Biology subjects

Loos, B.

Publications and source records attributed to Loos, B..

7 recordsLinked to original sources

bioRxiv2025

VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

Visual proteomics (VP) aims to allow researchers to visualise, measure and analyse proteins in the context of cell and tissue structure in health and disease. VP is becoming a reality through technological advances across several domains, including in situ structural biology and correlative light and electron microscopy (CLEM). However, widespread adoption remains limited due to the complexity and cost of the various VP approaches reported to date. Here we present the VP-CLEM-Kit, a disruptive cost-effective pipeline for super resolution volume CLEM (SR-vCLEM) that can be implemented with minimal advanced electron microscopy expertise and equipment, making it accessible to light microscopy facilities and research labs. SR-vCLEM is based on in-resin fluorescence (IRF), where fluorophores are preserved through processing into resin. The easyIRF protocol reported here reduces the requirement for complex costly sample preparation equipment and toxic chemicals compared to standard IRF protocols. easyIRF blocks are cut into ultrathin sections that are imaged using tomoSTORM, a new modular and cost-effective openFrame-based light microscope controlled by the open-source software package Micro-Manager that provides serial single molecule localisation microscopy in array tomography format. Sections are then post-stained and imaged using a tabletop scanning electron microscope controlled by open-source SBEMimage software to run in array tomography format. We demonstrate the potential of the VP-CLEM-Kit by imaging organelle reporters in human cell lines and stem cell derived neurons, fluorescently labelled protein in neurons, and immunolabelled cells in human kidney biopsy tissue from transplant patients. The VP-CLEM-Kit delivers a [~]5-fold improvement in resolution at a [~]7-fold lower cost, and thus provides new technical capability as well as a blueprint for more equitable access to advanced imaging workflows.

cell biology↗

bioRxiv2024

Mycobacterium tuberculosis ESX-T7SS impacts the 3D architecture of tuberculous lesion in susceptible mice

Tuberculosis (TB) is characterized by the formation of heterogenous, immune-rich granulomas present in various forms in the lungs. Both host and pathogen contribute to this heterogeneity however the molecular and cellular drivers of within-host granuloma heterogeneity remain to be fully elucidated. This knowledge gap is due to a lack of experimental approaches that can fully capture the complex dynamics of the lung architecture, dynamics of host-pathogen interplay and pathogenesis. Here, we developed an approach that combines PACT-based clearing with light sheet fluorescent microscopy for visualizing lesion architecture development and lung involvement in M. tuberculosis-infected C3HeB/FeJ susceptible mice. This 3D modelling of whole lung lobes approach revealed critical architectural features in lesion development and lung involvement that were not apparent using traditional thin section imaging. Wild type M. tuberculosis infection triggered a clear and well-organized granulomatous-like organization with substantial growth throughout the infection period that invaded a high percentage of the total lung volume. In contrast, infection with the avirulent ESX-1 deletion mutant strain Mtb {Delta}RD1 showed an altered growth pattern with diffuse and sparsely organized CD11b recruitment to sites of infection. Moreover, most of the Mtb {Delta}RD1-triggered lesions were present in the periphery of the lungs and did not display any formal organization. We further provide a novel way of interrogating PACT-cleared tissue for high ultrastructural content using volumetric correlative light and electron microscopy, allowing individual immune cell populations to be tracked and their fate within the granuloma captured. Ultimately, the combination of both modalities allowed an unprecedented view of the architectural distribution of M. tuberculosis in the lungs and the progression of lesion development over time. Our data highlight that ESX-1 from M. tuberculosis is required for lesion architecture progression in a susceptible mouse model of TB.

microbiology↗

bioRxiv2023

Altered neutrophil extracellular traps in response to Mycobacterium tuberculosis in persons living with HIV with no previous TB and negative TST and IGRA

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and test persistently negative in tests of immune sensitization tuberculin skin test (TST) and interferon gamma release assays (IGRA) for Mycobacterium tuberculosis (Mtb). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophil Mtb infection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected with Mtb H37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. At 1h of Mtb infection, PMNHITTIN displayed 151 significantly upregulated and 40 significantly downregulated differentially expressed genes (DEGs) and PMNHIT 98 significantly upregulated and 11 significantly downregulated DEGs. At the 6h timepoint, PMNHITTIN displayed 3106 significantly upregulated and 3548 significantly downregulated DEGs while PMNHIT had 3816 significantly up- and 3794 significantly downregulated DEGs. There was no significant differential transcriptional response at 1h between infected PMNHITTIN and PMNHIT. However, when contrasting the log2FC 6h infection response to Mtb from PMNHITTIN against PMNHIT, 2285 genes showed significant differential response between the two groups. Apoptosis and NETosis were key pathways linked to the enrichment of genes in PMNHITTIN when contrasted to PMNHIT after 6h infection with Mtb. Fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTIN compared to PMNHIT, showing that PMNHITTIN have a distinct response to Mtb.

genetics↗

bioRxiv2023

ALaSCA: A novel in silico simulation platform to untangle biological pathway mechanisms, with a case study in Type 1 Diabetes progression

IntroductionThe analysis of signaling pathways is a cornerstone in clarifying the biological mechanisms involved in complex genetic disorders. These pathways have intricate topologies, and the existing methods that are used for the interpretation of these pathways, remain limited. We have therefore developed the Adaptable Large-Scale Causal Analysis (ALaSCA) computational platform, which uses causal analysis and counterfactual simulation techniques. ALaSCA offers the ability to simulate the outcome of a number of different hypotheses to gain insight into the complex dynamics of biological mechanisms prior to, or even without, wet lab experimentation. ALaSCA is offered as a proprietary Python library for bioinformaticians and data scientists to use in their life sciences workflows. Here we demonstrate the ability of ALaSCA to untangle the pivots and redundancies within biological pathways of various drivers of a specific phenotypic process. This is achieved by studying a major disease of global relevance, namely Type 1 Diabetes (T1D), and quantifying causal relationships between antioxidant proteins and T1D progression. ALaSCA is also benchmarked against standard associative analysis methods. MethodsWe use our in silico simulation platform, ALaSCA, to apply both a number of machine learning (ML) and data imputation techniques, and perform causal inference and counterfactual simulation. ALaSCA uses standard ML and causal analysis libraries as well as custom code developed for data imputation and counterfactual simulation. Counterfactual simulation is a method for simulating potential or hypothetical model outcomes in the field of causal analysis (Glymour, Pearl and Jewell, 2016). We apply ALaSCA to T1D by using proteomic data from Liu et al. (2018), as the patients were selected based on the presence of T1D susceptible HLA (human leukocyte antigen)-DR/DQ alleles through genotyping at birth and followed prospectively. The genetic cause of T1D in this cohort is therefore known and the mechanism and proteins through which it causes T1D are well-characterized. This biological mechanism was converted into a directed acyclic graph (DAG) for the subsequent causal analyses. The dataset was used to benchmark the causal inference and counterfactual simulation capabilities of ALaSCA. Results and discussionAfter data imputation of the Liu, et al. (2018) dataset, causal inference and counterfactual simulation were completed. The causal inference output of the HLA, antioxidant, and non-causal proteins showed that the HLA proteins had the overall strongest causal effects on T1D, with antioxidant proteins having the overall second largest causal effects on T1D. The non-causal proteins showed negligibly small effects on T1D in comparison with the HLA and antioxidant proteins. With counterfactual simulation we were able to replicate evidence for and gain understanding into the protective effect that antioxidant proteins, specifically Superoxide dismutase 1 (SOD1), have in T1D, a trend which is seen in literature. We were also able to replicate an unusual case from literature where antioxidant proteins, specifically Catalase, do not have a protective effect on T1D. ConclusionBy analyzing the disease mechanism, with the inferred causal effects and counterfactual simulation, we identified the upstream HLA proteins, specifically the DR alpha chain and DR beta 4 chain proteins as causes of the protective effect of the antioxidant proteins on T1D. In contrast, through counterfactual simulation of the unusual case, in which the DR alpha chain and DR beta 4 chain proteins are not present in the model, we saw that the adverse effect which the antioxidant proteins have on T1D is due to the HLA protein, DQ beta 1 chain, and not the antioxidant proteins themselves. Future work would entail the application of the ALaSCA platform on various other diseases, and to integrate it into wet lab experimental design in a number of different biological study areas and topics.

bioinformatics↗

bioRxiv2022

ALaSCA: a computational platform for quantifying the effect of proteins using Pearlian causal inference, with an example application in Alzheimer's disease

IntroductionAn urgent need to delay the onset of aging-associated diseases has arisen due to increasing human lifespan. A dramatic surge in the number of identified potential molecular targets that could promote successful aging, has led to the challenge of prioritizing these targets for further research and drug development. In our previous work, we prioritized genes associated with aging processes based on their similarity to known aging-related genes and dysfunction marker genes in C. elegans. The goal of this study was to demonstrate the ability of our computational platform to identify molecular drivers of neuronal aging using specialized causal inference techniques. S6K was highly ranked in the previous study and here the nearby neighbors in its protein interaction network were selected to explore ALaSCAs (Adaptable Large-Scale Causal Analysis) ability to identify possible drivers of Alzheimers disease. MethodsUtilizing head and brain proteome data, two of ALaSCAs capabilities were used to understand how protein changes over the lifespan of Drosophila melanogaster affect a feature of neuronal aging, namely climbing ability: O_LIPearson correlation analysis was used to assess the relationship between the changes in abundance of specific proteins associated (through protein-protein interactions) with S6K and climbing ability. C_LIO_LIPearlian causal inference, required to achieve formal causal analysis, was used to determine which pathway, associated with proteins linked to S6K, has the largest effect on climbing ability and therefore to what degree these specific proteins are driving neuronal aging. C_LI Results and discussionBased on the correlation results, the proteins associated with fz, a gene encoding for the fz family of receptors that are involved in Wnt signaling, display an increase in abundance as climbing ability declines over time. When viewed together with the fz proteins strong negative causal value, it seems that their increased abundance over the lifespan of Drosophila is an important driver of the observed decrease in climbing ability. Additionally, expression of the genes FZD1 and FZD7 (fz orthologs) is altered in the hippocampus early on in Alzheimers disease human samples and in an amyloid precursor protein mouse model. ConclusionWe have demonstrated the potential of the ALaSCA platform to identify and provide evidence behind molecular mechanisms. This capability enables identification of possible drivers of Alzheimers disease - as the human orthologs of the proteins identified here, through its Pearlian causal inference capability, have been linked to Alzheimers disease progression.

bioinformatics↗

bioRxiv2021

Rapamycin induces autophagy and increases heat tolerance in Drosophila melanogaster

Mechanisms aimed at recovering from heat-induced damage are closely associated with the ability of ectotherms to survive exposition to stressful temperatures. Among these mechanisms the respective contribution of autophagy, a ubiquitous stress-responsive catabolic process, has more recently come to light. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to range of abiotic stressors in diverse ectothermic organisms. Since our understanding of the relationship between autophagy and heat-tolerance currently remains limited in insect models, we hypothesized that (1) heat-stress would cause an increase of autophagy in Drosophila melanogaster tissues and (2) rapamycin exposure would trigger a detectable autophagic response in flies and increase their heat-tolerance. In line with our hypothesis, we report that flies exposed to heat-stress present signs of protein aggregation and appears to trigger an autophagy-related homoeostatic response as a result. We further show that rapamycin feeding causes the systemic effect associated with TOR inhibition, induces autophagy at least locally in the fly gut, and increase the heat-stress tolerance of individuals. This points toward a likely substantial contribution of this autophagy to cope with stressful temperatures in insects.

physiology↗

bioRxiv2020

Mitochondrial event localiser (MEL) to quantitatively describe fission, fusion and depolarisation in the three-dimensional space

Mitochondrial fission and fusion play an important role not only in maintaining mitochondrial homeostasis but also in preserving overall cellular viability. However, quantitative analysis based on the three-dimensional localisation of these highly dynamic mitochondrial events in the cellular context has not yet been accomplished. Moreover, it remains largely uncertain where in the mitochondrial network depolarisation is most likely to occur. We present the mitochondrial event localiser (MEL), a method that allows high-throughput, automated and deterministic localisation and quantification of mitochondrial fission, fusion and depolarisation events in large three-dimensional microscopy time-lapse sequences. In addition, MEL calculates the number of mitochondrial structures as well as their combined and average volume for each image frame in the time-lapse sequence. The mitochondrial event locations can subsequently be visualised by superposition onto the fluorescence micrograph z-stack. We apply MEL to both control samples as well as cells that have been treated with hydroxychloroquine sulphate (HCQ) and observe that fission and fusion events mostly occur around the terminal branches of the mitochondrial network, with few events detected in the strongly networked areas such as the perinuclear region. Depolarisation was most abundant in the HCQ treated samples, with the majority of the depolarisation events occurring in the cell periphery.

bioinformatics↗