Editorial: Towards leaner and more effective value chain development

Value chain development (VCD) is a common term in today’s development lexicon1, where its use tends to conjure passionate ideas about how development programming can support smallholder participation in growing markets in the interest of economic growth, job creation, gender empowerment, and sustainable use of natural resources, among other goals. Since the early 2000s, Enterprise Development and Microfinance (EDM) has featured considerable debate on how to design market-oriented development interventions with smallholders, often based on positive experiences by a given NGO or project in a particular context. Early articles helped to put VCD on the development agenda, while advancing innovation in market-based project design and implementation. However, after more than a decade of it being firmly placed on the agenda, we still know relatively little about VCD. Apart from isolated case studies, the question of whether VCD has lived up to the expectations of smallholders, of the private sector, and of development agencies remains an open one. This double edition of EDM addresses the design, implementation, and impact of VCD support to smallholders and to small and medium enterprises (SMEs) as an important, yet under-researched dimension of VCD. The eight articles look into the needs and opportunities for increasing the effectiveness of VCD support services, with discussions on: the role of NGOs or governments in VCD; how large-scale buyers and certification programs shape VCD; and the role of finance and impact bonds in VCD. Advancing ideas on how to get the right mix of services, at the right time, to the right people, taking into account variations in the context in which livelihoods and business activities are embedded, will help stakeholders to effectively deliver on poverty and broader development goals

Evaluation of Parameters for Confident Phosphorylation Site Localization using an Orbitrap Fusion Tribrid Mass Spectrometer

Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high throughput phosphoproteomics. Here, we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform, using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. As well as evaluating multiple fragmentation regimes (HCD, EThcD and neutral loss triggered ET(ca/hc)D), and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with ptmRS, for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high resolution orbitrap-based MS/MS analysis, employing MASCOT/ptmRS for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphosites identified. Finally, our data highlights that a charge-state dependent fragmentation regime, and a multiple algorithm search strategy, are likely to be of benefit for confident large-scale phosphosite localization

Produção de edificações sustentáveis: desafios e alternativas

A discussão do projeto e execução de edificações sustentáveis vem sendo abordada por diferentes autores, em geral com foco no desempenho da edificação e vida útil dos materiais. Entretanto, para atender aos requisitos de desempenho ambiental das edificações, existe também a necessidade de se introduzir mudanças na organização e gestão do processo projetual. Este artigo apresenta os resultados de uma pesquisa que investigou como a incorporação dos requisitos da sustentabilidade ambiental podem influenciar as práticas de projeto de arquitetura e engenharia. Reconhecendo a influência francesa na produção de edificações sustentáveis no Brasil - em decorrência da adoção por algumas empresas brasileiras do referencial Aqua (adaptado do HQE® francês) - , a pesquisa foi realizada junto a empresas que atuam naquele país. Os resultados apontam que a melhoria do processo de projeto depende do estabelecimento de um sistema de gestão que possa auxiliar os profissionais a lidarem com o grande número requisitos relacionados com a edificação sustentável. Também indicam a necessidade de revisão dos esquemas adotados até então, de forma a viabilizar a introdução da interoperabilidade entre os diferentes profissionais de projeto, desde a concepção arquitetônica, que deve se tornar responsabilidade de toda a equipe de projeto

KinView: A visual comparative sequence analysis tool for integrated kinome research

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory

Split T Cell Tolerance against a Self/Tumor Antigen: Spontaneous CD4+ but Not CD8+ T Cell Responses against p53 in Cancer Patients and Healthy Donors

Analyses of NY-ESO-1-specific spontaneous immune responses in cancer patients revealed that antibody and both CD4+ and CD8+ T cell responses were induced together in cancer patients. To explore whether such integrated immune responses are also spontaneously induced for other tumor antigens, we have evaluated antibody and T cell responses against self/tumor antigen p53 in ovarian cancer patients and healthy individuals. We found that 21% (64/298) of ovarian cancer patients but no healthy donors showed specific IgG responses against wild-type p53 protein. While none of 12 patients with high titer p53 antibody showed spontaneous p53-specific CD8+ T cell responses following a single in vitro sensitization, significant p53-specific IFN-γ producing CD4+ T cells were detected in 6 patients. Surprisingly, similar levels of p53-specific CD4+ T cells but not CD8+ T cells were also detected in 5/10 seronegative cancer patients and 9/12 healthy donors. Importantly, p53-specific CD4+ T cells in healthy donors originated from a CD45RA− antigen-experienced T cell population and recognized naturally processed wild-type p53 protein. These results raise the possibility that p53-specific CD4+ T cells reflect abnormalities in p53 occurring in normal individuals and that they may play a role in processes of immunosurveillance or immunoregulation of p53-related neoplastic events

Covalent Aurora A regulation by the metabolic integrator coenzyme A

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues