Detection, infection dynamics and small RNA response against Culex Y virus in mosquito-derived cells.

Many insect cell lines are persistently infected with insect-specific viruses (ISV) often unrecognized by the scientific community. Considering recent findings showing the possibility of interference between arbovirus and ISV infections, it is important to pay attention to ISV-infected cell lines. One example is the Entomobirnavirus, Culex Y virus (CYV). Here we describe the detection of CYV using a combination of small RNA sequencing, electron microscopy and PCR in mosquito cell lines Aag2, U4.4 and C7-10. We found CYV-specific small RNAs in all three cell lines. Interestingly, the magnitude of the detected viral RNA genome is variable among cell passages and leads to irregular detection via electron microscopy. Gaining insights into the presence of persistent ISV infection in commonly used mosquito cells and their interactions with the host immune system is beneficial for evaluating the outcome of co-infections with arboviruses of public health concern.

Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses.

SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3' rapid amplification of cDNA ends (RACE), we mapped the 3' end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3' end of the N mRNA terminates upstream of a 5'-GCCAGCC-3' motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies.IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5'-GCCAGCC-3' motif present in the virus genomic S RNA.

Synthetic Lethal Screen Demonstrates That a JAK2 Inhibitor Suppresses a BCL6-dependent IL10RA/JAK2/STAT3 Pathway in High Grade B-cell Lymphoma.

We demonstrate the usefulness of synthetic lethal screening of a conditionally BCL6-deficient Burkitt lymphoma cell line, DG75-AB7, with a library of small molecules to determine survival pathways suppressed by BCL6 and suggest mechanism-based treatments for lymphoma. Lestaurtinib, a JAK2 inhibitor and one of the hits from the screen, repressed survival of BCL6-deficient cells in vitro and reduced growth and proliferation of xenografts in vivo BCL6 deficiency in DG75-AB7 induced JAK2 mRNA and protein expression and STAT3 phosphorylation. Surface IL10RA was elevated by BCL6 deficiency, and blockade of IL10RA repressed STAT3 phosphorylation. Therefore, we define an IL10RA/JAK2/STAT3 pathway each component of which is repressed by BCL6. We also show for the first time that JAK2 is a direct BCL6 target gene; BCL6 bound to the JAK2 promoter in vitro and was enriched by ChIP-seq. The place of JAK2 inhibitors in the treatment of diffuse large B-cell lymphoma has not been defined; we suggest that JAK2 inhibitors might be most effective in poor prognosis ABC-DLBCL, which shows higher levels of IL10RA, JAK2, and STAT3 but lower levels of BCL6 than GC-DLBCL and might be usefully combined with novel approaches such as inhibition of IL10RA.

Harm reduction in name, but not substance: a comparative analysis of current Canadian provincial and territorial policy frameworks.

BACKGROUND: In Canada, funding, administration, and delivery of health services-including those targeting people who use drugs-are primarily the responsibility of the provinces and territories. Access to harm reduction services varies across jurisdictions, possibly reflecting differences in provincial and territorial policy commitments. We examined the quality of current provincial and territorial harm reduction policies in Canada, relative to how well official documents reflect internationally recognized principles and attributes of a harm reduction approach. METHODS: We employed an iterative search and screening process to generate a corpus of 54 provincial and territorial harm reduction policy documents that were current to the end of 2015. Documents were content-analyzed using a deductive coding framework comprised of 17 indicators that assessed the quality of policies relative to how well they described key population and program aspects of a harm reduction approach. RESULTS: Only two jurisdictions had current provincial-level, stand-alone harm reduction policies; all other documents were focused on either substance use, addiction and/or mental health, or sexually transmitted and/or blood-borne infections. Policies rarely named specific harm reduction interventions and more frequently referred to generic harm reduction programs or services. Only one document met all 17 indicators. Very few documents acknowledged that stigma and discrimination are issues faced by people who use drugs, that not all substance use is problematic, or that people who use drugs are legitimate participants in policymaking. A minority of documents recognized that abstaining from substance use is not required to receive services. Just over a quarter addressed the risk of drug overdose, and even fewer acknowledged the need to apply harm reduction approaches to an array of drugs and modes of use. CONCLUSIONS: Current provincial and territorial policies offer few robust characterizations of harm reduction or go beyond rhetorical or generic support for the approach. By endorsing harm reduction in name, but not in substance, provincial and territorial policies may communicate to diverse stakeholders a general lack of support for key aspects of the approach, potentially challenging efforts to expand harm reduction services.

Enhancing recovery after minimally invasive repair of pectus excavatum.

