Trifluoromethylthiolation, Trifluoromethylation, and Arylation Reactions of Difluoro Enol Silyl Ethers.

This study reports a new application area of difluoro enol silyl ethers, which can be easily obtained from trifluoromethyl ketones. The main focus has been directed to the electrophilic fluoroalkylation and arylation methods. The trifluoromethylthiolation of difluoro enol silyl ethers can be used for the construction of a novel trifluoromethylthio-α,α-difluoroketone (-COCF2SCF3) functionality. The -CF2SCF3 moiety has interesting properties due to the electron-withdrawing, albeit lipophilic, character of the SCF3 group, which can be combined with the high electrophilicity of the difluoroketone motif. The methodology could also be extended to difluoro homologation of the trifluoromethyl ketones using the Togni reagent. In addition, we presented a method for transition-metal-free arylation of difluoro enol silyl ethers based on hypervalent iodines.

Reemergence of Classical Swine Fever, Japan, 2018.

In September 2018, classical swine fever reemerged in Japan after 26 years, affecting domestic pigs and wild boars. The causative virus belongs to the 2.1 subgenotype, which caused repeated outbreaks in eastern and Southeast Asia. Intensive surveillance of swine and vaccination of wild boars will help control and eradicate this disease in Japan.

Impact of Thrombus Length on Outcomes After Intra-Arterial Aspiration Thrombectomy in the THERAPY Trial.

BACKGROUND AND PURPOSE: Increasing thrombus length (TL) impedes recanalization after intravenous (IV) thrombolysis. We sought to determine whether the clinical benefit of aspiration thrombectomy relative to IV r-tPA (recombinant tissue-type plasminogen activator) may be greater at longer TL. METHODS: THERAPY was a randomized trial of aspiration thrombectomy plus IV r-tPA versus IV r-tPA alone in large-vessel stroke patients with prospective TL measurement ≥8 mm. In this post hoc study, we evaluated the association of TL with trial end points and potential endovascular treatment effect, using univariate, multivariable, and multiplicative interaction analyses. RESULTS: TL data were available for all 108 patients (28% internal carotid artery, 62% M1, and 10% M2). Median TL was 14.0 mm (interquartile range, 9.7-19.5 mm). Longer TL was associated with worse outcome (90-day modified Rankin Scale score: odds ratio, 1.24 per 5-mm TL increment; 95% confidence interval, 1.04-1.52; P=0.02), even after adjusting for key outcome predictors (adjusted P=0.004). Longer TL was also associated with more serious adverse events (adjusted P=0.01), more symptomatic hemorrhages (adjusted P=0.03), and increased mortality (adjusted P=0.01). No significant relationship was observed between TL and angiographic reperfusion (modified thrombolysis in cerebral ischemia 2b-3), but greater TL was associated with longer endovascular procedural times (ρ=0.36; P=0.045). Increasing TL was associated with greater aspiration thrombectomy treatment effect (interaction term P=0.03). This might be related to a potentially stronger adverse effect of increasing TL on 90-day modified Rankin Scale for patients treated with IV r-tPA (ρ=0.39; P=0.01) compared with intra-arterial therapy (ρ=0.20; P=0.165). CONCLUSIONS: Ischemic stroke patients with longer symptomatic thrombi have worse 90-day clinical outcomes but may have a greater relative benefit of aspiration thrombectomy over IV r-tPA alone. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01429350.

High Abundance and Genetic Variability of Atypical Porcine Pestivirus in Pigs from Europe and Asia.

Atypical porcine pestivirus (APPV) was recently reported to be associated with neurologic disorders in newborn piglets. Investigations of 1,460 serum samples of apparently healthy pigs from different parts of Europe and Asia demonstrate a geographically wide distribution of genetically highly variable APPV and high APPV genome and antibody detection rates.

Development of a new LAMP assay for the detection of CSFV strains from Cuba: a proof-of-concept study.

