Biology subjects

Brandt, C.

Publications and source records attributed to Brandt, C..

At least 19 recordsLinked to original sources

bioRxiv2026

In vivo screening reveals new regulators of Natural Killer cell development and functional response to acute cytomegalovirus infection

Natural killer (NK) cells are critical innate immune effectors in antiviral and antitumour responses. However, the factors governing NK cell development and function remain incompletely understood. To address this, we adopted an in vivo screening approach, generating a panel of mice deficient in genes differentially expressed across NK cell maturation stages or following viral infection. We performed a combinational screen comprising baseline immunophenotyping and mouse cytomegalovirus (MCMV) challenge. We identified novel regulators of NK cell development, including transcriptional regulators and proteins with putative trafficking functions. MCMV challenge studies additionally identified proteins that impacted antiviral immunity independently of NK cell phenotypes, including a novel regulator of NK cell degranulation, Synaptotagmin-like protein 3. Identified genes exhibited reduced tolerance to loss-of-function in large scale human sequencing studies and evidence for reduced NK cell numbers in a cohort of immunodeficiency patients. Together, these findings provide a resource of NK-expressed genes important for NK cell maturation and function, with relevance to human health. SummaryPring et al. conduct an in vivo screen in mice identifying novel regulators of natural killer (NK) cell development and/or antiviral functionality. Large-scale human sequencing and human immunodeficiency patients revealed evidence for the relevance of identified genes in human health.

immunology↗

bioRxiv2025

Cure of experimental Trypanosoma vivax infection with a single dose of an unmodified antibody-based drug targeting the invariant flagellum cell surface protein IFX

Animal African trypanosomiasis (AAT) is an infectious wasting disease of economically important livestock caused by Trypanosoma spp. parasites. The disease is primarily caused by two species: T. congolense and T. vivax which are endemic in many African countries. AAT is managed by therapeutic and prophylactic drugs; however, resistance is now widely reported and the development of new drugs has been impeded due to a chronic lack of investment. Recently, we identified an invariant flagellar-associated cell surface protein (IFX) that could elicit protective immune responses when used as a vaccine against T. vivax. We showed that a complement-recruiting anti-IFX monoclonal antibody can prevent infection when used prophylactically. Here, we show that this same unmodified antibody can be used to cure T. vivax infections in a murine experimental model. Importantly, we show that infections can be treated with a single dose and demonstrate full cure by the lack of detectable parasites in peripheral tissues even after immunosuppression. Using structural modelling and site-directed mutagenesis, we localise the protective antibody epitope thereby identifying targetable regions on IFX to improve vaccine design. Together, these findings validate IFX as both a prophylactic and curative drug target that could be useful in the management of AAT. ImportanceTrypanosoma vivax is a parasite that causes animal African trypanosomiasis (AAT), a chronic wasting disease that infects economically-important livestock animals which is a particular problem in African countries south of the Sahara. The impact of this disease is significant: it is responsible for over 3 million cattle deaths and an estimated $4.5 billion of annual lost productivity. There is a desperate need to develop new control measures because resistance is now widely reported to the drugs commonly used to treat this infection. We show here that a single dose of an unmodified monoclonal antibody that recognises IFX - a parasite cell surface protein localised to the flagellum - is sufficient to cure an established T. vivax infection with no parasite reservoirs detectable in peripheral tissues. Our finding validates IFX as a new drug target and provides a rationale route to the development of new drugs to target AAT.

microbiology↗

bioRxiv2025

The metabolic and molecular mechanisms underlying running-induced energy compensation

Exercise not only regulates energy expenditure but also appetite, yet the underpinnings remain unclear. We describe that increased energy intake is a defense against energy loss that depends on initial running distance and operates independently of diet and age. Running caused a rapid circadian-dependent fat remodeling leading to a decline in circulating leptin accompanied by the activation of hypothalamic neurons. We discovered that the activation of the {beta}3-adrenergic receptor drives running-induced fat loss and the lower leptin triggers energy compensation by upregulating Neuropeptide Y. Once energy compensation is achieved, running is associated with molecular changes in hypothalamic signaling related to appetite and functional adaptations, such as enhanced sensitivity to hunger and satiety signals and increased responsiveness to appetite suppression induced by -Melanocyte-Stimulating Hormone. The increased food intake persisted without fat rebound beyond running in both lean and obese young mice, uncovering a new homeostatic balance in young mice.

physiology↗

bioRxiv2025

Host microbiome determines host specificity of the human whipworm, Trichuris trichiura

