Seroprevalence of Alphavirus Antibodies in a Cross-Sectional Study in Southwestern Tanzania Suggests Endemic Circulation of Chikungunya

Background

To date, Alphavirus infections and their most prominent member, chikungunya fever, a viral disease which first became apparent in Tanzania in 1953, have been very little investigated in regions without epidemic occurrence. Few data exist on burden of disease and socio-economic and environmental covariates disposing to infection.

Methods

A cross-sectional seroprevalence study was undertaken in 1,215 persons from Mbeya region, South-Western Tanzania, to determine the seroprevalence of anti-Alphavirus IgG antibodies, and to investigate associated risk factors.

Results

18% of 1,215 samples were positive for Alphavirus IgG. Seropositivity was associated with participant age, low to intermediate elevation, flat terrain and with IgG positivity for Rift Valley fever, Flaviviridae, and rickettsiae of the spotted fever group. When comparing the geographical distribution of Alphavirus seropositivity to that of Rift Valley fever, it was obvious that Alphaviruses had spread more widely throughout the study area, while Rift Valley fever was concentrated along the shore of Lake Malawi.

Conclusion

Alphavirus infections may contribute significantly to the febrile disease burden in the study area, and are associated with several arthropod-borne infections. Their spread seems only limited by factors affecting mosquitoes, and seems less restricted than that of Rift Valley fever.     

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.     

Centrosome linker protein C‐Nap1 maintains stem cells in mouse testes

The centrosome linker component C‐Nap1 (encoded by CEP250) anchors filaments to centrioles that provide centrosome cohesion by connecting the two centrosomes of an interphase cell into a single microtubule organizing unit. The role of the centrosome linker during development of an animal remains enigmatic. Here, we show that male CEP250 ^−/− mice are sterile because sperm production is abolished. Premature centrosome separation means that germ stem cells in CEP250 ^−/− mice fail to establish an E‐cadherin polarity mark and are unable to maintain the older mother centrosome on the basal site of the seminiferous tubules. This failure prompts premature stem cell differentiation in expense of germ stem cell expansion. The concomitant induction of apoptosis triggers the complete depletion of germ stem cells and consequently infertility. Our study reveals a role for centrosome cohesion in asymmetric cell division, stem cell maintenance, and fertility.     

Breaking the ties that bind: new advances in centrosome biology

The centrosome, which consists of two centrioles and the surrounding pericentriolar material, is the primary microtubule-organizing center (MTOC) in animal cells. Like chromosomes, centrosomes duplicate once per cell cycle and defects that lead to abnormalities in the number of centrosomes result in genomic instability, a hallmark of most cancer cells. Increasing evidence suggests that the separation of the two centrioles (disengagement) is required for centrosome duplication. After centriole disengagement, a proteinaceous linker is established that still connects the two centrioles. In G2, this linker is resolved (centrosome separation), thereby allowing the centrosomes to separate and form the poles of the bipolar spindle. Recent work has identified new players that regulate these two processes and revealed unexpected mechanisms controlling the centrosome cycle.     

AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Can spelt wheat be used as heterotic group for hybrid wheat breeding?

Key message

Spelt wheat is a distinct genetic group to elite bread wheat, but heterosis for yield and protein quality is too low for spelt to be recommended as heterotic group for hybrid breeding in wheat.

Abstract

The feasibility to switch from line to hybrid breeding is currently a hot topic in the wheat community. One limitation seems to be the lack of divergent heterotic groups within wheat adapted to a certain region. Spelt wheat is a hexaploid wheat that can easily be crossed with bread wheat and that forms a divergent genetic group when compared to elite bread wheat. The aim of this study was to investigate the potential of Central European spelt as a heterotic group for Central European bread wheat. We performed two large experimental field studies comprising in total 43 spelt lines, 14 wheat lines, and 273 wheat–spelt hybrids, and determined yield, heading time, plant height, resistance against yellow rust, leaf rust, and powdery mildew, as well as protein content and sedimentation volume. Heterosis of yield was found to be lower than that of hybrids made between elite wheat lines. Moreover, heterosis of the quality trait sedimentation volume was negative. Consequently, spelt wheat does not appear suited to be used as heterotic group in hybrid wheat breeding. Nevertheless, high combining abilities of a few spelt lines with elite bread wheat lines make them interesting resources for pre-breeding in bread wheat. Thereby, the low correlation between line per se performance and combining ability of these spelt lines shows the potential to unravel the breeding value of genetic resources by crossing them to an elite tester.