Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Neat plasma proteomics: getting the best out of the worst.

Plasma proteomics holds immense potential for clinical research and biomarker discovery, serving as a non-invasive "liquid biopsy" for tissue sampling. Mass spectrometry (MS)-based proteomics, thanks to improvement in speed and robustness, emerges as an ideal technology for exploring the plasma proteome for its unbiased and highly specific protein identification and quantification. Despite its potential, plasma proteomics is still a challenge due to the vast dynamic range of protein abundance, hindering the detection of less abundant proteins. Different approaches can help overcome this challenge. Conventional depletion methods face limitations in cost, throughput, accuracy, and off-target depletion. Nanoparticle-based enrichment shows promise in compressing dynamic range, but cost remains a constraint. Enrichment strategies for extracellular vesicles (EVs) can enhance plasma proteome coverage dramatically, but current methods are still too laborious for large series. Neat plasma remains popular for its cost-effectiveness, time efficiency, and low volume requirement. We used a test set of 33 plasma samples for all evaluations. Samples were digested using S-Trap and analyzed on Evosep One and nanoElute coupled to a timsTOF Pro using different elution gradients and ion mobility ranges. Data were mainly analyzed using library-free searches using DIA-NN. This study explores ways to improve proteome coverage in neat plasma both in MS data acquisition and MS data analysis. We demonstrate the value of sampling smaller hydrophilic peptides, increasing chromatographic separation, and using library-free searches. Additionally, we introduce the EV boost approach, that leverages on the extracellular vesicle fraction to enhance protein identification in neat plasma samples. Globally, our optimized analysis workflow allows the quantification of over 1000 proteins in neat plasma with a 24SPD throughput. We believe that these considerations can be of help independently of the LC-MS platform used.

Effect of urine alkalization on urinary inflammatory markers in cystinuric patients.

Background: Cystinuria is associated with a high prevalence of chronic kidney disease (CKD). We previously described a urinary inflammatory-protein signature (UIS), including 38 upregulated proteins, in cystinuric patients (Cys-patients), compared with healthy controls (HC). This UIS was higher in Cys-patients with CKD. In the present observational study, we aimed to investigate the UIS in Cys-patients without CKD and patients with calcium nephrolithiasis (Lith-patients), versus HC and the effect of urine alkalization on the UIS of Cys-patients. Methods: UIS was evaluated by nano-liquid chromatography coupled to high-resolution mass spectrometry in adult HC, Lith-patients and non-treated Cys-patients with an estimated glomerular filtration rate >60 mL/min/1.73 m2, and after a 3-month conventional alkalizing treatment in Cys-patients. Results: Twenty-one Cys-patients [12 men, median age (interquartile range) 30.0 (25.0-44.0) years], 12 Lith-patients [8 men, 46.2 (39.5-54.2) years] and 7 HC [2 men, 43.1 (31.0-53.9) years] were included. Among the 38 proteins upregulated in our previous work, 11 proteins were also upregulated in Cys-patients compared with HC in this study (5 circulating inflammatory proteins and 6 neutrophil-derived proteins). This UIS was also found in some Lith-patients. Using this UIS, we identified two subclusters of Cys-patients (5 with a very high/high UIS and 16 with a moderate/low UIS). In the Cys-patients with very high/high UIS, urine alkalization induced a significant decrease in urinary neutrophil-derived proteins. Conclusion: A high UIS is present in some Cys-patients without CKD and decreases under alkalizing treatment. This UIS could be a prognostic marker to predict the evolution towards CKD in cystinuria.

mTOR inhibition suppresses salinomycin-induced ferroptosis in breast cancer stem cells by ironing out mitochondrial dysfunctions.

Ferroptosis constitutes a promising therapeutic strategy against cancer by efficiently targeting the highly tumorigenic and treatment-resistant cancer stem cells (CSCs). We previously showed that the lysosomal iron-targeting drug Salinomycin (Sal) was able to eliminate CSCs by triggering ferroptosis. Here, in a well-established breast CSCs model (human mammary epithelial HMLER CD24low/CD44high), we identified that pharmacological inhibition of the mechanistic target of rapamycin (mTOR), suppresses Sal-induced ferroptosis. Mechanistically, mTOR inhibition modulates iron cellular flux and thereby limits iron-mediated oxidative stress. Furthermore, integration of multi-omics data identified mitochondria as a key target of Sal action, leading to profound functional and structural alteration prevented by mTOR inhibition. On top of that, we found that Sal-induced metabolic plasticity is mainly dependent on the mTOR pathway. Overall, our findings provide experimental evidence for the mechanisms of mTOR as a crucial effector of Sal-induced ferroptosis pointing not only that metabolic reprogramming regulates ferroptosis, but also providing proof-of-concept that careful evaluation of such combination therapy (here mTOR and ferroptosis co-targeting) is required in the development of an effective treatment.

Using the Intrinsic Geometry of Binodal Curves to Simplify the Computation of Ternary Liquid-Liquid Phase Diagrams.

Phase diagrams are powerful tools to understand the multi-scale behaviour of complex systems. Yet, their determination requires in practice both experiments and computations, which quickly becomes a daunting task. Here, we propose a geometrical approach to simplify the numerical computation of liquid-liquid ternary phase diagrams. We show that using the intrinsic geometry of the binodal curve, it is possible to formulate the problem as a simple set of ordinary differential equations in an extended 4D space. Consequently, if the thermodynamic potential, such as Gibbs free energy, is known from an experimental data set, the whole phase diagram, including the spinodal curve, can be easily computed. We showcase this approach on four ternary liquid-liquid diagrams, with different topological properties, using a modified Flory-Huggins model. We demonstrate that our method leads to similar or better results comparing those obtained with other methods, but with a much simpler procedure. Acknowledging and using the intrinsic geometry of phase diagrams thus appears as a promising way to further develop the computation of multiphase diagrams.

Propranolol reduces the accumulation of cytotoxic aggregates in C9orf72-ALS/FTD in vitro models.

Mutations in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathogenetic mechanisms linked to this gene are a direct consequence of an aberrant intronic expansion of a GGGGCC hexanucleotide located between the 1a and 1b non-coding exons, which can be transcribed to form cytotoxic RNA foci or even translated into aggregation-prone dipeptide repeat proteins. Importantly, the abnormal length of these repeats affects also the expression levels of C9orf72 itself, which suggests haploinsufficiency as additional pathomechanism. Thus, it appears that both toxic gain of function and loss of function are distinct but still coexistent features contributing to the insurgence of the disease in case of C9orf72 mutations. In this study, we aimed at identifying a strategy to address both aspects of the C9orf72-related pathobiochemistry and provide proof-of-principle information for a better understanding of the mechanisms leading to neuronal loss. By using primary neurons overexpressing toxic poly(GA), the most abundant protein product of the GGGGCC repeats, we found that the antiarrhythmic drug propranolol could efficiently reduce the accumulation of aberrant aggregates and increase the survival of C9orf72-related cultures. Interestingly, the improved catabolism appeared to not depend on major degradative pathways such as autophagy and the proteasome. By analyzing the proteome of poly(GA)-expressing neurons after exposure to propranolol, we found that the drug increased lysosomal degradation through a mechanism directly involving C9orf72 protein, whose levels were increased after treatment. Further confirmation of the beneficial effect of the beta blocker on aggregates' accumulation and survival of hiPSC-derived C9orf72-mutant motoneurons strengthened the finding that addressing both facets of C9orf72 pathology might represent a valid strategy for the treatment of these ALS/FTD cases.

Nanoprecipitation through solvent-shifting using rapid mixing: Dispelling the Ouzo boundary to reach large solute concentrations.

HYPOTHESIS: The addition of a non-solvent to a solute in good solvent solution leads to nanoprecipitation, which is the spontaneous formation of nanodomains. Yet, increasing solute concentration usually leads to the formation of macrodomains that quickly separate into a bulk phase, which is a severe process limitation. The corresponding concentration threshold, often termed as the Ouzo boundary, remains a mystery that could find its origin in the complex interplay between nanoprecipitation and mixing. EXPERIMENTS: We performed a systematic investigation of nanoprecipitation thermodynamics and kinetics as well as its interplay with mixing hydrodynamics for the hexadecane-acetone-water system, in the presence of the non-ionic C16EO8 surfactant. The binodal curve and its underlying tie-lines were obtained using Raman spectroscopy, allowing the computation of the spinodal curve. Kinetics were probed using a continuous flow setup that combines two sequential rapid mixers. The impact of mixing efficiency was probed systematically by varying the oil concentration for respectively slow and rapid mixing, while the uncoupling from mixing and nanoprecipitation was quantified by modifying systematically the flow rate in a continuous flow approach. FINDINGS: We elucidate the nature of the Ouzo boundary that marks the maximal solute concentration leading to nanoobjects. Rather than a thermodynamic boundary, as evidenced by its uncorrelation to the spinodal curve, it results from the coupling of nanoprecipitation and mixing when both processes occur within the same time range, leading to heterogeneous conditions and the escape of some objects to the macroscale. Increasing the solute concentration speeds up nanoprecipitation and thus requires increasingly faster mixing times to uncouple both processes. Accordingly, if the mixing efficiency is large enough, it is possible to dispel the Ouzo boundary and reach very large solute concentrations. Implementing rapid mixing strategies in continuous flow approaches is thus the solution to overcome the most stringent condition of nanoprecipitation and open the way to scale-up, while also providing efficient means to probe its fast mechanism. Overall, the simultaneous control of hydrodynamics and physical chemistry is thus key to boost up the Ouzo effect.

Pancreatic islet cell stress induced by insulin-degrading enzyme deficiency promotes islet regeneration and protection from autoimmune diabetes.

Appropriate tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with highly variable demand for insulin synthesis. An efficient UPR ensures a sufficient beta cell mass and secretory output but can also affect beta cell resilience to autoimmune aggression. The factors regulating protein homeostasis in the face of metabolic and immune challenges are insufficiently understood. We examined beta cell adaptation to stress in mice deficient for insulin-degrading enzyme (IDE), a ubiquitous protease with high affinity for insulin and genetic association with type 2 diabetes. IDE deficiency induced a low-level UPR in both C57BL/6 and autoimmune non-obese diabetic (NOD) mice, associated with rapamycin-sensitive beta cell proliferation strongly enhanced by proteotoxic stress. Moreover, in NOD mice, IDE deficiency protected from spontaneous diabetes and triggered an additional independent pathway, conditional on the presence of islet inflammation but inhibited by proteotoxic stress, highlighted by strong upregulation of regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. Our findings establish a key role of IDE in islet cell protein homeostasis, identify a link between low-level UPR and proliferation, and reveal an UPR-independent anti-inflammatory islet cell response uncovered in the absence of IDE of potential interest in autoimmune diabetes.

An Alkanethiol-Surfactant Bilayer To Prevent Shape Conversion of Anisotropic Silver Nanoparticles and Probe Their Formation Mechanisms.

Anisotropic nanoparticles can be synthesized under kinetic control but may undergo subsequent shape changes due to atomic reorganization. Furthermore, their synthesis involves rapid steps, which are challenging to monitor in situ. Here, we show how a nanoemulsion of alkanethiols with an ethoxylated surfactant, easily prepared and metastable for months, can simultaneously prevent shape reorganization and arrest reaction kinetics. We illustrate this generic method on the silver nanoplates synthesized in concentrated acetic acid aqueous solutions, in which rapid shape reorganization occurs. We show that there exists an optimum thiol concentration corresponding to full coverage of all silver surface atoms, which can be simply calculated from particle dimensions. Furthermore, we demonstrate that arresting nanoparticle formation can be achieved within milliseconds using a tandem rapid mixers scheme in a continuous flow setup, allowing ex situ monitoring of the reaction.

Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts with the v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7 knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7.

Proteomic profiling of sweat in patients with cystic fibrosis provides new insights into epidermal homoeostasis.

Background: A high proportion of patients with Cystic Fibrosis (CF) also present the rare skin disease aquagenic palmoplantar keratoderma. A possible link between this condition and absence of a functional CF Transmembrane conductance Regulator protein in the sweat acinus and collecting duct remains unknown. Methods: In-depth characterization of sweat proteome profiles was performed in 25 CF patients compared to 12 healthy controls. A 20 µL sweat sample was collected after pilocarpine iontophoresis and liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomic analysis was performed. Results: Sweat proteome profile of CF patients was significantly different from that of healthy subjects with 57 differentially expressed proteins. Cystic Fibrosis sweat proteome was characterized by an increase in 25 proteins including proteases (Kallikrein 7 and 13, Phospholipase B domain containing 1, Cathepsin A L2 and B, Lysosomal Pro-X carboxypeptidase); proinflammatory proteins (Annexin A2, Chitinase-3-like protein 1); cytochrome c and transglutaminases. Thirty-two proteins were downregulated in CF sweat including proteases (Elastase 2), antioxidative protein FAM129 B; membrane-bound transporter SLC6A14 and regulator protein Sodium-hydrogen antiporter 3 regulator 1. Conclusion: This study is the first to report in-depth characterization of endogenous peptides in CF sweat and could help understand the complex physiology of the sweat gland. The proteome profile highlights the unbalanced proteolytic and proinflammatory activity of sweat in CF. These results also suggest a defect in pathways involved in skin barrier integrity in CF patients. Sweat proteome profile could prove to be a useful tool in the context of personalized medicine in CF.

Measuring mutual diffusion coefficients in aqueous binary mixtures with unidimensional drying cells.

Chemical diffusion is a mass transport process caused by thermally generated motions of species. In a binary mixture, the diffusion of one species in one direction involves the diffusion of another species in the opposite direction, which corresponds to a single mutual diffusion coefficient. Here, we report a simple and general method to measure such coefficients in binary liquid mixtures, using the PNIPAM/water system as a study case. Experimentally, we show how a simple unidirectional drying cell coupled with a spatially-resolved characterization method such as Raman microscopy can yield concentration gradients developing in between two boundaries of known and constant chemical potential. Acquiring such gradients over time leads to a time-set that is shown to collapse to a single master curve using a change of variable. Such a scaling law offers a self-checking frame for solving analytically the diffusion-advection equation. As a result, we show that a simple analytical formula relates the measured concentration gradient with the concentration-dependent mutual diffusion coefficient. In the PNIPAM/water system, the mutual diffusion coefficient sharply decreases at low water content. Our work thus highlights the importance of considering the concentration-dependence of the mutual diffusion coefficient in complex aqueous solutions and provides a method to measure it.

Promitotic Action of Oenothera biennis on Senescent Human Dermal Fibroblasts.

Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. The aim of this work is to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of 'senescence-associated-ß-galactosidase', the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significative reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific promitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.

Assessing suspension and infectivity times of virus-loaded aerosols involved in airborne transmission.

Airborne transmission occurs through droplet-mediated transport of viruses following the expulsion of an aerosol by an infected host. Transmission efficiency results from the interplay between virus survival in the drying droplet and droplet suspension time in the air, controlled by the coupling between water evaporation and droplet sedimentation. Furthermore, droplets are made of a respiratory fluid and thus, display a complex composition consisting of water and nonvolatile solutes. Here, we quantify the impact of this complex composition on the different phenomena underlying transmission. Solutes lead to a nonideal thermodynamic behavior, which sets an equilibrium droplet size that is independent of relative humidity. In contrast, solutes do not significantly hinder transport due to their low initial concentration. Realistic suspension times are computed and increase with increasing relative humidity or decreasing temperature. By uncoupling drying and suspended stages, we observe that enveloped viruses may remain infectious for hours in dried droplets. However, their infectivity decreases with increasing relative humidity or temperature after dozens of minutes. Examining expelled droplet size distributions in the light of these results leads to distinguishing two aerosols. Most droplets measure between 0 and 40 µm and compose an aerosol that remains suspended for hours. Its transmission efficiency is controlled by infectivity, which decreases with increasing humidity and temperature. Larger droplets form an aerosol that only remains suspended for minutes but corresponds to a much larger volume and thus, viral load. Its transmission efficiency is controlled by droplet suspension time, which decreases with increasing humidity and decreasing temperature.

BLI-MS: Combining biolayer interferometry and mass spectrometry.

Biolayer interferometry (BLI) is a technology which allows to study the affinity between two interacting macro-molecules and to visualize their kinetic of interaction in real time. In this work, we combine BLI interaction measurement with mass spectrometry in order to identify the proteins interacting with the bait. We provide for the first time the proof of concept of the feasibility of BLI-MS in complex biological mixtures.

Controlling nanoparticle formation from the onset of nucleation through a multi-step continuous flow approach.

HYPOTHESIS: Metallic nanoparticles of various shapes and sizes can be synthesised through a diversity of bottom-up pathways, such as precipitation induced by chemical reduction. Varying composition, by adjusting concentrations or adding/replacing species, is the predominant strategy to tune nanoparticles structures. However, controlling time down to the onset of precipitation, nucleation, should also provide a powerful means to control nanostructuration. EXPERIMENTS: We perform sequential reagent additions with a time resolution down to the millisecond. We use a millifluidic continuous flow setup consisting of tangential mixers in series, which allows flow rates up to dozens of litres per hour. We systematically vary both addition order and delay for each reagent involved in the synthesis of silver nanoplates. The resulting dispersions are compared using UV-visible spectroscopy, transmission electron microscopy and small-angle X-ray scattering. FINDINGS: We show that synthesis pathways differing only in the order of sub-second additions lead to drastically different synthetic outcomes. Silver nanoparticles of different shapes and sizes, displaying an array of plasmonic colours, are synthesised at the same final composition by tuning the composition pathways along time. Our results unlock a previously inaccessible portion of the space of parameters, which will lead to an enhanced structural diversity, control and understanding of nanoparticles syntheses.

How the interplay of molecular and colloidal scales controls drying of microgel dispersions.

Bringing an aqueous dispersion or solution into open air leads to water evaporation. The resulting drying process initiates the buildup of spatial heterogeneities, as nonvolatile solutes and colloids concentrate. Such composition gradients associate with mesostructure gradients, which, in turn, impact flows within these multicomponent systems. In this work, we investigate the drying of microgel dispersions in respect to two reference systems, a colloidal dispersion and a polymer solution, which, respectively, involve colloidal and molecular length scales. We evidence an intermediate behavior in which a film forms at the air/liquid interface and is clearly separated from bulk by a sharp drying front. However, complex composition and mesostructure gradients develop throughout the drying film, as evidenced by Raman and small-angle X-ray scattering mapping. We show that this results from the soft colloidal structure of microgel, which allows them to interpenetrate, deform, and deswell. As a result, water activity and water transport are drastically decreased in the vicinity of the air/liquid interface. This notably leads to diffusional drying kinetics that are nearly independent on the air relative humidity. The interplay between water fraction, water activity, and mesostructure on water transport is generic and, thus, shown to be pivotal in order to master evaporation in drying complex fluids.

Two Dimensional Oblique Molecular Packing within a Model Peptide Ribbon Aggregate.

A10 K (A=alanine, K=lysine) model peptides self-assemble into ribbon-like ß-sheet aggregates. Here, we report an X-ray diffraction investigation on a flow-aligned dispersion of these self-assembly structures. The two-dimensional wide-angle X-ray scattering pattern suggests that peptide pack in a two-dimensional oblique lattice, essentially identical to the crystalline packing of polyalanine, An (for n>4). One side of the oblique unit cell, corresponding to the anti-parallel ß-sheet, is oriented along the ribbon's axis. Together with recently published small angle X-ray scattering data of the same system, this work thus yields a detailed description of the self-assembled ribbon aggregates, down to the molecular length scale. Notably, our results highlight the importance of the crystalline peptide packing within its self-assembly aggregates, which is often neglected.

Polyvinylpyrrolidone (PVP) impurities drastically impact the outcome of nanoparticle syntheses.

HYPOTHESIS: Polymer additives such as Polyvinylpyrrolidone (PVP) are ubiquitously used in wet chemical reduction methods to tune nanoparticle sizes and shapes. However, all polymers retain some traces of their synthetic history through their end-groups and impurities. These impurities may thus impact redox and interfacial processes occurring during the formation of nanocolloids. EXPERIMENTS: We report a systematic comparison of four representative silver nanoparticle syntheses in the presence of either commercial PVP or its purified version, obtained through dialysis or filtration. We characterized the resulting nanoparticle dispersions through UV-visible spectroscopy, electron microscopy, X-ray scattering and Raman spectroscopy. FINDINGS: For all syntheses and methods, the simple removal of PVP molecular impurities drastically modifies nanoparticle size, shape and formation kinetic. Impurities from additives thus play a pivotal role in nanoparticle syntheses and must be systematically evaluated for relevant mechanistic investigations and robust process engineering.