Biology subjects

Mueller, M.

Publications and source records attributed to Mueller, M..

5 recordsLinked to original sources

bioRxiv2018

A cost-effective approach to Super-resolution Optical Fluctuation (SOFI) microscopy using an industry-grade CMOS camera

Super-resolution (SR) fluorescence microscopy, especially at high speeds, is typically carried out on high-performance research microscopes. The substantial cost of such equipment, combined with the limited distribution of such instruments in imaging facilities, have complicated access to super-resolution imaging for research groups in the biosciences. In this work we promote the accessibility of Super-resolution Optical Fluctuation Imaging (SOFI) to the scientific community by demonstrating its flexibility in terms of the minimal required performance of the imaging system. We show that SOFI data can be acquired using a very cost-efficient industrial-grade detector on both a standard research microscope and an entirely home-built wide-field system. This enables more scientists to enter the field of live-cell super-resolution imaging, as it provides access to a robust and fast SR imaging modality at comparatively modest cost.

biophysics↗

bioRxiv2018

Homeostatic and Interferon-induced gene expression represent different states of promoter-associated transcription factor ISGF3

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signalling cascades, including the JAK-STAT pathway. Our understanding how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. We used an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and, unexpectedly, IFN{gamma} cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.

immunology↗

bioRxiv2018

Longitudinal tracking of Plasmodium falciparum clones in complex infections by amplicon deep sequencing

BackgroundLongitudinal tracking of individual Plasmodium falciparum strains in multi-clonal infections is essential for investigating infection dynamics of malaria. The traditional genotyping techniques did not permit tracking changes in individual clone density during persistent natural infections. Amplicon deep sequencing (Amp-Seq) offers a tool to address this knowledge gap.\n\nMethodsThe sensitivity of Amp-Seq for relative quantification of clones was investigated using three molecular markers, ama1-D2, ama1-D3, and cpmp. Amp-Seq and length-polymorphism based genotyping were compared for their performance in following minority clones in longitudinal samples from Papua New Guinea.\n\nResultsAmp-Seq markers were superior to length-polymorphic marker msp2 in detecting minority clones (sensitivity Amp-Seq: 95%, msp2: 85%). Multiplicity of infection (MOI) by Amp-Seq was 2.32 versus 1.73 for msp2. The higher sensitivity had no effect on estimates of force of infection because missed minority clones were detected in preceding or succeeding bleeds. Individual clone densities were tracked longitudinally by Amp-Seq despite MOI>1, thus providing an additional parameter for investigating malaria infection dynamics.\n\nConclusionAmp-Seq based genotyping of longitudinal samples improves detection of minority clones and estimates of MOI. Amp-Seq permits tracking of clone density over time to study clone competition or the dynamics of specific, i.e. resistance-associated genotypes.

microbiology↗

bioRxiv2018

Human Papillomavirus Type 16 L1/L2 VLP Experimental Internalisation by Human Peripheral Blood Leukocytes

Human papillomavirus (HPV) accounts for hundreds of thousands of new cases of cervical cancer yearly, and half of these women die of this neoplasia. This study investigates the possibility of HPV16 L1/L2VLP to be internalized by human peripheral blood leukocytes in ex vivo assays. We have developed a leukocyte separation method from heparinized blood samples aiming cellular integrity and viability. We have expressed humanized L1 and L2 viral capsid proteins in HEK293T epithelial human cells, transiently transfecting them with vectors encoding humanized HPV16 L1 and L2 genes. Recombinant L1/L2 capsid proteins and structured virus-like particles interacted with human peripheral blood mononuclear cells, lymphocytes and monocytes, and were internalized through a pathway involving CD71 transferrin receptors. This was observed, at a percentile of about 54% T- CD4, 47% T-CD8, 48% B-CD20, and 23% for monocytes-CD14. The group of polymorph nuclear cells: neutrophils-eosinophils-basophils group did not internalize any VLPs. Blockage assays with biochemical inhibitors of distinct pathways, like chlorpromazine, rCTB, filipin, nystatin, liquemin, and sodium azide also evidentiated the occurrence of virus-like particles indiscriminate entrance via membrane receptor on mononuclear cells. This study shows that HPV16 L1/L2 VLPs can interact with the plasma membrane surface and successfully enter lymphocytes without requiring a specific receptor. Legend of the Graphical AbstractGraphical abstract showing ex vivo and in vitro internalization between VLPs and host cells. After leukocytes separation from human whole blood, it was performed the identification of human peripheral blood leukocytes in ex vivo interactions with VLPs. The graph shows that of the cells that interacted with VLPs, 52% corresponded to lymphocytes T-CD4, 47% lymphocytes T-CD8, 48% lymphocytes B-CD20, and only 23% of the monocytes CD14 interacted with these particles. However, monocytes apparently internalized larger amounts of particles when compared to lymphocytes. It is probable that in some T lymphocytes the amount of internalized particles has been imperceptible to the confocal microscope, since the VLPs produced in this research are around 50 nm in diameter. These results lead to two important implications. First, the interaction of VLPs with lymphocytes may result in the activation of these cells and, consequently, increase the population of these circulating cells, this being crucial in the induction of specific immune response. In the second implication, these lymphocytes would internalize small amounts of virus, insufficient to activate the immune system. Here it is important to note that lymphocytes are cells capable of dividing and it is estimated that the half-life of these inactive cells in humans is of some years. In addition, as it is known, inactive lymphocytes continually re-circulate through the bloodstream and lymphatic vessels. The percentage of cells that interacted with the HPV16 L1/L2 VLPs was calculated by the number of cells recognized by the anti-CD antibodies, which internalized these particles. The result corresponds to the analysis in duplicates, being representative of at least four tests. All images are original and cells were processed by Cianciarullo AM et al., at the Butantan Institute, Sao Paulo - SP, Brazil. Electron micrographs of human leukocytes, HEK293T and HPV16 L1/L2 VLPs were obtained in a Zeiss EM109 transmission electron microscope. The blue color of the VLPs and colored leukocytes were virtually attributed. Leukocyte and HEK293T present filamentous actin (red) and HPV16 L1/L2 VLPs (green), by fluorescence in a Confocal Zeiss LSM 510 Meta Microscope O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/299214v2_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@1ee89b8org.highwire.dtl.DTLVardef@193d0a6org.highwire.dtl.DTLVardef@1674526org.highwire.dtl.DTLVardef@ecd80d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2018

Estimating the Contribution of Proteasomal Spliced Peptides to the HLA-I Ligandome

Spliced peptides are short protein fragments spliced together in the proteasome by peptide bond formation. True estimation of the contribution of proteasome-spliced peptides (PSPs) to the global Human Leukocyte Antigen (HLA) ligandome is critical. A recent study suggested that PSPs contribute up to 30% of the HLA ligandome. We performed a thorough reanalysis of the reported results using multiple computational tools and various validation steps and concluded that only a fraction of the proposed PSPs passes the quality filters. To better estimate the actual number of PSPs, we present an alternative workflow. We performed de-novo sequencing of the HLA-peptide spectra and discarded all de-novo sequences found in the UniProt database. We checked whether the remaining de-novo sequences could match spliced peptides from human proteins. The spliced sequences were appended to the UniProt fasta file, which was searched by two search tools at a FDR of 1%. We find that maximally 2-4% of the HLA ligandome could be explained as spliced protein fragments. The majority of these potential PSPs have good peptide-spectrum match properties and are predicted to bind the respective HLA molecules. However, it remains to be shown how many of these potential PSPs actually originate from proteasomal splicing events.

bioinformatics↗