Biology subjects

Brown, H. G.

Publications and source records attributed to Brown, H. G..

5 recordsLinked to original sources

bioRxiv2026

Whole cryo-FIB lamella acquisition at high magnification with rectangular beam montage cryo-electron tomography

The visualisation of large biological samples in cryo-electron tomography requires both few-nanometre resolution and a wide, ideally >10 micrometrre, field of view. For a single image, increasing magnification trades increased resolution for a smaller field of view. Montaging, where high-magnification images are stitched together to increase field of view, addresses the trade-off but is dose-inefficient with round beams that are standard in transmission electron microscopy (TEM), since this illumination shape does not tessellate. Our workflow greatly simplifies montaging schemes for dose-sensitive specimens, allowing large montages of any shape - from individual cells or other features of interest up to whole lamellae - to be collected at high resolution without compromising electron dose efficiency. We demonstrate the workflow by acquiring montage tomograms of whole yeast (Saccharomyces cerevisiae) cells in cryo-FIB lamellae and of human neurons.

biophysics↗

bioRxiv2025

Amniotic fluid extracellular vesicle properties evolve with gestational age and reflect fetal development

Amniotic fluid (AF) is a valuable source of extracellular vesicles (EVs) derived from the fetoplacental unit. Preclinical and clinical studies have highlighted promising applications of AF-EVs and their role in cellular communication, yet our understanding of AF-EV physiology is limited. This study aimed to examine the physiological importance of AF-EVs in fetal development from the second trimester to term gestation. We obtained AF samples from routine second-trimester amniocentesis and prelabour Caesarean section at term. We isolated EVs using a combination of differential centrifugation, filtration, and ultracentrifugation and characterised them using nanoparticle tracking analysis, cryoelectron microscopy, and Western blotting. The differential EV proteome was analysed using label-free proteomics. We assessed the second trimester and term AF-EV properties through an enrichment analysis. The EV size and protein enrichment difference revealed a gestational-age-dependent variation in the predominant EV subtype. Second-trimester-derived EVs were enriched in ectosomes, while term EVs contained a significant proportion of exosomes. We identified several morphologies of AF-EVs, including unilamellar, multilamellar, multicompartmental and granular-centred EVs, across gestations. Proteomics analysis of AF-EVs identified 4137 proteins with high confidence, of which 1099 exhibited significant differential expression between the two groups. Second-trimester-enriched AF-EV proteins represented molecule assembly processes, metabolism and organogenesis. At term, AF-EV proteins corresponded to impending newborn functions such as immunity and digestion. In conclusion, we provide compelling evidence that EV biogenesis and secretion in the fetoplacental unit undergo significant alterations across gestation, revealing a complex and dynamic physiology and intercellular communication that adapts to the needs of the developing fetus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/649052v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@3358c4org.highwire.dtl.DTLVardef@108ec96org.highwire.dtl.DTLVardef@e49b91org.highwire.dtl.DTLVardef@a1888b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO This figure summarises the studys main findings, including the shift in the predominant EV subtype, the different organs and biofluids represented by the enriched proteins at each gestation, and the main biological pathways. C_FIG

developmental biology↗

bioRxiv2023

Vgamma9Vdelta2 T cells recognize butyrophilin 2A1 and 3A1 heteromers

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity, however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell receptor ({gamma}{delta}TCR) in complex with BTN member 2A1 (BTN2A1) revealed that BTN2A1 engages the side of the {gamma}{delta}TCR, leaving the apical TCR surface bioavailable. We reveal that BTN3A1 is a second {gamma}{delta}TCR ligand, that co-engages {gamma}{delta}TCR via binding to this accessible apical surface. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to engage {gamma}{delta}TCR; indeed, either forced separation or locking together of BTN2A1 and BTN3A1 resulted in enhanced or abrogated {gamma}{delta}TCR interaction, respectively. Our findings reveal a new paradigm in immune activation, whereby {gamma}{delta}TCRs recognize dual epitopes on BTN2A1 and BTN3A1 complexes.

immunology↗

bioRxiv2023

Fitting a square beam in a square camera: novel condensor apertures for low-dose transmission electron microscopy

In transmission electron microscopy (TEM) cameras are square or rectangular but beams are round. With a beam size chosen to fill the camera at a given image magnification, the circular lobes of the beam will extend beyond the cameras field of view and irradiate areas that are not acquired on the camera, damaging and precluding them from future acquisitions if the sample is beam sensitive. In this paper we present development of condenser aperture plates for TEM that have square and rectangular apertures which improve the efficiency of sample area usage by 44% or greater in low dose TEM applications. We demonstrate that the use of these apertures is compatible with high-resolution cryogenic (cryo) TEM by reconstructing sub 2 [A] apo-ferritin models from a datasets recorded with both square and rectangular apertures. Moreover the design of our aperture plates should improve the flexibility of 2 condenser systems for cryo-TEM acquisitions with multiple shots per hole by tailored matching of beam sizes to camera sizes at each magnification.

biophysics↗

bioRxiv2021

MeasureIce: Accessible on-the-fly measurement of ice thickness in cryo-electron microscopy

Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo electron microscopy. The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some addition noise in the TEM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the "ice-channel" technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 [A]ngstrom resolution in subsequent refinement of the atomic map.

biophysics↗