Long-term whipworm-host co-evolution has resulted in tropism for the caecum of specific hosts, an organ with the densest microbial population in the body. Here, we demonstrate that the host specificity of human whipworms (Trichuris trichiura) is host microbiome-driven. We successfully establish a T. trichiura infection in a non-primate host using a humanised-microbiota mouse model. We further show, in vitro, that hatching of T. trichiura was triggered by mucosal scrapings of the caecum of human microbiota-associated mice, but not from wild-type mice, which only induced T. muris hatching. Comparative metagenomic analysis of the murine versus humanised microbiomes directly implicated specific bacterial species in T. trichiura egg hatching. Additionally, we demonstrate that host tissue does not directly determine host specificity, as T. trichiura readily infected mouse caecaloids. Our findings indicate that host-microbiome-whipworm co-evolution has resulted in exquisite bacterial-whipworm egg interactions critical for hatching and development of these parasites in their definitive hosts.

microbiology↗

bioRxiv2025

Accurate and Reproducible Whole-Genome Genotyping for Bacterial Genomic Surveillance with Nanopore Sequencing Data

Despite recent advances in error rate reduction, until recently Oxford Nanopore Technology (ONT) sequences lacked the accuracy required for fine scale bacterial genomic analysis. Here, recent software improvements of ONT and the ONT-cgMLST-Polisher within the SeqSphere+ software were evaluated. We used short-(Illumina) and long-read ONT sequences of 80 multidrug-resistant bacteria (MDROs) for benchmarking. Illumina reads were de-novo-assembled using SKESA. For ONT, Dorado super accurate (SUP) model 4.3 or 5.0 basecalled reads were assembled with Flye and then polished with Medaka v1.12 m4.3 or Medaka v2.0 bacterial methylation model. In addition, the ONT-cgMLST-Polisher was run over all assemblies. The ground truth (GT) hybrid assemblies were created using Hybracter v0.10.0. Sixteen isolates from four species out of the original 80 isolates were sent to six laboratories for a ring trial. The 80 MDROs basecalled with SUP m4.3 had an average cgMLST allele distance (AD) to the GT of 4.94 with Medaka v1.12 and 1.78 with Medaka v2.0, respectively. After further polishing the Medaka v2.0 data with the ONT-cgMLST-Polisher, the AD dropped to 0.09. Using data basecalled with SUP m5.0 with Medaka v2.0 further reduced the AD significantly to 0.04. While the ring trial data basecalled with Dorado SUP m4.3 showed more variability and insufficient results for some samples, model 5.0 data resulted in average ADs of 0.36 and 0.17 without and with the ONT-cgMLST-Polisher, respectively. In conclusion, recent ONT Dorado and Medaka models combined with the ONT-cgMLST-Polisher improved ONT sequencing accuracy and made it sufficiently reproducible for genomic surveillance of bacteria. Importance Oxford Nanopore Technologies (ONT) sequencing methodology is especially attractive for small and medium-sized laboratories due to its relatively low capital investment and price per sample consumable costs. However, until recently it lacked accuracy and reproducibility for bacterial genomic genotyping. Here, we present an evaluation of the most recent ONT bioinformatic (basecalling and polishing of consensus) improvements and a new ONT-cgMLST-Polisher tool. We demonstrate that by applying those procedures ONT whole-genome genotyping-based surveillance of bacteria is finally accurate and reproducible enough for routine application even in small laboratories

bioinformatics↗

bioRxiv2025

Decoding bacterial methylomes in four public health-relevant microbial species: Nanopore sequencing enables reproducible analysis of DNA modifications

Investigating bacterial methylation profiles provides essential complementary information to the native DNA sequence, significantly extending our understanding of how DNA modifications influence virulence, antibiotic resistance, and the ability of bacteria to evade the immune system. Recent advancements in real-time Nanopore sequencing and basecalling algorithms have enabled the direct detection of modified bases from raw signal data, eliminating the need for bisulfite treatment of DNA. However, decoding methylation signals remains challenging due to rapid technological and methodological progress. In this study, we focus on public health-relevant bacterial strains to analyze their methylation profiles and identify methylation motifs. Our dataset includes samples from Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecium, and Klebsiella pneumoniae, sequenced on the Nanopore GridION platform using the latest flow cell chemistry (R10.4.1) and modification basecalling models (Dorado basecalling SUP model v5). We investigated distinct methylation patterns within and between species, focusing on heavily modified genes or genomic regions. Our results reveal distinct species-specific methylation profiles, with each strain exhibiting unique modification patterns. We developed a modular pipeline using Nextflow and the Nanopore Modkit tool to streamline the detection of methylated motifs. We compared the results with outputs from MicrobeMod, a recent toolkit for exploring prokaryotic methylation and base modifications in nanopore sequencing. Our pipeline is publicly available for further use (github.com/rki-mf1/ont-methylation). We identified known methylation motifs already described in the literature and novel de novo motifs, providing deeper insights into the diversity of bacterial DNA modifications. Furthermore, we identified genomic regions that are extensively methylated, which could have implications for bacterial behavior and pathogenicity. We also assess improvements in basecalling accuracy, specifically how methylated bases can influence neighboring basecalls. Recent advances in basecalling models, particularly v5 models as part of Dorado, have reduced these issues, improving the reliability of methylation detection in bacterial genomes. In conclusion, our study highlights the potential of current nanopore sequencing tools for detecting DNA modifications in prokaryotes. By making our pipeline and results publicly available, we facilitate further research into bacterial DNA modifications and their role in microbial pathogenesis.

bioinformatics↗

bioRxiv2024

Diversification of Pseudomonas aeruginosa biofilm populations under repeated phage exposures decreases the efficacy of the treatment

Phage therapy has been proposed as a therapeutic alternative to antibiotics for treatment of chronic, biofilm-related P. aeruginosa infections. To get a deeper insight into the complex biofilm-phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/ml) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic background. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to recruitment of different resistance and tolerance mechanisms.

microbiology↗

bioRxiv2024

Cell Maps for Artificial Intelligence: AI-Ready Maps of Human Cell Architecture from Disease-Relevant Cell Lines

This article describes the Cell Maps for Artificial Intelligence (CM4AI) project and its goals, methods, standards, current datasets, software tools, status, and future directions. CM4AI is the Functional Genomics Data Generation Project in the U.S. National Institute of Healths (NIH) Bridge2AI program. Its overarching mission is to produce ethical, AI-ready datasets of cell architecture, inferred from multimodal data collected for human cell lines, to enable transformative biomedical AI research.

systems biology↗

bioRxiv2024

Inferring the composition of the blood plasma proteome by a human proteome distribution atlas

The plasma proteome is maintained by the influx and efflux of proteins from surrounding organs and cells. To quantify the extent different organs and cells contribute to the plasma proteome composition, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in human plasma in health and disease. First, we constructed an extensive human proteome atlas from 18 vascularized organs and the most abundant cell types in blood. Second, the atlas was interfaced with previous RNA/protein atlases to objectively define proteome wide protein-organ associations to enable both the inference of origin and the reproducible quantification of organ-specific proteins in plasma. We demonstrate that the resource can determine disease-specific quantitative changes of organ-enriched protein panels in three separate patient cohorts with infection, pancreatitis, and myocardial injury. The strategy can be extended to other diseases to advance our understanding of the processes contributing to plasma proteome dynamics.

systems biology↗

bioRxiv2023

Integrated organismal responses induced by ecologically-relevant pCO2 and temperature exposures in developing lake sturgeon

Atmospheric CO2 and temperature are rising concurrently, and may have profound impacts on the transcriptional, physiological, and behavioral responses of aquatic organisms. Further, spring snow melt may cause transient increases of pCO2 in freshwater systems. Lake sturgeon (Acipenser fulvescens) groups were raised in current and projected levels of warming and pCO2. Following an overwintering period, lake sturgeon were exposed to a transient increase in pCO2, simulating a spring melt. Diverging transcriptional patterns were found in each group and metabolic rate was lower in the combined stressor group compared to others. Behavioral assays revealed no effect of environment on alarm cue responses or boldness, but there was a decrease in total activity following an acute CO2 exposure. These results demonstrate compensatory and compounding mechanisms of pCO2 and warming dependent on developmental conditions of a freshwater fish, and provide key information for responses to future climate change.

zoology↗

bioRxiv2023

Nanopore sequencing for accurate bacterial outbreak tracing

Our study investigated the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a three-year-long Klebsiella pneumoniae outbreak with Illumina short read sequencing data as the point of reference. We detected considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besides K. pneumoniae. Based on this data, we explored PCR-based sequencing and a masking strategy, which both successfully addressed these inaccuracies and ensured accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA is freely available on GitHub under the GNUv3 license: github.com/replikation/MPOA) to identify and mask problematic genome positions in a reference-free manner. Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investing outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either PCR-based sequencing or using our provided bioinformatic workflow. We would advise that read mapping-based quality control of genomes should be provided when publishing results.

molecular biology↗

bioRxiv2023

Targeted decontamination of sequencing data with CLEAN

Many biological and medical questions are answered based on the analysis of sequence data. However, we can find contamination, artificial spike-ins, and overrepresented rRNA sequences in various read collections and assemblies. In particular, spike-ins used as controls, as those known from Illumina or Nanopore data, are often not considered as contaminants and also not appropriately removed during analyses. Additionally, removing human host DNA may be necessary for data protection and ethical considerations to ensure that individuals cannot be identified. We developed CLEAN, a pipeline to remove unwanted sequences from both long- and short-read sequencing techniques. While focusing on Illumina and Nanopore data with their technology-specific control sequences, the pipeline can also be used for host decontamination of metagenomic reads and assemblies, or the removal of rRNA from RNA-Seq data. The results are the purified sequences and sequences identified as contaminated with statistics summarized in a report. The output can be used directly in subsequent analyses, resulting in faster computations and improved results. Although decontamination seems mundane, many contaminants are routinely overlooked, cleaned by steps that are not fully reproducible or difficult to trace. CLEAN facilitates reproducible, platform-independent data analysis in genomics and transcriptomics and is freely available at https://github.com/rki-mf1/clean under a BSD3 license. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/552089v3_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@133c638org.highwire.dtl.DTLVardef@1ab448org.highwire.dtl.DTLVardef@1320f21org.highwire.dtl.DTLVardef@17f7a9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

A drug repurposing screen for whipworms informed by comparative genomics

Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of[≤] 50 M against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug repurposing against whipworms. Therefore, we recommend that prior to in vivo studies, future researchers first assess drug absorption by the host, for example, using human intestinal organoids or cell lines, and drug uptake by whipworms using intestinal organoids infected with T. muris.

pharmacology and toxicology↗

bioRxiv2021

EROS-mediated control of NOX2 and P2X7 biosynthesis

EROS (Essential for Reactive Oxygen Species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease (CGD), but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase (OST) machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions and P2X7 is almost absent in EROS deficient mouse and human primary cells. Accordingly, lack of EROS results in markedly abnormal P2X7 signalling, inflammasome activation and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation and possibly gene therapy.

immunology↗

bioRxiv2021

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore's adaptive sequencing

Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopores adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective human host depletion resulted in a 1.70 fold ({+/-} 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

molecular biology↗

bioRxiv2021

Adaptive nanopore sequencing on miniature flow cell detects extensive antimicrobial resistence

Rapid screening of hospital admissions to detect asymptomatic carriers of resistant bacteria can prevent pathogen outbreaks. However, the resulting isolates rarely have their genome sequenced due to cost constraints and long turn-around times to get and process the data, limiting their usefulness to the practitioner. Here we use real-time, on-device target enrichment ("adaptive") sequencing as a highly multiplexed assay covering 1,147 antimicrobial resistance genes. We compare its utility against standard and metagenomic sequencing, focusing on an isolate of Raoultella ornithinolytica harbouring three carbapenemases (NDM, KPC, VIM). Based on this experimental data, we then model the influence of several variables on the enrichment results and predict a large effect of nucleotide identity (higher is better) and read length (shorter is better). Lastly, we show how all relevant resistance genes are detected using adaptive sequencing on a miniature ("Flongle") flow cell, motivating its use in a clinical setting to monitor similar cases and their surroundings.

microbiology↗

bioRxiv2021

Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae

Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening more than ten thousand genomes, 500 thousand metagenomes, and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.

microbiology↗

bioRxiv2021

poreCov - an easy to use, fast, and robust workflow for SARS-CoV-2 genome reconstruction via nanopore sequencing

In response to the SARS-CoV-2 pandemic, a highly increased sequencing effort has been established worldwide to track and trace ongoing viral evolution. Technologies such as nanopore sequencing via the ARTIC protocol are used to reliably generate genomes from raw sequencing data as a crucial base for molecular surveillance. However, for many labs that perform SARS-CoV-2 sequencing, bioinformatics is still a major bottleneck, especially if hundreds of samples need to be processed in a recurring fashion. Pipelines developed for short-read data cannot be applied to nanopore data. Therefore, specific long-read tools and parameter settings need to be orchestrated to enable accurate genotyping and robust reference-based genome reconstruction of SARS-CoV-2 genomes from nanopore data. Here we present poreCov, a highly parallel workflow written in Nextflow, using containers to wrap all the tools necessary for a routine SARS-CoV-2 sequencing lab into one program. The ease of installation, combined with concise summary reports that clearly highlight all relevant information, enables rapid and reliable analysis of hundreds of SARS-CoV-2 raw sequence data sets or genomes. poreCov is freely available on GitHub under the GNUv3 license: github.com/replikation/poreCov.

bioinformatics↗