A Synthetic HIV-1 Subtype C Backbone Generates Comparable PR and RT Resistance Profiles to a Subtype B Backbone in a Recombinant Virus Assay

In order to determine phenotypic protease and reverse transcriptase inhibitor-associated resistance in HIV subtype C virus, we have synthetically constructed an HIV-1 subtype C (HIV-1-C) viral backbone for use in a recombinant virus assay. The in silico designed viral genome was divided into 4 fragments, which were chemically synthesized and joined together by conventional subcloning. Subsequently, gag-protease-reverse-transcriptase (GPRT) fragments from 8 HIV-1 subtype C-infected patient samples were RT-PCR-amplified and cloned into the HIV-1-C backbone (deleted for GPRT) using In-Fusion reagents. Recombinant viruses (1 to 5 per patient sample) were produced in MT4-eGFP cells where cyto-pathogenic effect (CPE), p24 and Viral Load (VL) were monitored. The resulting HIV-1-C recombinant virus stocks (RVS) were added to MT4-eGFP cells in the presence of serial dilutions of antiretroviral drugs (PI, NNRTI, NRTI) to determine the fold-change in IC50 compared to the IC50 of wild-type HIV-1 virus. Additionally, viral RNA was extracted from the HIV-1-C RVS and the amplified GPRT products were used to generate recombinant virus in a subtype B backbone. Phenotypic resistance profiles in a subtype B and subtype C backbone were compared. The following observations were made: i) functional, infectious HIV-1 subtype C viruses were generated, confirmed by VL and p24 measurements; ii) their rate of infection was slower than viruses generated in the subtype B backbone; iii) they did not produce clear CPE in MT4 cells; and iv) drug resistance profiles generated in both backbones were very similar, including re-sensitizing effects like M184V on AZT

Interobserver Agreement of PD-L1/SP142 Immunohistochemistry and Tumor-Infiltrating Lymphocytes (TILs) in Distant Metastases of Triple-Negative Breast Cancer: A Proof-of-Concept Study. A Report on Behalf of the International Immuno-Oncology Biomarker Working Group

Patients with advanced triple-negative breast cancer (TNBC) benefit from treatment with atezolizumab, provided that the tumor contains 651% of PD-L1/SP142-positive immune cells. Numbers of tumor-infiltrating lymphocytes (TILs) vary strongly according to the anatomic localization of TNBC metastases. We investigated inter-pathologist agreement in the assessment of PD-L1/SP142 immunohistochemistry and TILs. Ten pathologists evaluated PD-L1/SP142 expression in a proficiency test comprising 28 primary TNBCs, as well as PD-L1/SP142 expression and levels of TILs in 49 distant TNBC metastases with various localizations. Interobserver agreement for PD-L1 status (positive versus negative) was high in the proficiency test: the corresponding scores as percentages showed good agreement with the consensus diagnosis. In TNBC metastases, there was substantial variability in PD-L1 status at the individual patient level. For one in five patients, the chance of treatment was essentially random, with half of the pathologists designating them as positive and half negative. Assessment of PD-L1/SP142 and TILs as percentages in TNBC metastases showed poor and moderate agreement, respectively. Additional training for metastatic TNBC is required to enhance interobserver agreement. Such training, focusing on metastatic specimens, seems worthwhile, since the same pathologists obtained high percentages of concordance (ranging from 93% to 100%) on the PD-L1 status of primary TNBCs

The co-evolution of policy mixes and socio-technical systems: towards a conceptual framework of policy mix feedback in sustainability transitions

Understanding how policymaking processes can influence the rate and direction of socio-technical change towards sustainability is an important, yet underexplored research agenda in the field of sustainability transitions. Some studies have sought to explain how individual policy instruments can influence transitions, and the politics surrounding this process. We argue that such individual policy instruments can cause wider feedback mechanisms that influence not only their own future development, but also other instruments in the same area. Consequently, by extending the scope of analysis to that of a policy mix allows us to account for multiple policy effects on socio-technical change and resultant feedback mechanisms influencing the policy processes that underpin further policy mix change. This paper takes a first step in this regard by combining policy studies and innovation studies literatures to conceptualise the co-evolutionary dynamics of policy mixes and socio-technical systems. We focus on policy processes to help explain how policy mixes influence socio-technical change, and how changes in the socio-technical system also shape the evolution of the policy mix. To do so we draw on insights from the policy feedback literature, and propose a novel conceptual framework. The framework highlights that policy mixes aiming to foster sustainability transitions need to be designed to create incentives for beneficiaries to mobilise further support, while overcoming a number of prevailing challenges which may undermine political support over time. In the paper, we illustrate the framework using the example of the zero carbon homes policy mix in the UK. We conclude with deriving research and policy implications for analysing and designing dynamic policy mixes for sustainability transitions