Classical swine fever (CSF) is a devastating animal disease of great economic impact worldwide. In many countries, CSF has been endemic for decades, and vaccination of domestic pigs is one of the measures to control the disease. Consequently, differentiating infected from vaccinated animals by antibody ELISA screening is not applicable. In some countries, such as Cuba, lack of molecular techniques for sensitive, rapid and reliable detection of virus genomes is a critical point. To overcome this problem, an easy-to-use one-tube assay based on the loop-mediated isothermal amplification (LAMP) principle has been developed for detection of the genome of CSF virus (CSFV) of endemic Cuban genotype 1.4 isolates. The assay reliably detected recent isolates from three different regions of Cuba with an analytical sensitivity 10-100 times lower than that of quantitative reverse transcription RT-qPCR. Diagnostic test sensitivity was examined using reference sera from two groups of pigs experimentally infected with Cuban virulent strain CSF0705 "Margarita" and the recent field isolate CSF1058 "Pinar del Rio". Differences in pathogenicity of the two viruses were reflected in the clinical course of disease as well as in virus loads of blood samples. Low viral RNA loads in samples from pigs infected with the field isolate caused serious detection problems in RT-LAMP as well as in RT-qPCR. Thus, it will be necessary in future research to focus on targeted sampling of diseased animals and to restrict diagnosis to the herd level in order to establish LAMP as an efficient tool for diagnosing CSF under field conditions.

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis.

The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers' yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations and RNA interference-mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammonium stress.

Arabidopsis SET DOMAIN GROUP2 is required for H3K4 trimethylation and is crucial for both sporophyte and gametophyte development.

Histone H3 lysine 4 trimethylation (H3K4me3) is abundant in euchromatin and is in general associated with transcriptional activation in eukaryotes. Although some Arabidopsis thaliana SET DOMAIN GROUP (SDG) genes have been previously shown to be involved in H3K4 methylation, they are unlikely to be responsible for global genome-wide deposition of H3K4me3. Most strikingly, sparse knowledge is currently available about the role of histone methylation in gametophyte development. In this study, we show that the previously uncharacterized SDG2 is required for global H3K4me3 deposition and its loss of function causes wide-ranging defects in both sporophyte and gametophyte development. Transcriptome analyses of young flower buds have identified 452 genes downregulated by more than twofold in the sdg2-1 mutant; among them, 11 genes, including SPOROCYTELESS/NOZZLE (SPL/NZZ) and MALE STERILITY1 (MS1), have been previously shown to be essential for male and/or female gametophyte development. We show that both SPL/NZZ and MS1 contain bivalent chromatin domains enriched simultaneously with the transcriptionally active mark H3K4me3 and the transcriptionally repressive mark H3K27me3 and that SDG2 is specifically required for the H3K4me3 deposition. Our data suggest that SDG2-mediated H3K4me3 deposition poises SPL/NZZ and MS1 for transcriptional activation, forming a key regulatory mechanism in the gene networks responsible for gametophyte development.

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus.

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

Prevention of early flowering by expression of FLOWERING LOCUS C requires methylation of histone H3 K36.

Flowering represents a crucial transition from a vegetative to a reproductive phase of the plant life cycle. Despite extensive studies, the molecular mechanisms controlling flowering remain elusive. Although the enzymes involved are unknown, methylation of histone H3 K9 and K27 correlates with repression of FLOWERING LOCUS C (FLC), an essential transcriptional repressor involved in flowering time control in Arabidopsis thaliana; in contrast, methylation of H3K4 correlates with FLC activation. Here we show that loss-of-function of SET DOMAIN GROUP 8 (SDG 8), which encodes a homologue of the yeast SET2 histone methyltransferase, results in reduced dimethylation of histone H3K36, particularly in chromatin associated with the FLC promoter and the first intron, regions that contain essential cis-elements for transcription. sdg8 mutants display reduced FLC expression and flower early, establishing SDG8-mediated H3K36 methylation as a novel epigenetic memory code required for FLC expression in preventing early flowering. This is the first demonstrated role of H3K36 methylation in eukaryote development.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protects Pigs against PCV2b Challenge and Induces Serum Neutralizing Antibody Response against CSFV.

Porcine circovirus type 2 (PCV2) is endemic worldwide. PCV2 causes immunosuppressive infection. Co-infection of pigs with other swine viruses, such as pseudorabies virus (PRV) and classical swine fever virus (CSFV), have fatal outcomes, causing the swine industry significant economic losses in many if not all pig-producing countries. Currently available inactivated/modified-live/vectored vaccines against PCV2/CSFV/PRV have safety and efficacy limitations. To address these shortcomings, we have constructed a triple gene (thymidine kinase, glycoprotein E [gE], and gG)-deleted (PRVtmv) vaccine vector expressing chimeric PCV2b-capsid, CSFV-E2, and chimeric Erns-fused with bovine granulocytic monocyte-colony stimulating factor (Erns-GM-CSF), designated as PRVtmv+, a trivalent vaccine. Here we compared this vaccine's immunogenicity and protective efficacy in pigs against wild-type PCV2b challenge with that of the inactivated Zoetis Fostera Gold PCV commercial vaccine. The live PRVtmv+ prototype trivalent subunit vaccine is safe and highly attenuated in pigs. Based on PCV2b-specific neutralizing antibody titers, viremia, viral load in lymphoid tissues, fecal-virus shedding, and leukocyte/lymphocyte count, the PRVtmv+ yielded better protection for vaccinated pigs than the commercial vaccine after the PCV2b challenge. Additionally, the PRVtmv+ vaccinated pigs generated low to moderate levels of CSFV-specific neutralizing antibodies.

Comparative Analysis of Tunisian Sheep-like Virus, Bungowannah Virus and Border Disease Virus Infection in the Porcine Host.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Base-catalysed 18F-labelling of trifluoromethyl ketones. Application to the synthesis of 18F-labelled neutrophil elastase inhibitors.

A new method for the fluorine-18 labelling of trifluoromethyl ketones has been developed. This method is based on the conversion of a-COCF3 functional group to a difluoro enol silyl ether followed by halogenation and fluorine-18 labelling. The utility of this new method was demonstrated by the synthesis of fluorine-18 labelled neutrophil elastase inhibitors, which are potentially useful for detection of inflammatory disorders.

Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus.

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana.

Post-translational modifications of proteins by addition of ubiquitin can regulate protein degradation and localization, protein-protein interactions and transcriptional activation. In the ubiquitylation system, substrate specificity is primarily determined by the E2 ubiquitin-conjugating enzyme (UBC) and the E3 ubiquitin ligase. The Arabidopsis thaliana genome contains 37 genes encoding UBC homologs. However, the biological functions of these genes remain largely uncharacterized. Here, we report reverse genetic characterization of AtUBC1 and AtUBC2. While the loss-of-function single mutants Atubc1-1 and Atubc2-1 only show weak phenotypes, the double mutant Atubc1-1 Atubc2-1 shows a dramatically reduced number of rosette leaves and an early-flowering phenotype. Consistent with these results, the transcript levels of the floral repressor genes FLOWERING LOCUS C (FLC), MADS ASSOCIATED FLOWERING 4 (MAF4) and MAF5 are reduced in the double mutant. Loss-of-function mutants of HISTONE MONOUBIQUITINATION 1 (HUB1) and HUB2, which were previously reported to encode an E3 involved in histone H2B ubiquitylation, also show an early-flowering phenotype and reduced levels of FLC, MAF4 and MAF5 transcripts. In both Atubc1-1 Atubc2-1 and hub2-2 mutants, H2B mono-ubiquitylation is drastically reduced. Taken together, our results indicate that E2s AtUBC1/AtUBC2 and E3s HUB1/HUB2 together mediate H2B ubiquitylation, which is involved in the activation of floral repressor genes as well as in other processes as indicated by the pleiotropic phenotypes of the mutants.

Inactivation of Classical Swine Fever Virus in Porcine Serum Samples Intended for Antibody Detection.

Shipping of serum samples that were taken from pigs infected with classical swine fever (CSF) virus is frequently requested with the objective of serological analyses, not only for diagnostic purposes but also for exchange of reference materials that are used as control material of diagnostic assays. On the basis of the fact that an outbreak with CSF is associated with enormous economic losses, biological safety during the exchange of reference material is of great importance. The present study aimed to establish a pragmatic approach for reliable CSF virus (CSFV) inactivation in serum without impairing antibody detection. Considering the fact that complement inactivation through heating is routinely applied, the basic idea was to combine heat treatment with the dilution of serum in a detergent containing buffer in order to facilitate the inactivation process. The results show that treatment of serum samples with phosphate buffered saline-Tween20 (final concentration = 0.15%) along with incubation at 56 °C for 30 min inactivated CSFV and such treatment with ≤ 0.25% PBS-Tween20 does not impair subsequent antibody detection by ELISA or virus neutralization test. This minimizes the risk of virus contamination and represents a valuable contribution to a safer CSF diagnosis on a national and international level.

Characterization of the Humoral Immune Response Induced after Infection with Atypical Porcine Pestivirus (APPV).

Atypical porcine pestivirus (APPV) is a widely distributed pathogen causing congenital tremor (CT) in piglets. So far, no data are available regarding the humoral immune response against APPV. In this study, piglets and their sows from an affected herd were tested longitudinally for viral genome and antibodies. APPV genome was detected in the majority of the piglets (14/15) from CT affected litters. Transient infection of gilts was observed. Kinetics of Erns- and E2-specific antibodies and their neutralizing capacity were determined by recently (Erns) and newly (E2) developed antibody ELISAs and virus neutralization assays. Putative maternally derived antibodies (MDA) were detected in most piglets, but displayed only low to moderate neutralizing capacity (ND50 ≤ 112). Horizontal APPV transmission occurred when uninfected and infected piglets were mingled on the flat deck. Horizontally infected piglets were clinically inapparent and showed only transient viremia with subsequently consistently high E2 antibody levels. For piglets from CT affected litters, significantly lower neutralizing antibody titers were observed. Results indicate that E2 represents the main target of neutralizing antibodies. Characterization of the humoral immune response against APPV will help to provide valuable serological diagnosis, to understand the epidemiology of this novel pathogen, and to implement tailored prevention strategies.

Further characterization of bovine hepacivirus: Antibody response, course of infection, and host tropism.

Bovine hepacivirus (BovHepV) is a recently added member to the growing genus Hepacivirus within the family Flaviviridae. Animal hepaciviruses are rarely characterized so far. Apart from norway rat hepacivirus which represents a promising HCV surrogate model, only equine hepaciviruses have been studied to some extent. BovHepV has been initially identified in bovine samples and was shown to establish persistent infections in cattle. However, consequences of those chronic infections, humoral immune response and the possibility of an extended host spectrum have not been explored so far. Therefore, we here investigated (a) the presence of anti-NS3-antibodies and viral RNA in cattle herds in Germany, (b) the course of infection in cattle, and (c) the host tropism including zoonotic potential of bovine hepaciviruses. Our results show that 19.9% of investigated bovine serum samples had antibodies against BovHepV. In 8.2% of investigated samples, viral RNA was detected. Subsequent genetic analysis revealed a novel genetic cluster of BovHepV variants. For 25 selected cattle in a BovHepV positive herd the presence of viral genomic RNA was monitored over one year in two to three months intervals by RT-PCR in order to discriminate acute versus persistent infection. In persistently infected animals, no serum antibodies were detected. Biochemical analyses could not establish a link between BovHepV infection and liver injury. Apart from a single sample of a pig providing a positive reaction in the antibody test, neither BovHepV-specific antibodies nor viral RNA were detected in porcine, equine or human samples implying a strict host specificity of BovHepV.

[18F]fluoro-benziodoxole: a no-carrier-added electrophilic fluorinating reagent. Rapid, simple radiosynthesis, purification and application for fluorine-18 labelling.

Operationally simple radiosynthesis and purification of [18F]fluoro-benziodoxole was developed starting from a cyclotron produced [18F]F- precursor, [18F]TBAF, and tosyl-benziodoxole. The synthetic utility of [18F]fluoro-benziodoxole was demonstrated by electrophilic fluorocyclization of o-styrilamides proceeding with high RCC (typically 50-90%) and high molar activity (up to 396 GBq µmol-1).

Copper-catalyzed synthesis of allenylboronic acids. Access to sterically encumbered homopropargylic alcohols and amines by propargylboration.

Tri- and tetrasubstituted allenylboronic acids were prepared via a new versatile copper-catalyzed methodology. The densely functionalized allenylboronic acids readily undergo propargylboration reactions with ketones and imines without any additives. Catalytic asymmetric propargylborylation of ketones is demonstrated with high stereoselectivity allowing for the synthesis of highly enantioenriched tertiary homopropargyl alcohols. The reaction is suitable for kinetic resolution of racemic allenylboronic acids affording alkynes with adjacent quaternary stereocenters.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins.