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Schilling, M.

Publications and source records attributed to Schilling, M..

31 records · Page 2Linked to original sources

Amplification and extraction free quantitative detection of viral nucleic acids and single-base mismatches using magnetic signal amplification circuit

Established nucleic acid detection assays require extraction and purification before sequence amplification and/or enzymatic reactions, hampering their widespread applications in point-of-care (POC) formats. Magnetic immunoassays based on magnetic particle spectroscopy and magnetic nanoparticles (MNPs) are isothermal, extraction- and purification-free, and can be quantitative and benchtop, making them suitable for POC settings. Here, we demonstrate a Magnetic signal Amplification Circuit (MAC) that combines specificity of toehold-mediated DNA strand displacement with magnetic response of MNPs to a clustering/declustering process. Our MAC assays require neither amplification nor extraction of target nucleic acids, and reveal four times better sensitivity than that of a magnetic circuit without signal amplification. Using MAC, we detect a highly specific 43 nucleotides sequence of SARS-CoV-2 virus. The MAC enables sensing both DNA and RNA targets with varying lengths and resolving single-base mismatches. Our MAC can be a powerful tool for translating research of nucleic acids detection to the clinic.

biochemistry↗

Circadian regulation of human immunodeficiency virus type 1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks curative therapies or vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organisms response to its environment and invading pathogens. Recent reports of diurnal variation in peripheral viral load in HIV-1 infected subjects highlights the need for mechanistic studies. Here, we demonstrate rhythmic HIV-1 replication in circadian-synchronised model systems and show the circadian transcription factors, BMAL1 and REV-ERB, bind conserved motifs in the HIV-1 promoter. REV-ERB competes with ROR for binding to the same regulatory motif, and we uncover a role for ROR inhibitors to perturb rhythmic HIV-1 replication and host gene expression. We demonstrate that many HIV-1 host factors are circadian regulated and likely to define rhythmic viral replication. In summary, this study increases our understanding of the mechanisms underlying the circadian regulation of HIV that can inform HIV therapy and management.

microbiology↗

neuroWalknet, a controller for hexapod walking allowing for context dependent behavior

Decentralized control has been established as a key control principle in insect walking and has been successfully leveraged to account for a wide range of walking behaviors in the proposed neuroWalknet architecture. This controller allows for walking patterns with different velocities in forward and backward direction -- quite similar to the behavior shown in stick insects --, for negotiation of curves, and for robustly dealing with various disturbances. While these simulations focus on the cooperation of different, decentrally controlled legs, here we consider a set of biological experiments not yet been tested by neuroWalknet, that focus on the function of the individual leg and are context dependent. These intraleg studies deal with four groups of interjoint reflexes. The reflexes are elicited by either a stimulation of the femoral chordotonal organ (fCO) or a specific stimulation of campaniform sensilla (CS). Motor output signals are recorded from the alpha-joint, the beta-joint or the gamma-joint of the leg. Furthermore, such reflexes have been studied while the ganglion was treated with pilocarpine. Although these biological data represent results obtained from different local reflexes in different contexts, they fit with and are embedded into the behavior shown by the global structure of neuroWalknet. In particular, a specific and intensively studied behavior, active reaction, has since long been assumed to represent a separate behavioral element, from which it is not clear why it occurs in some situations, but not in others. This question could now be explained as an emergent property of the holistic structure of neuroWalknet. When experimenting with pilocarpine, oscillations were induced in neuroWalknet even though this does not include an explicit central pattern generator and in this way provides a simpler model as a functional explanation. As the simulation data result from a holistic system, further results were obtained that could be used as predictions to be tested in further biological experiments. AUTHOR SUMMARYBehavior of animals can be studied by detailed observation, but observation alone does not explain the function of the underlying neuronal controller structures. To better understand this function, an important tool can be to develop an artificial structure based on simulated neurons and a simulated or physical body. Although typical animal behavior appears complex, the corresponding neuronal structures may be comparatively simple. The goal for such a hypothetical structure should be to include as many different behaviors as possible, and, at the same time, search for a simple explanation consisting of a minimum of neuronal elements. Furthermore, such a simulation system, e.g. an artificial neuronal network, should contain hypotheses that can be tested in biological experiments. We propose an extension to such a network that is based on a decentralized neuronal structure, using a neural network as a scaffold, that enables various combinations of local neuronal elements that allow for emergent, i.e. not explicitly designed properties. Indeed, neuroWalknet contains further abilities not yet recognized in the earlier version. For instance, neither explicit structures like central pattern generators nor explicit Active Reaction are required to reproduce typical intraleg reactions. Therefore, neuroWalknet presents a holistic approach enabling emergent properties out of the cooperation of small neuronal elements that are context dependent instead of explicit, dedicated elements.

neuroscience↗

Heavy metal pollution impacts soil bacterial community structure and antimicrobial resistance at the Birmingham 35th Avenue Superfund Site

Heavy metals (HMs) are known to modify bacterial communities both in the laboratory and in situ. As a consequence, soils in HM contaminated sites like the U.S. Environmental Protection Agency (EPA) Superfund sites are predicted to have altered ecosystem functioning, with potential ramifications for the health of organisms, including humans, that live nearby. Further, several studies have shown that metal tolerant bacteria are often also resistant to antimicrobial agents (AMR), and therefore HM contaminated soils could potentially act as reservoirs that could disseminate AMR genes into human-associated pathogenic bacteria. To explore this possibility soil samples were collected from six public locations in the zip code 35207 (the home of the North Birmingham 35th Avenue Superfund site) and in six public areas in a neighboring reference zip code (35214). Sequencing of the V4 region of the bacterial 16S rRNA gene revealed that elevated concentrations of HMs As, Mn, Pb, and Zn reduced microbial diversity and altered community structure within each zip code. While there was no difference between zip codes in the proportion of total culturable microbes that survived antimicrobial or metal exposure, bacterial isolates with HMR almost always also exhibited AMR. Metagenomes inferred using PICRUST2 also predicted significantly higher mean relative frequencies in 35207 for several AMR genes related to both specific and broad-spectrum AMR phenotypes. Together, these results support the hypothesis that chronic HM pollution alters soil bacterial community structure in ecologically meaningful ways and may also select for bacteria with increased potential to contribute to AMR in human bacterial disease.

microbiology↗

Common signatures of differential microRNA expression in Parkinson's and Alzheimer's disease brains

BackgroundDysregulation of microRNA (miRNA)-mediated gene expression has been implicated in the pathogenesis and course of many neurodegenerative diseases including Parkinsons disease (PD). However, the functionally relevant miRNAs remain largely unknown. Previous meta-analyses on differential miRNA expression data in post-mortem PD brains have highlighted several miRNAs showing consistent and statistically significant effects. However, these meta-analyses were based on exceedingly small sample sizes. MethodsIn this study, we quantified the expression of the four most compelling PD candidate miRNAs from these meta-analyses in the superior temporal gyrus (STG) of one of the largest case-control post-mortem brain datasets available (261 samples), thereby quadruplicating previously investigated sample sizes. Furthermore, we probed for common differential miRNA expression signatures with Alzheimers disease (AD) by also analyzing these miRNAs in post-mortem STG of 190 AD patients and controls and by testing six top AD miRNAs in the PD brains. ResultsOf all ten analyzed miRNAs, PD candidate miRNA homo sapiens (hsa-) miR-132-3p showed evidence for differential expression in both PD (p=4.89E-06) and AD (p=3.20E-24), and AD miRNAs hsa-miR-132-5p (p=4.52E-06) and hsa-miR-129-5p (p=0.0379) showed evidence for differential expression in PD. Combining these novel data with previously published data substantially improved the statistical support (=3.85E-03 using Bonferroni correction) of the corresponding meta-analyses clearly and compellingly implicating these miRNAs in both PD and AD. Furthermore, hsa-miR-132-3p/-5p (but not hsa-miR-129-5p) showed association with neuropathological Braak PD staging (p=3.51E-03/p=0.0117), suggesting that these miRNAs may play a role in -synuclein aggregation beyond the early disease phase. ConclusionsOur study represents the largest independent assessment of recently highlighted candidate brain miRNAs in PD and AD post-mortem brain samples, to date. Our results implicate hsa-miR-132-3p/-5p and hsa-miR-129-5p to be differentially expressed in both PD and AD brains, potentially pinpointing shared pathogenic mechanisms across these neurodegenerative diseases.

neuroscience↗

Entorhinal cortex EWAS meta-analysis highlights four novel loci showing differential methylation in Alzheimer's disease

BackgroundStudies on DNA methylation (DNAm) in Alzheimers disease (AD) have recently highlighted several genomic loci showing association with disease onset and progression. MethodsHere, we conducted an epigenome-wide association study (EWAS) using DNAm profiles in entorhinal cortex (EC) from 149 AD patients and control brains and combined these with two previously published EC datasets by meta-analysis (total n=337). ResultsWe identified 12 cytosine-phosphate-guanine (CpG) sites showing epigenome-wide significant association with either case-control status or Braaks tau-staging. Four of these CpGs, located in proximity to CNFN/LIPE, TENT5A, PALD1/PRF1, and DIRAS1, represent novel findings. Integrating DNAm levels with RNA sequencing-based mRNA expression data generated in the same individuals showed significant DNAm-mRNA correlations for 6 of the 12 significant CpGs. Lastly, by calculating rates of epigenetic age acceleration using two recently proposed "epigenetic clock" estimators we found a significant association with accelerated epigenetic aging in AD patients vs. controls. ConclusionIn summary, our study represents the hitherto most comprehensive EWAS in AD using EC and highlights several novel differentially methylated loci with potential effects on gene expression.

genomics↗

Differential microRNA expression analyses across two brain regions in Alzheimer's disease

BackgroundDysregulation of microRNAs (miRNAs) is involved in the pathogenesis of neurodegenerative diseases, including Alzheimers disease (AD). Hitherto, sample sizes from differential miRNA expression studies in AD are exceedingly small aggravating any biological inference. To overcome this limitation, we investigated six candidate miRNAs in a large collection of brain samples. MethodsBrain tissue was derived from superior temporal gyrus (STG) and entorhinal cortex (EC) from 99 AD patients and 91 controls. Expression of six miRNAs was examined by qPCR (STG) or small RNA sequencing (EC). Brain region-dependent differential miRNA expression was investigated in a transgenic AD mouse model using qPCR and FISH. Total RNA sequencing was used to assess differential expression of miRNA target genes. ResultsMiR-129-5p, miR-132-5p, and miR-138-5p were significantly downregulated in AD vs. controls both in STG and EC, while miR-125b-5p and miR-501-3p showed no evidence for differential expression in this dataset. In addition, miR-195-5p was significantly upregulated in EC but not STG in AD patients. The brain region-specific pattern of miR-195-5p expression was corroborated in vivo in transgenic AD mice. Total RNA sequencing identified several novel and functionally interesting target genes of these miRNAs involved in synaptic transmission (GABRB1), the immune-system response (HCFC2) or AD-associated differential methylation (SLC16A3). ConclusionsUsing two different methods (qPCR and small RNA-seq) in two separate brain regions in 190 individuals we more than doubled the available sample size for most miRNAs tested. Differential gene expression analyses confirm the likely involvement of miR-129-5p, miR-132-5p, miR-138-5p, and miR-195-5p in AD pathogenesis and highlight several novel potentially relevant target mRNAs. FundingThis work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the National Science Foundation China (NSFC) as a Joint Sino-German research project ("MiRNet-AD", #391523883). Additional support was provided by the DFG Research Infrastructure NGS_CC (project 407495230) as part of the Next Generation Sequencing Competence Network (#423957469) and the Cure Alzheimer s Fund (CAF) as part of the CIRCUITS consortium project.

neuroscience↗

Identification of SARS-CoV2-mediated suppression of NRF2 signaling reveals a potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here we demonstrate that the NRF2 anti-oxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular anti-viral program, which potently inhibits replication of SARS-CoV2 across cell lines. The anti-viral program extended to inhibit the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, induction of NRF2 by 4-OI and DMF limited host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2. One Sentence SummaryNRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate inhibited SARS-CoV2 replication and virus-induced inflammatory responses, as well as replication of other human pathogenic viruses.

microbiology↗

Pharmacological activation of the circadian component REV-ERB inhibits HIV-1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks a curative therapy or vaccine. Despite the reduction in AIDS-related deaths achieved by current antiretroviral therapies, drawbacks including drug resistance and the failure to eradicate infection highlight the need to identify new pathways to target the infection. Circadian rhythms are endogenous 24-hour oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components participating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, CLOCK and BMAL1 activate rhythmic transcription of genes including the nuclear receptor REV-ERB, which represses BMAL1 and plays an essential role in sustaining a functional clock. We investigated whether REV-ERB activity regulates HIV-1 replication, and found REV-ERB agonists inhibited HIV-1 promoter activity in cell lines, primary human CD4 T cells and macrophages, whilst antagonism or genetic disruption of REV-ERB increased promoter activity. Furthermore, the REV-ERB agonist SR9009 inhibited promoter activity of different HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic ligands to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock transcription factors in regulating HIV-1 replication.

microbiology↗

Identification of Interleukin1β as an Amplifier of Interferon alpha-induced Antiviral Responses

The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFN)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFN-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFN-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFN-induced antiviral genes can be mimicked by co-application of IFN and interleukin1 beta (IL1{beta}). Consistently, we found that IL1{beta} enhances IFN-mediated repression of viral replication. Conversely, we observed that in IL1{beta} receptor knock-out mice replication of viruses sensitive to IFN is increased. Thus, IL1{beta} is capable to potentiate IFN-induced antiviral responses and could be exploited to improve antiviral therapies. Author SummaryInnate immune responses contribute to the control of viral infections and the induction of interferon alpha (IFN)-mediated antiviral responses is an important component. However, IFN induces a multitude of antiviral response genes and the expression dynamics of these genes can be classified as early, intermediate and late. Here we show, based on a mathematical modeling approach, that mRNA stability as well as the negative regulator IRF2 control the expression dynamics of IFN-induced antiviral genes. Knock-down of IRF2 resulted in the amplified IFN-mediated induction of the antiviral genes and this amplified expression of antiviral genes could be mimicked by co-stimulation with IFN and IL1{beta}. We observed that co-stimulation with IFN and IL1{beta} enhanced the repression of virus replication and that knock-out of the IL1 receptor in mice resulted in increased replication of a virus sensitive to IFN. In sum, our studies identified IL1{beta} as an important amplifier of IFN-induced antiviral responses.

systems biology↗

Cell-to-cell variability in JAK2/STAT5 pathway components and cytoplasmic volumes define survival threshold in erythroid progenitor cells

Survival or apoptosis is a binary decision in individual cells. Yet, at the cell population level, a graded increase in survival of CFU-E cells is observed upon stimulation with Erythropoietin (Epo). To identify components of JAK2/STAT5 signal transduction that contribute to the graded population response, a cell population-level model calibrated with experimental data was extended to study the behavior in single cells. The single-cell model showed that the high cell-to-cell variability in nuclear phosphorylated STAT5 is caused by variability in the amount of EpoR:JAK2 complexes and of SHP1 as well as the extent of nuclear import due to the large variance in the cytoplasmic volume of CFU-E cells. 24 to 118 pSTAT5 molecules in the nucleus for 120 min are sufficient to ensure cell survival. Thus, variability in membrane-associated processes are responsible to convert a switch-like behavior at the single-cell level to a graded population level response. HighlightsO_LIMathematical modeling enables integration of heterogeneous data C_LIO_LISingle-cell modeling captures binary decision process C_LIO_LIMultiple sources of cell-to-cell variability in erythroid progenitor cells C_LIO_LIMinimal amount of active STAT5 sufficient for survival of erythroid progenitor cells C_LI

systems biology↗

Decentralized Control of Insect Walking - a simple neural network explains a wide range of behavioral and neurophysiological results

Control of walking with six or more legs in an unpredictable environment is a challenging task, as many degrees of freedom have to be coordinated. Generally, solutions are proposed that rely on (sensory-modulated) CPGs, mainly based on data from neurophysiological studies. Here, we are introducing a sensor based controller operating on artificial neurons, being applied to a (simulated) hexapod robot with a morphology adapted to Carausius morosus. We show that such a decentralized solution leads to adaptive behavior when facing uncertain environments which we demonstrate for a large range of behaviors - slow and fast walking, forward and backward walking, negotiation of curves and walking on a treadmill with various treatment of individual legs. This approach can as well account for these neurophysiological results without relying on explicit CPG-like structures, but can be complemented with these for very fast walking.

animal behavior and cognition↗

Regulation of spatial and temporal gene expression in an animal germline

In animal germlines, regulation of cell proliferation and differentiation is particularly important but poorly understood. Here, using a cryo-cut approach, we mapped RNA expression along the Caenorhabditis elegans germline and, using mutants, dissected gene regulatory mechanisms that control spatio-temporal expression. We detected, at near single-cell resolution, > 10,000 mRNAs, > 300 miRNAs and numerous novel miRNAs. Most RNAs were organized in distinct spatial patterns. Germline-specific miRNAs and their targets were co-localized. Moreover, we observed differential 3 UTR isoform usage for hundreds of mRNAs. In tumorous gld-2 gld-1 mutants, gene expression was strongly perturbed. In particular, differential 3 UTR usage was significantly impaired. We propose that PIE-1, a transcriptional repressor, functions to maintain spatial gene expression. Our data also suggest that cpsf-4 and fipp-1 control differential 3 UTR usage for hundreds of genes. Finally, we constructed a \"virtual gonad\" enabling \"virtual in situ hybridizations\" and access to all data (https://shiny.mdc-berlin.de/spacegerm/).

systems biology↗