Policy mixes for incumbency: the destructive recreation of renewable energy, shale gas 'fracking,' and nuclear power in the United Kingdom

The notion of a ‘policy mix’ can describe interactions across a wide range of innovation policies, including ‘motors for creation’ as well as for ‘destruction’. This paper focuses on the United Kingdom’s (UK) ‘new policy direction’ that has weakened support for renewables and energy efficiency schemes while strengthening promotion of nuclear power and hydraulic fracturing for natural gas (‘fracking’). The paper argues that a ‘policy apparatus for incumbency’ is emerging which strengthens key regimebased technologies while arguably damaging emerging niche innovations. Basing the discussion around the three technology-based cases of renewable energy and efficiency, fracking, and nuclear power, this paper refers to this process as “destructive recreation”. Our study raises questions over the extent to which policymaking in the energy field is not so much driven by stated aims around sustainability transitions, as by other policy drivers. It investigates different ‘strategies of incumbency’ including ‘securitization’, ‘masking’, ‘reinvention’, and ‘capture.’ It suggests that analytical frameworks should extend beyond the particular sectors in focus, with notions of what counts as a relevant ‘policy maker’ correspondingly also expanded, in order to explore a wider range of nodes and critical junctures as entry points for understanding how relations of incumbency are forged and reproduced

UDP-glucuronosyltransferase-mediated metabolic activation of the tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole

International audience2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3- b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by 1H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N 2-(β- D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N 2-Gl) and N 2-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b] indole (AαC-HON 2-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN 2-O-Gl). AαC-HN 2-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20-50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (V max/K m) at forming AαC-N 2-Gl (0.67 μl*min -1*mg of protein -1), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μl*min -1*mg of protein -1), whereas UGT1A1 was most efficient at forming AαC-HON 2-Gl (5.0 μl*min -1*mg of protein -1). Human hepatocytes produced AαC-N 2-Gl and AαC-HN 2-O-Gl in abundant quantities, but AαC-HON 2-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Development of a real-time multiplex RSV detection assay for difficult respiratory samples, using ultrasone waves and MNAzyme technology

Background: Elderly infected with Human Respiratory Syncytial Virus (RSV) often bear low viral loads that stay below the detection limits of commercial assays. A more sensitive detection of RSV infections can improve patient management, guide containment strategies, and possibly prevent morbidity and mortality among populations most severely affected by RSV. Objective: To test the sensitivity for RSV detection by using an alternative extraction method in combination with a new amplification procedure. Study design: Nasopharyngeal washes and sputum samples (n = 78) form clinical cases, and broncheo-alveolar lavages (n = 27) from an experimental RSV rat model were obtained. An ultrasone-based RNA extraction method was combined with a multi-component Nucleic Acid enzymes (MNAzyme) amplification procedure for simultaneous detection of RSV-A, RSV-B, and an Internal Extraction control IEC. Results: Compared to standard real-time PCR technology, this method resulted in an increased detection sensitivity, ranging from 0.9 to 4.93 log (average 2.05 +/- 1.01) for RSV-A and 0.76 to 4.28 log (average 1.30 +/- 0.92) for RSV-B. Conclusions: An ultrasone-based extraction method with MNAzyme amplification resulted in improved detection of RSV in different respiratory samples, including sputum. This generic method for nucleic acid extraction should be readily applicable for any other respiratory pathogen. (C) 2009 Elsevier B.V. All rights reserved

Pharmacokinetics-Pharmacodynamics of a Respiratory Syncytial Virus Fusion Inhibitor in the Cotton Rat Model▿

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, young children, elderly persons, and severely immunocompromised patients. Effective postinfection treatments are not widely available, and currently there is no approved vaccine. TMC353121 is a potent RSV fusion inhibitor in vitro, and its ability to reduce viral loads in vivo was demonstrated in cotton rats following prophylactic intravenous administration. Here, the pharmacokinetics of TMC353121 in the cotton rat, which is semipermissive for RSV replication, were further explored to build a pharmacokinetic-pharmacodynamic (PK-PD) model and to estimate the plasma drug levels needed for significant antiviral efficacy. TMC353121 reduced the viral titers in bronchoalveolar lavage fluid in a dose-dependent manner after a single subcutaneous administration and intranasal RSV inoculation 24 h after compound administration. The viral titer reduction and plasma TMC353121 concentration at the time of RSV inoculation were well described using a simple Emax model with a maximal viral titer reduction (Emax) of 1.5 log10. The plasma drug level required to achieve 50% of the Emax (200 ng/ml) was much higher than the 50% inhibitory concentration observed in vitro in HeLaM cells (0.07 ng/ml). In conclusion, this simple PK-PD approach may be useful in predicting efficacious exposure levels for future RSV inhibitors