Biology subjects

Marsh, M.

Publications and source records attributed to Marsh, M..

9 recordsLinked to original sources

bioRxiv2024

A Deep Learning Model for Accurate Segmentation of the Drosophila melanogaster Brain from Micro-CT Imaging

The use of microcomputed tomography (Micro-CT) for imaging biological samples has burgeoned in the past decade, due to increased access to scanning platforms, ease of operation, isotropic three-dimensional image information, and the ability to derive accurate quantitative data. However, manual data analysis of Micro-CT images can be laborious and time intensive. Deep learning offers the ability to streamline this process, but historically has included caveats--namely, the need for a large amount of training data, which is often limited in many Micro-CT studies. Here we show that accurate deep learning models can be trained using only 1-3 Micro-CT images of the adult Drosophila melanogaster brain using Dragonflys pre-trained neural networks and minimal user knowledge. We further demonstrate the power of our model by showing that it can accurately segment the brain across different tissue contrast stains, scanner models, and genotypes. Finally, we show how the model can assist in identifying morphological similarities and differences between mutants based on volumetric quantification, facilitating a rapid assessment of novel phenotypes. Our models are freely available and can be refined based on individual user needs. SummaryMicro-CT data can be automatically segmented and quantified using a deep learning model trained on as few as 3 samples, facilitating rapid comparison of developmental phenotypes.

developmental biology↗

bioRxiv2023

Seasonal fluctuations in the bone microstructure of Sciurus vulgaris fuscoater humeri: a case study using phenomics on μCT-scans

Sciurus vulgaris Linnaeus, 1758, the red squirrel, is a small, mostly arboreally living rodent, spread across the Palearctic. It is mostly vegetarian, feeding on plants, fungi and seeds, and is less active in the winter months, but does not hibernate. In this lateral study, the humeri of the subspecies Sciurus vulgaris fuscoater, the Central European red squirrel, were analysed to uncover potential intraspecific variation between individuals found in different seasons. The {micro}CT-scans were obtained with a resolution of 26 microns. Five bone parameters were calculated and statistically evaluated with regards to seasonal variations: total volume, bone volume, endocortical surface, cortical thickness, and average trabecular thickness. Bone volume, trabecular thickness and endocortical thickness correlate with bone size, whereas cortical thickness does not. Seasonal differences were observed between the warmer summer and autumn months versus the colder winter and spring months for all parameters. We, speculatively, relate the observed seasonal variation to nutrient intake, notably calcium. These results offer a deeper understanding of intraindividual variation in red squirrels, that may be useful in further ecological, taxonomic, and paleontological research.

zoology↗

bioRxiv2021

Deep learning-based segmentation of high-resolution computed tomography image data outperforms commonly used automatic bone segmentation methods

Segmenting bone from background is required to quantify bone architecture in computed tomography (CT) image data. A deep learning approach using convolutional neural networks (CNN) is a promising alternative method for automatic segmentation. The study objectives were to evaluate the performance of CNNs in automatic segmentation of human vertebral body (micro-CT) and femoral neck (nano-CT) data and to investigate the performance of CNNs to segment data across scanners. Scans of human L1 vertebral bodies (microCT [North Star Imaging], n=28, 53m3) and femoral necks (nano-CT [GE], n=28, 27m3) were used for evaluation. Six slices were selected for each scan and then manually segmented to create ground truth masks (Dragonfly 4.0, ORS). Two-dimensional U-Net CNNs were trained in Dragonfly 4.0 with images of the [FN] femoral necks only, [VB] vertebral bodies only, and [F+V] combined CT data. Global (i.e., Otsu and Yen) and local (i.e., Otsu r = 100) thresholding methods were applied to each dataset. Segmentation performance was evaluated using the Dice coefficient, a similarity metric of overlap. Kruskal-Wallis and Tukey-Kramer post-hoc tests were used to test for significant differences in the accuracy of segmentation methods. The FN U-Net had significantly higher Dice coefficients (i.e., better performance) than the global (Otsu: p=0.001; Yen: p=0.001) and local (Otsu [r=100]: p=0.001) thresholding methods and the VB U-Net (p=0.001) but there was no significant difference in model performance compared to the FN + VB U-net (p=0.783) on femoral neck image data. The VB U-net had significantly higher Dice coefficients than the global and local Otsu (p=0.001 for both) and FN U-Net (p=0.001) but not compared to the Yen (p=0.462) threshold or FN + VB U-net (p=0.783) on vertebral body image data. The results demonstrate that the U-net architecture outperforms common thresholding methods. Further, a network trained with bone data from a different system (i.e., different image acquisition parameters and voxel size) and a different anatomical site can perform well on unseen data. Finally, a network trained with combined datasets performed well on both datasets, indicating that a network can feasibly be trained with multiple datasets and perform well on varied image data.

bioengineering↗

bioRxiv2021

A tyrosine-based trafficking signal in the simian immunodeficiency virus envelope cytoplasmic domain is strongly selected for in pathogenic SIV infection

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239{Delta}GY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239{Delta}GY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the {Delta}GY deletion and a nine nucleotide deletion that encodes amino acids 734-736 ({Delta}QTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naive PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

cell biology↗

bioRxiv2021

Single-molecule super-resolution imaging of T-cell plasma membrane CD4 redistribution upon HIV-1 binding

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For the first time, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

biophysics↗

bioRxiv2020

The Nef protein of the macrophage tropic HIV-1 strain AD8 counteracts human Bst-2/tetherin

Bst-2/tetherin inhibits the release of numerous enveloped viruses by physically attaching nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency viruses (HIV), and pandemic main (M) group HIV-1s are thought to exclusively rely on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 was able to use its Nef protein to compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous tetherin levels from the cell surface, physically separating it from the site of viral budding and thus preventing HIV retention. Mechanistically, AD8 Nef enhances l-tetherin internalisation, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, this is the first report showing that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

cell biology↗

bioRxiv2020

Super-Beacons: open-source probes with spontaneous tunable blinking compatible with live-cell super-resolution microscopy

Localization based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states. These transitions are commonly regulated by high-intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single-molecule self-quenching as a mechanism to generate spontaneous photoswitching independent of illumination. To demonstrate this principle, we developed a new class of DNA-based open-source Super-Resolution probes named Super-Beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live-cell friendly Super-Resolution Microscopy without high-illumination or toxic imaging buffers is revealed by imaging Interferon Inducible Transmembrane proteins (IFITMs) at sub-100nm resolutions.

cell biology↗

bioRxiv2019

Comparative analysis reveals adaptive evolution of bat IFITMs and a novel antiviral determinant

Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site - codon 70 - within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72 and C105, mutation of each cysteine residue individually impairs virus restriction, and a triple C71-C72-C105 mutant loses all restriction, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation.

immunology↗

bioRxiv2018

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

cell biology↗