Analysing and Applying Stakeholder Perceptions to Improve Protected Area Governance in Ugandan Conservation Landscapes

Given the diversity of active institutions and stakeholders in a landscape, and the difficulties in ensuring inclusive decision-making, evaluating landscape governance can help surface and address underlying issues. In the context of two protected area landscapes in Uganda, where landscape approaches are being implemented through a wider project on landscape governance, we analyse stakeholder perceptions of inclusive decision-making and then use this evaluation to stimulate dialogue amongst stakeholder groups in each landscape. We ask, how can capturing, analysing, and collaboratively applying people’s perceptions address inclusive decision-making in landscape governance? We collected and analysed perceptions using SenseMaker®, a software package that enables analysis of micronarratives (stories) from the field based on how respondents classify their own stories, using triads, dyads, stones, and multiple-choice questions. This self-categorisation by the respondent reduces bias in the analysis and allows the micronarrative to be cross-examined in a variety of ways when analysed using Sensemaker. This analysis created an integrated view of the stakeholder’s perceptions about inclusive decision-making in landscape governance. The results show large portions of the respondents feel their voices are neglected, and management of the landscape is poor in Mount Elgon, while in Agoro-Agu, it is the opposite trend. During a community feedback process, reasons for these trends were discussed and solutions proposed. Some of the underlying factors include historical relationships with park authorities and displacement during park creation. To more precisely answer our research question, one could have extended stays in the communities studied in these landscapes, using ethnographic methods including interviews and participant observation; nonetheless, our method, including the feedback process, was an innovative and important way to confront our findings with the informants directly and foster collaborative action. We conclude that understanding people’s perceptions, including through participatory feedback, can significantly inform and improve management decisions, help resolve conflicts, and facilitate dialogue between different stakeholders in the landscape

Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

Conceived and designed the experiments: FS PC BC ES AM. Performed the experiments: FS RS ES PF SR. Analyzed the data: FS BC ES PF GEK PFC AM. Contributed reagents/materials/analysis tools: PC PB GC. Wrote the paper: FS GEK BC AM.The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.Yeshttp://www.plosone.org/static/editorial#pee

Multigene RNA Vector Based on Coronavirus Transcription

Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on human coronavirus 229E. We have constructed a prototype RNA vector containing the 5′ and 3′ ends of the human coronavirus genome, the entire human coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a human coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and human coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that human coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the human coronavirus 229E and have the ability to transduce human dendritic cells

Characterization of White Bream Virus Reveals a Novel Genetic Cluster of Nidoviruses

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5′ leader sequence that is identical to the 5′-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses

Human Coronavirus 229E Papain-Like Proteases Have Overlapping Specificities but Distinct Functions in Viral Replication

Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PL(pro)s). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PL(pro) activities. Reverse-genetics data revealed that replacement of the PL2(pro) active-site cysteine was lethal. By contrast, the PL1(pro) activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1(pro) active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1(pro) activity. Further experiments showed that both PL1(pro) and PL2(pro) were able to cleave the nsp1-nsp2 cleavage site, with PL2(pro) cleaving the site less efficiently. The PL1(pro)-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1(pro) mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PL(pro) paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2(pro) plays a universal and essential proteolytic role that appears to be assisted by the PL1(pro) paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed

Fachliche Lernprozesse in Interaktionen: Wissenssoziologische Modellierung und dokumentarische Rekonstruktion

Asbrand B, Hackbarth A. Fachliche Lernprozesse in Interaktionen: Wissenssoziologische Modellierung und dokumentarische Rekonstruktion. In: Martens M, Rabenstein K, Bräu K, et al., eds. Konstruktionen von Fachlichkeit. Ansätze, Erträge und Diskussionen in der empirischen Unterrichtsforschung. Bad Heilbrunn: Klinkhardt; 2018: 139-151

Recombinant Mouse Hepatitis Virus Strain A59 from Cloned, Full-Length cDNA Replicates to High Titers In Vitro and Is Fully Pathogenic In Vivo

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture (∼10(9) PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus