Biology subjects

Paris, G.

Publications and source records attributed to Paris, G..

7 recordsLinked to original sources

bioRxiv2026

Rapid Production and Cryo-EM Structure Determination of GPCR-Nanodisc Complexes via Cell-Free Synthesis

G protein-coupled receptors (GPCRs) represent major therapeutic targets, yet cryo-EM structure determination of unliganded or antagonist-bound GPCRs remains severely bottlenecked by small receptor mass, detergent-induced instability, and multi-week cell-based production pipelines. Here, we combine intracellular loop 3 (ICL3) rigid fiducial engineering with a high-throughput, cell-free protein synthesis workflow to rapidly generate cryo-EM-ready GPCR-nanodisc assemblies. Using the eProtein Discovery(TM) digital microfluidics system, we performed automated nanoliter-scale screening of construct fusions and nanodisc lipid compositions, establishing that co-translational insertion into nanodiscs is strictly required for receptor solubilization and affinity purification. Direct scale-up of screening conditions yielded structural-grade protein within four days from gene assembly to grid vitrification. We demonstrate this workflow with a 3.38 Angstrom cryo-EM structure of antagonist-bound human adenosine A2A receptor (A2AR)-beta-lactamase fusion in complex with beta-lactamase inhibitory protein II (BLIP-II). To demonstrate generalizability of protein quality, we extended this detergent-free co-translational pipeline to an expanded panel of 12 wild-type GPCRs spanning Classes A, B, and C, as well as six orphan receptors. All targets were purified with biophysical quality comparable to the A2AR benchmark. By removing the reliance on cellular expression or agonist/G-protein stabilization and compressing construct assembly, nanodisc screening, biophysical quality control, and grid vitrification into a four-day turnaround, this detergent-free cell-free pipeline provides a rapid Design-Build-Test-Learn (DBTL) engine to accelerate structure-based drug discovery for challenging and unliganded GPCR targets.

biochemistry↗

bioRxiv2026

IsdH binding to hemoglobin:haptoglobin is decoupled from iron acquisition in Staphylococcus aureus

Staphylococcus aureus requires iron for proliferation during infection and acquires it mainly from host hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The hemophores IsdB and IsdH mediate the initial steps of Hb recognition. Notably, IsdH also recognizes the hemoglobin:haptoglobin (HbHp) complex, although the molecular determinants and physiological relevance of this interaction remain unclear. Here, combining cryo-electron microscopy and biochemical and cellular assays, we define the basis of HbHp recognition by IsdH. The structure reveals how IsdH engages HbHp and captures the intrinsic conformational flexibility of the HbHp assembly. Functional analyses demonstrate that, despite retaining the ability to extract heme from HbHp, IsdH does not support S. aureus growth under iron-restricted conditions when HbHp is the sole iron source. These findings suggest that HbHp recognition by IsdH may serve functions beyond nutrient iron acquisition, contributing to modulation of host-pathogen interactions.

biochemistry↗

bioRxiv2026

Substrate induced activation in the conserved ribonuclease YicC

YicC is a conserved protein, recently discovered to have hydrolytic endoribonuclease activity, with a phylogenetic distribution implicating an ancestry deeper than that for most extant ribonucleases central to RNA metabolism. We present evidence that Escherichia coli YicC can cleave the small regulatory RNAs (sRNAs), RyhB and RprA, even when they are sequestered in otherwise protective complexes with the RNA chaperone Hfq. Nonetheless, YicC activity is markedly diminished when RyhB and other sRNAs are paired with cognate mRNA. Our cryoEM structures of catalytically inactive YicC in complex with RyhB and in the apo state reveal quaternary and tertiary structural switches, triggered by substrate engagement, that engulf and ratchet a stem loop element of the sRNA into an internal channel, where metal-assisted hydrolytic action occurs. Based on these findings, we propose that the enzyme favours specific stem-loop structures and may discriminate between pools of active, target-engaged sRNAs and those that are inactive. The mechanism for substrate-triggered conformational switching could represent an ancient strategy for selective RNA degradation.

molecular biology↗

bioRxiv2025

Reproducibility and reliability of Free-Water-corrected Diffusion Tensor Imaging of the brain: Revisited

Diffusion tensor imaging (DTI) corrected for the free-water (FW) enables the separation of a hindered Gaussian-like profile from an isotropic component, which represents diffusion found in cerebrospinal and interstitial fluids within the extracellular space of grey and white matter. The assessment of the reproducibility and reliability properties of FW-corrected DTI is a crucial factor in demonstrating the potential clinical utility of this refinement, particularly considering the examinations across multiple medical centres. This paper explores the variability, reliability, and separability properties of free-water volume fraction (FWVF) and FW-corrected DTI-based measures in healthy human brain white matter using publicly available test-retest databases acquired in 1) intra-scanner, 2) intra-scanner longitudinal and 3) inter-scanner settings under varying acquisition schemes. Three different estimation techniques to retrieve the FW-corrected DTI parameters tailored to single- or multiple-shell diffusion-sensitizing magnetic resonance (MR) acquisitions are investigated: i) a direct optimization of bi-tensor signal representation in the variational framework, ii) the region contraction-based approach, and iii) the spherical means technique combined with a correction of diffusion-weighted MR signal prior to DTI estimation. We found the previous suggestion (Hum. Brain Mapp., 38(1), 2017, 10.1002/hbm.23350) that the FW correction to DTI in a single-shell diffusion-weighted MR acquisition improves the repeatability and reliability of DTI-based measures may be data- and methodology-dependent, and does not generalise to multiple-shell scenario. Our experiments have shown that the most reliable and repeatable/reproducible measures, while preserving a moderate separability property, are fractional anisotropy and axial diffusivity estimated in a multiple-shell variant under a combined FW-correction scheme. On the contrary, our results show evidence that the least reliable measures are the mean diffusivity estimated using any FW-correction procedure, as well as the FWVF parameter itself. These results can be used to establish the direction for selecting the most attractive FW-correction DTI scheme for clinical applications in terms of the variability-reliability-separability criterion.

bioengineering↗

bioRxiv2024

Plastid translocon recycling in dinoflagellates demonstrates the portability of complex plastids between hosts

The plastids of photosynthetic organisms on land are predominantly primary plastids derived from an ancient endosymbiosis of a cyanobacterium. Conversely, marine photosynthetic diversity is dominated by plastids gained by subsequent endosymbioses of photosynthetic eukaryotes, so-called complex plastids. The plastids of major eukaryotic lineages including cryptophytes, haptophytes, stramenopiles, dinoflagellates and apicomplexans, were originally all posited to derive from a single secondary endosymbiosis of a red alga--the chromalveloate hypothesis 1. Subsequent phylogenetic resolution of eukaryotes indicated that separate events of plastid acquisition must have occurred to account for this distribution of plastids 2,3. The number of such events, however, and the donor organisms for the new plastid endosymbioses are still not resolved. A perceived bottleneck of endosymbiotic plastid gain is the development of protein targeting from the hosts into new plastids, and this supposition has often driven hypotheses towards minimising the number of plastid-gain events to explain plastid distribution in eukaryotes. But how plastid protein-targeting is established for new endosymbionts is often unclear, which makes it difficult to assess the likelihood of plastid transfers between lineages. Here we show that Kareniaceae dinoflagellates, that possess complex plastids known to be derived from haptophytes, acquired all the necessary protein import machinery from these haptophytes. Furthermore, cryo-electron tomography revealed that no additional membranes were added to the Kareniaceae complex plastid during serial endosymbiosis, suggesting that the haptophyte-derived import processes were sufficient. Our analyses suggests that complex red plastids are preadapted for horizontal transmission, potentially explaining their widespread distribution in aquatic algal diversity.

evolutionary biology↗

bioRxiv2022

Free-water volume fraction increases non-linearly with age in the white matter of the healthy human brain

The term free-water volume fraction (FWVF) refers to the cerebrospinal and interstitial fluids in the extracellular space of the white matter (WM) of the brain, which has been demonstrated as a sensitive biomarker that correlates with the cognitive performance and the neuropathological processes modifying the interstitial extracellular spaces. It can be quantified by properly fitting the isotropic compartment of the magnetic resonance (MR) signal in diffusion-sensitized sequences. Using N = 287 healthy subjects aged 25-94, this study examines in detail the evolution of the FWVF in the human brain WM across the adult lifespan, which has been previously reported to exhibit a positive trend. We found evidence of a noticeably non-linear gain after the sixth decade of life, with a region-specific variate and varying change rate of the FWVF parameter with age, at the same time a heteroskedastic pattern across the adult lifespan is suggested. On the other hand, the FW-compensated MR signal leads to a region-dependent flattened age-related evolution of the mean diffusivity (MD) and fractional anisotropy (FA), along with a considerable reduction in their variability, as compared to standard studies conducted over the raw MR signal. This way, our study provides a new perspective on the trajectory-based assessment of the brain and explains the source of the variations observed in FA and MD parameters across the lifespan with previous studies with the standard diffusion tensor imaging.

neuroscience↗

bioRxiv2021

Anisotropy Measure from Three Diffusion-Encoding Gradient Directions

PurposeWe propose a method that can provide information about the anisotropy and orientation of diffusion in the brain from only 3 orthogonal gradient directions without imposing additional assumptions. MethodsThe method is based on the Diffusion Anisotropy (DiA) that measures the distance from a diffusion signal to its isotropic equivalent. The original formulation based on a Spherical Harmonics basis allows to go down to only 3 orthogonal directions in order to estimate the measure. In addition, an alternative simplification and a color-coding representation are also proposed. ResultsAcquisitions from a publicly available database are used to test the viability of the proposal. The DiA succeeded in providing anisotropy information from the white matter using only 3 diffusion-encoding directions. The price to pay for such reduced acquisition is an increment in the variability of the data and a subestimation of the metric. ConclusionsThe calculation of anisotropy information from DMRI is feasible using fewer than 6 gradient directions by using DiA. The method is totally compatible with existing acquisition protocols and it may provide complementary information about orientation in fast diffusion acquisitions.

neuroscience↗