PURPOSE: There are variations in the perioperative management of patients who undergo minimally invasive repair of pectus excavatum (MIRPE). The purpose is to analyze the change in resource utilization after implementation of a standardized practice plan and describe an enhanced recovery pathway. METHODS: A standardized practice plan was implemented in 2013. A retrospective review of patients who underwent MIRPE from 2012 to 2015 was performed to evaluate the trends in resource utilization. A pain management protocol was implemented and a retrospective review was performed of patients who underwent repair before (2010-2012) and after (2014-2015) implementation. RESULTS: There were 71 patients included in the review of resource utilization. After implementation, there was a decrease in intensive care unit length of stay (LOS), and laboratory and radiologic studies ordered. There were 64 patients included in the pain protocol analysis. After implementation, postoperative morphine equivalents (3.3 ± 1.4 vs 1.2 ± 0.5 mg/kg, p < 0.01), urinary retention requiring catheterization (33 vs 14%, p = 0.07), and LOS (4 ± 1 vs 2.8 ± 0.8 days, p < 0.01) decreased. CONCLUSION: The implementation of an enhanced recovery pathway is a feasible and effective way to reduce resource utilization and improve outcomes in pediatric patients who undergo minimally invasive repair of pectus excavatum.

Evolution of the Bunyamwera virus polymerase to accommodate deletions within genomic untranslated region sequences.

UNLABELLED: The untranslated regions (UTR) present at the ends of bunyavirus genome segments are required for essential steps in the virus life cycle and provide signals for encapsidation by nucleocapsid protein and the promoters for RNA transcription and replication as well as for mRNA transcription termination. For the prototype bunyavirus, Bunyamwera virus (BUNV), only the terminal 11 nucleotides (nt) of the segments are identical. Thereafter, the UTRs are highly variable both in length and in sequence. Furthermore, apart from the conserved termini, the UTRs of different viruses are highly variable. We previously generated recombinant BUNV carrying the minimal UTRs on all three segments that were attenuated for growth in cell culture. Following serial passage of these viruses, the viruses acquired increased fitness, and amino acid changes were observed to accumulate in the viral polymerase (L protein) of most mutant viruses, with the vast majority of the amino acid changes occurring in the C-terminal region. The function of this domain within L remains unknown, but by using a minigenome assay we showed that it might be involved in UTR recognition. Moreover, we identified an amino acid mutation within the polymerase that, when introduced into an otherwise wild-type BUNV, resulted in a virus with a temperature-sensitive phenotype. Viruses carrying temperature-sensitive mutations are good candidates for the design of live attenuated vaccines. We suggest that a combination of stable deletions of the UTRs together with the introduction of temperature-sensitive mutations in both the nucleocapsid and the polymerase could be used to design live attenuated vaccines against serious pathogens within the family Bunyaviridae. IMPORTANCE: Virus growth in tissue culture can be attenuated by introduction of mutations in both coding and noncoding sequences. We generated attenuated Bunyamwera viruses by deleting sequences within both the 3' and 5' untranslated regions (UTR) on each genome segment and showed that the viruses regained fitness following serial passage in cell culture. The fitter viruses had acquired amino acid changes predominantly in the C-terminal domain of the viral polymerase (L protein), and by using minigenome assays we showed that the mutant polymerases were better adapted to recognizing the mutant UTRs. We suggest that deletions within the UTRs should be incorporated along with other specific mutations, including deletion of the major virulence gene encoding the NSs protein and introduction of temperature-sensitive mutations, in the design of attenuated bunyaviruses that could have potential as vaccines.

Flexibility of bunyavirus genomes: creation of an orthobunyavirus with an ambisense S segment.

UNLABELLED: The Bunyamwera (BUNV) orthobunyavirus NSs protein has proven a challenge to study in the context of viral infection. NSs is encoded in a reading frame that overlaps that of the viral nucleocapsid (N) protein thus limiting options for mutagenesis. In addition, NSs is poorly immunogenic, and antibodies only work in certain techniques while the protein itself is subject to proteasomal degradation. In order to generate a virus that expresses NSs independently of N, an ambisense S RNA segment was designed by mutating the 5'- and 3'-terminal nucleotide sequences. These mutations were previously shown to alter promoter activity so that both replication and transcription were promoted from both the genome and the antigenome RNAs (J. N. Barr et al., J. Virol. 79: 12602-12607, 2005). As proof of principle, a recombinant BUNV was created that expressed green fluorescent protein (GFP) in the ambisense orientation. GFP expression was detected throughout at least 10 passages. Recombinant BUNV encoding epitope-tagged versions of NSs in the ambisense orientation expressed NSs via a subgenomic mRNA, and two viruses grew to titers only modestly lower than parental rBUNdelNSs2 virus. The ambisense viruses were temperature sensitive, and NSs was shown to localize to both the nucleus and the cytoplasm during infection. These viruses will be useful in further studies on structure-function relationships of the orthobunyavirus NSs protein. IMPORTANCE: Bunyamwera virus (BUNV) is the type species and model system for both the family Bunyaviridae and the genus Orthobunyavirus, a group that includes many significant human and animal pathogens. Studying the basic molecular biology of these viruses is of great importance to underpin research into vaccines and antivirals. We demonstrate here the plasticity of the BUNV genome by generating recombinant viruses where the normal negative-sense S segment has been converted into an ambisense segment, allowing independent expression of either a foreign gene (green fluorescent protein) or the viral nonstructural NSs protein. These new reagents will allow detailed investigation of NSs, the orthobunyavirus interferon antagonist.

Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist.

UNLABELLED: Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. IMPORTANCE: Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses.