TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat

The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.     

Evaluation of Two Influenza Surveillance Systems in South Africa

Background

The World Health Organisation recommends outpatient influenza-like illness (ILI) and inpatient severe acute respiratory illness (SARI) surveillance. We evaluated two influenza surveillance systems in South Africa: one for ILI and another for SARI.

Methodology

The Viral Watch (VW) programme has collected virological influenza surveillance data voluntarily from patients with ILI since 1984 in private and public clinics in all 9 South African provinces. The SARI surveillance programme has collected epidemiological and virological influenza surveillance data since 2009 in public hospitals in 4 provinces by dedicated personnel. We compared nine surveillance system attributes from 2009–2012.

Results

We analysed data from 18,293 SARI patients and 9,104 ILI patients. The annual proportion of samples testing positive for influenza was higher for VW (mean 41%) than SARI (mean 8%) and generally exceeded the seasonal threshold from May to September (VW: weeks 21–40; SARI: weeks 23–39). Data quality was a major strength of SARI (most data completion measures >90%; adherence to definitions: 88–89%) and a relative weakness of the VW programme (62% of forms complete, with limited epidemiologic data collected; adherence to definitions: 65–82%). Timeliness was a relative strength of both systems (e.g. both collected >93% of all respiratory specimens within 7 days of symptom onset). ILI surveillance was more nationally representative, financially sustainable and expandable than the SARI system. Though the SARI programme is not nationally representative, the high quality and detail of SARI data collection sheds light on the local burden and epidemiology of severe influenza-associated disease.

Conclusions

To best monitor influenza in South Africa, we propose that both ILI and SARI should be under surveillance. Improving ILI surveillance will require better quality and more systematic data collection, and SARI surveillance should be expanded to be more nationally representative, even if this requires scaling back on information gathered.     

HCV NS5B polymerase-bound conformation of a soluble sulfonamide inhibitor by 2D transferred NOESY

HCV NS5B RNA-dependent RNA polymerase (NS5B) is essential for viral replication and is therefore considered a target for antiviral drug development. From our ongoing screening effort in the search for new anti-HCV agents, a novel inhibitor 1 with low microM activity against the HCV NS5B polymerase was identified. SAR analysis indicated the optimal substitution pattern required for activity, for example, carboxylic acid group at 2-position of thiophene ring. We describe the steps taken to identify and solve the bioactive conformation of derivative 6 through the use of the transferred NOE method (trNOE).     

Three-dimensional reconstruction of the flagellar hook from Caulobacter crescentus

The structure of the bacterial flagellar hook produced by a mutant of Caulobacter crescentus was studied by electron microscopy, optical diffraction, and digital image processing techniques. The helical surface lattice of the hook is defined by a single, right-handed genetic helix having a pitch of about 23 Å, an axial rise per subunit of 4 Å and an azimuthal angle between subunits of 64·5 °. The lattice is also characterized by intersecting families of 5-start, 6-start and long-pitch 11-start helices. These helical parameters are remarkably similar to those determined for the flagellar filaments from several strains of gram-negative bacteria. The technique of three-dimensional image reconstruction (DeRosier & Klug, 1968) was applied to nine of the better preserved specimens and the diffraction data from five of these were correlated and averaged and used to generate an average three-dimensional model of the hook. The pattern of density modulations in the three-dimensional model is suggestive of an elongated, curved shape for the hook subunit (100 Å × 25 Å × 25 Å). The subunits are situated in the lattice of the polyhook such that their long axes are tilted about 45 ° with respect to the hook axis. The subunits appear to make contact with each other along the 6-start helices at a radius of 80 Å and also along the 11-start helices at a radius of 65 Å. Few structural features are revealed at radii between 15 å and 45 Å and, therefore, we are unable to decide to what extent the hook subunits extend into this region. The most striking characteristic of the model is the presence of deep, broad, continuous 6-start helical grooves extending from an inner radius of about 50 Å to the perimeter of the particle at 105 Å radius. Normal hooks usually appear curved in electron micrographs and sometimes so are the mutant hooks; the prominent 6-start grooves appear to allow for bending with minimal distortion of matter in the outer regions of the hook. A round stain-filled channel about 25 Å in diameter runs down the center of the polyhook. Such a channel supports a model for flagellar assembly in which flagellin subunits travel through the interior of the flagellum to the growing distal end of the filament.     

Eosinophils preserve parasitic nematode larvae by regulating local immunity

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.     

Site-specific 3D imaging of cells and tissues with a dual beam microscope

Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.     

Preferential mutation of the neurofibromatosis type 1 gene in paternally derived chromosomes

Summary An interesting feature of neurofibromatosis type 1 (NF1) is its high mutation rate of 1×10^–4 per gamete per generation. The molecular basis for frequent NF1 mutation in unknown; the gene is not deletion prone. We have found that in all ten families examined, the apparent new NF1 mutation occurred on the paternally-derived chromosome. The probability of observing this result by chance is less than 0.001 assuming an equal frequency of mutation of paternal and maternal NF1 genes. We hypothesize a role for genomic imprinting that may either enhance mutation of the paternal NF1 gene or confer protection from mutation to the maternal NF1 gene.     

Measles Outbreak in South Africa: Epidemiology of Laboratory-Confirmed Measles Cases and Assessment of Intervention, 2009–2011

Background

Since 1995, measles vaccination at nine and 18 months has been routine in South Africa; however, coverage seldom reached >95%. We describe the epidemiology of laboratory-confirmed measles case-patients and assess the impact of the nationwide mass vaccination campaign during the 2009 to 2011 measles outbreak in South Africa.

Methods

Serum specimens collected from patients with suspected-measles were tested for measles-specific IgM antibodies using an enzyme-linked immunosorbent assay and genotypes of a subset were determined. To estimate the impact of the nationwide mass vaccination campaign, we compared incidence in the seven months pre- (1 September 2009–11 April 2010) and seven months post-vaccination campaign (24 May 2010–31 December 2010) periods in seven provinces of South Africa.

Results

A total of 18,431 laboratory-confirmed measles case-patients were reported from all nine provinces of South Africa (cumulative incidence 37 per 100,000 population). The highest cumulative incidence per 100,000 population was in children aged <1 year (603), distributed as follows: <6 months (302/100,000), 6 to 8 months (1083/100,000) and 9 to 11 months (724/100,000). Forty eight percent of case-patients were ≥5 years (cumulative incidence 54/100,000). Cumulative incidence decreased with increasing age to 2/100,000 in persons ≥40 years. A single strain of measles virus (genotype B3) circulated throughout the outbreak. Prior to the vaccination campaign, cumulative incidence in the targeted vs. non-targeted age group was 5.9-fold higher, decreasing to 1.7 fold following the campaign (P<0.001) and an estimated 1,380 laboratory-confirmed measles case-patients were prevented.

Conclusion

We observed a reduction in measles incidence following the nationwide mass vaccination campaign even though it was conducted approximately one year after the outbreak started. A booster dose at school entry may be of value given the high incidence in persons >5 years.     

Deoxyribonucleic Acid Sequence Homologies Among Bacterial Insertion Sequence Elements and Genomes of Various Organisms

Plasmid and phage deoxyribonucleic acid (DNA) harboring bacterial insertion sequence (IS) elements IS1, IS2, and IS5 were characterized and used as probes to detect homologous sequences in various procaryotic and eucaryotic genomes. The hybridization method used permits the detection of sequences partially homologous to the elements. Hybridization of the IS-containing probes to each other revealed a region of limited homology between IS1 and IS2. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. The homologous sequence contains a tandemly repeated tetranucleotide sequence which resembles the repeated sequence at the hot spot for spontaneous mutations in the lacI gene (P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. Miller, J. Mol. Biol. 126:847-863, 1978). Homology between the IS elements and various genomes was determined by hybridizing labeled DNA containing IS1, IS2, and IS5 sequences to Southern blots of chromosomal DNA cleaved with restriction endonucleases. IS1 and IS5 appear limited to the enteric bacteria, whereas IS2 sequences can also be detected in Pseudomonas putida, Pseudomonas aeruginosa, and Serratia marcescens. Bacteria which appear not to possess extrachromosomal elements, e.g., Caulobacter crescentus, did not show homology with any insertion sequences tested. In addition, sequences homologous to IS1, IS2, or IS5 were not detected in Saccharomyces cerevisiae, Dictyostelium discoideum, or calf thymus DNA.     

De novo structure determination of 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid,a novel and abundant metabolite in Acinetobacter baylyi ADP1

Metabolomics - Macrophage metabolism contributes to the progression of metabolic diseases, and peroxisome proliferator-activated receptors (PPARs) play vital roles in macrophage metabolism and the...