Biology subjects

Mueller, M.

Publications and source records attributed to Mueller, M..

88 records · Page 5Linked to original sources

bioRxiv2020

Active focus stabilization for fluorescence microscopy by tracking actively generated infrared spots.

Fluorescent time-lapse experiments often suffer from focus drift, regularly rendering long measurements partially unusable. Frequently, this instability can be traced back to the specific mechanical components of the setup, but even in highly robust implementations z-drift occurs due to small temperature fluctuations which are hard to avoid. To resolve this issue, microscope manufacturers often offer their own interpretation of out-of-focus correction modules for their flagship instruments. However, self-assembled or older systems typically have to fend for their own or adapt their measurements to circumvent drift effects. In this manuscript, we propose a cost-efficient z-drift detection- and correction system that, due to its modular design, can be attached to any fluorescence microscope with an actuated stage or objective, be it in a custom or commercial setup. The reason for this wide applicability is specific to the design, which has a straightforward alignment procedure and allows sharing optics with the fluorescent emission path. Our system employs an infrared (IR) laser that is passed through a double-hole mask to achieve two parallel beams which are made to reflect on the coverslip and subsequently detected on an industrial sCMOS camera. The relative position of these beams is then uniquely linked to the z-position of a microscope-mounted sample. The system was benchmarked by introducing temperature perturbations, where it was shown to achieve a stable focus, and by scanning different positions while simulating a perturbation in the z-position of the stage, where we show that a lost focus can be recovered within seconds.

biochemistry↗

bioRxiv2020

Thin Film Metallization Stacks Serve as Reliable Conductors on Ceramic-based Substrates for Active Implants

Hermetic and non-hermetic packages of active implantable medical devices are often fabricated of ceramics like alumina. Screen printed PtAu paste is the state of the art metallization for functional structures. Due to solid state and liquid diffusion of Au at thermal exposure, solder times are limited; otherwise metal structures tend to delaminate. Moreover, it was shown that PtAu with solder fails after 37.4 years. We established a thin film metallization on alumina process to overcome these disadvantages. The metallization consists of sputtered platinum with an underlying adhesion layer made of tungsten-titanium to increase the adhesion strength to the alumina substrate. We avoided using gold in this work due to its high diffusion tendency. Instead, the materials in use provide relatively low diffusion properties, which potentially increases the long term mechanical performance and usability during assembly and packaging.Utilizing the Design of Experiment (DoE) methodology, we derived an optimal Pt thickness of 500 nm with 43 nm of WTi as adhesion promoting layer. After accelerated aging at 150 °C, corresponding to 125 years at body temperature (37 °C), the contact pad adhesion strength was with 32.75 MPa ± 7.08 MPa still significantly higher than the safety limit of 17 MPa, following the recommendations for a robust screen-printing metallization process. Moreover, soldering times of up to 120 s did not influence the adhesive strength. The new process reduced the minimum track distance to 50% of screen printing values and is capable of rapid prototyping. It helps to make the assembly process independent of the manufacturing person, to increase the yield of device fabrication and -most important in implantable device manufacturing-to make it more robust and thereby more safe for the patient.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2020

Dendritic integration in olfactory bulb granule cells: Thresholds for lateral inhibition and role of active conductances upon simultaneous activation.

The inhibitory axonless olfactory bulb granule cells (GCs) form reciprocal dendrodendritic synapses with mitral and tufted cells via large spines, mediating recurrent and lateral inhibition. Rat GC dendrites are excitable by local Na+ spine spikes and global Ca2+- and Na+-spikes. To investigate the transition from local to global signaling without Na+ channel inactivation we performed simultaneous holographic two-photon uncaging in acute brain slices, along with whole-cell recording and dendritic Ca2+ imaging. Less than 10 coactive reciprocal spines were sufficient to generate diverse regional and global signals that also included local dendritic Ca2+- and Na+-spikes (D-spikes). Individual spines could sense the respective signal transitions as increments in Ca2+ entry. Dendritic integration was mostly linear until a few spines below global Na+-spike threshold, where often D-spikes set in. NMDARs strongly contributed to active integration, whereas morphological parameters barely mattered. In summary, thresholds for GC-mediated bulbar lateral inhibition are low.

neuroscience↗

bioRxiv2019

Ibotenic acid biosynthesis in the fly agaric is initiated by glutamate hydroxylation

The fly agaric, Amanita muscaria, is widely known for its content of the psychoactive metabolites ibotenic acid and muscimol. 50 years ago, their biosynthesis was hypothesized to start with 3-hydroxyglutamate. Here, we build on this hypothesis by the identification and recombinant production of a glutamate hydroxylase from A. muscaria. The corresponding gene is surrounded by six other genes, which we link to ibotenic acid production using recent genetic data. Our data provide new insights into a decades-old question concerning a centuries-old drug.

biochemistry↗

bioRxiv2019

Rapid and Sustained Homeostatic Control of Presynaptic Exocytosis at a Central Synapse

Animal behavior is remarkably robust despite constant changes in neural activity. Homeostatic plasticity stabilizes central nervous system (CNS) function on time scales of hours to days. If and how CNS function is stabilized on more rapid time scales remains unknown. Here we discovered that mossy fiber synapses in the mouse cerebellum homeostatically control synaptic efficacy within minutes after pharmacological glutamate receptor impairment. This rapid form of homeostatic plasticity is expressed presynaptically. We show that modulations of readily-releasable vesicle pool size and release probability normalize synaptic strength in a hierarchical fashion upon acute pharmacological and prolonged genetic receptor perturbation. Presynaptic membrane capacitance measurements directly demonstrate regulation of vesicle pool size upon receptor impairment. Moreover, presynaptic voltage-clamp analysis revealed increased calcium-current density under specific experimental conditions. Thus, homeostatic modulation of presynaptic exocytosis through specific mechanisms stabilizes synaptic transmission in a CNS circuit on time scales ranging from minutes to months. Rapid presynaptic homeostatic plasticity may ensure stable neural circuit function in light of rapid activity-dependent plasticity.

neuroscience↗

bioRxiv2019

Surface proteome dynamics during neuronal development and synaptic plasticity

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization. While broad brain transcriptome, connectome and global proteome maps are being generated, system-wide analysis of temporal protein dynamics at the subcellular level are currently lacking for neuronal development and synapse formation. We performed a temporally-resolved surfaceome analysis of developing primary neuron cultures to a depth of 1000 bona fide surface proteins and reveal dynamic surface protein clusters that reflect the functional requirements during distinct stages of neuronal development. Moreover, our data shows that synaptic proteins are globally trafficked to the surface prior to synapse formation. Direct comparison of surface and total protein pools demonstrates that, depending on the time scale, surface abundance changes can correlate or differ from total protein abundance. The uncoupling of surface and total abundance changes has direct functional implications as shown in the context of synaptic vesicle transport. To demonstrate the utility of our approach we analyzed the surfaceome modulation in response to homeostatic synaptic scaling and found dynamic remodeling of the neuronal surface, which was largely independent of global proteostasis, indicative of wide-spread regulation on the level of surface trafficking. Finally, we present a quantitative analysis of the neuronal surface during early-phase long-term potentiation (LTP) and reveal fast externalization of diverse classes of surface proteins beyond the AMPA receptor, providing new insights into the requirement of exocytosis for LTP. Our resource and finding of organizational principles highlight the importance of subcellular resolution for systems-level understanding of cellular processes, which are typically masked by broad omics-style approaches.

neuroscience↗

bioRxiv2019

On the Reliability of Chronically Implanted Thin-Film Electrodes in Human Arm Nerves for Neuroprosthetic Applications

Direct stimulation of peripheral nerves can successfully provide sensory feedback to amputees while using hand prostheses. Recent clinical studies have addressed this important limitation of current prostheses solutions using different implantable electrode concepts. Longevity of the electrodes is key to success. We have improved the long-term stability of the polyimide-based transverse intrafascicular multichannel electrode (TIME) that showed promising performance in clinical trials by integration of silicon carbide adhesion layers. The TIMEs were implanted in the median and ulnar nerves of three trans-radial amputees for up to six months. Here, we present the characterization of the electrical properties of the thin-film metallization as well as material status post explantationem for the first time. The TIMEs showed reliable performance in terms of eliciting sensation and stayed within the electrochemical safe limits maintaining a good working range with respect to amplitude modulation. After termination of the trials and explantation of the probes, no signs of corrosion or morphological change to the thin-film metallization was observed by means of electrochemical and optical analysis. Damage to the metallization was assigned exclusively to mechanical impacts during explantation and handling. The results indicate that thin-film metallization on polymer substrates is applicable in permanent implant system.

bioengineering↗

bioRxiv2019

De novo Classification of Mouse B Cell Types using Surfaceome Proteotype Maps

System-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when sample is limiting. We miniaturized and automated the previously described Cell Surface Capture technology increasing sensitivity, reproducibility, and throughput. We used this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and used targeted flow cytometry to uncover developmental cell subpopulations.

biochemistry↗

bioRxiv2019

Quantification and selection of ictogenic zones in epilepsy surgery

Network models of brain dynamics provide valuable insight into the healthy functioning of the brain and how this breaks down in disease. A pertinent example is the use of network models to understand seizure generation (ictogenesis) in epilepsy. Recently, computational models have emerged to aid our understanding of seizures and to predict the outcome of surgical perturbations to brain networks. Such approaches provide the opportunity to quantify the effect of removing regions of tissue from brain networks and thereby search for the optimal resection strategy.\n\nHere, we use computational models to elucidate how sets of nodes contribute to the ictogenicity of networks. In small networks we fully elucidate the ictogenicity of all possible sets of nodes and demonstrate that the distribution of ictogenicity across sets depends on network topology. However, the full elucidation is a combinatorial problem that becomes intractable for large networks. Therefore, we develop a global optimisation approach to search for minimal sets of nodes that contribute significantly to ictogenesis. We demonstrate the potential applicability of these methods in practice by identifying optimal sets of nodes to resect in networks derived from 20 individuals who underwent resective surgery for epilepsy.

neuroscience↗

bioRxiv2019

Notch signalling mediates secondary senescence

Oncogene induced senescence (OIS) is a tumour suppressive response to oncogene activation that can be transmitted to neighbouring cells through secreted factors of the senescence associated secretory phenotype (SASP). Using single-cell transcriptomics we observed two distinct endpoints, a primary marked by Ras and a secondary by Notch. We find that secondary senescence in vitro and in vivo requires Notch, rather than SASP alone as previously thought. Currently, primary and secondary senescent cells are not thought of as functionally distinct endpoints. A blunted SASP response and the induction of fibrillar collagens in secondary senescence compared to OIS point towards a functional diversification. One Sentence SummaryNotch signalling is an essential driver of secondary senescence with primary and secondary senescence being distinct molecular endpoints.

genomics↗

bioRxiv2018

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

biochemistry↗

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↗