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Gianni Ciofani1, Serena Danti2, Giada Graziana Genchi1,3, Delfo D'Alessandro2, Jean-Luc Pellequer4, Michaël Odorico4, Virgilio Mattoli1, Mario Giorgi51Italian Institute of Technology, Center of MicroBioRobotics co Scuola Superiore Sant'Anna, 2Department of Neuroscience, University of Pisa, 3The BioRobotics Institute, Scuola Superiore Sant'Anna, Pisa, Italy; 4Commissariat à l'Energie Atomique, Institut de Biologie Environnementale et Biotechnologie, Department of Biochemistry and Nuclear Toxicology, Bagnols-sur-Cèze, France; 5Division of Pharmacology and Toxicology, Veterinary Clinics Department, University of Pisa, Pisa, ItalyAbstract: Boron nitride nanotubes (BNNTs) have attracted huge attention in many different research fields thanks to their outstanding chemical and physical properties. During recent years, our group has pioneered the use of BNNTs for biomedical applications, first of all assessing their in vitro cytocompatibility on many different cell lines. At this point, in vivo investigations are necessary before proceeding toward realistic developments of the proposed applications. In this communication, we report a pilot toxicological study of BNNTs in rabbits. Animals were injected with a 1 mg/kg BNNT solution and blood tests were performed up to 72 hours after injection. The analyses aimed at evaluating any acute alteration of hematic parameters that could represent evidence of functional impairment in blood, liver, and kidneys. Even if preliminary, the data are highly promising, as they showed no adverse effects on all the evaluated parameters, and therefore suggest the possibility of the realistic application of BNNTs in the biomedical field.Keywords: boron nitride nanotubes, in vivo testing, toxicology

After the recent double revolutions in structural biology, which include the use of direct detectors for cryo-electron microscopy resulting in a significant improvement in the expected resolution of large macromolecule structures, and the advent of AlphaFold which allows for near-accurate prediction of any protein structures, the field of structural biology is now pursuing more ambitious targets, including several MDa assemblies. But complex target systems cannot be tackled using a single biophysical technique. The field of integrative structural biology has emerged as a global solution. The aim is to integrate data from multiple complementary techniques to produce a final three-dimensional model that cannot be obtained from any single technique. The absence of atomic force microscopy data from integrative structural biology platforms is not necessarily due to its nm resolution, as opposed to Å resolution for x-ray crystallography, nuclear magnetic resonance, or electron microscopy. Rather a significant issue was that the AFM topographic data lacked interpretability. Fortunately, with the introduction of the AFM-Assembly pipeline and other similar tools, it is now possible to integrate AFM topographic data into integrative modeling platforms. The advantages of single molecule techniques, such as AFM, include the ability to confirm experimentally any assembled molecular models or to produce alternative conformations that mimic the inherent flexibility of large proteins or complexes. The review begins with a brief overview of the historical developments of AFM data in structural biology, followed by an examination of the strengths and limitations of AFM imaging, which have hindered its integration into modern modeling platforms. This review discusses the correction and improvement of AFM topographic images, as well as the principles behind the AFM-Assembly pipeline. It also presents and discusses a series of challenges that need to be addressed in order to improve the incorporation of AFM data into integrative modeling platform., (© 2024 The Author(s). Journal of Molecular Recognition published by John Wiley & Sons Ltd.)

Background: Atomic force microscopy (AFM) is one of the main techniques used to characterize the mechanical properties of soft biological samples and biomaterials at the nanoscale. Despite efforts made by the AFM community to promote open-source data analysis tools, standardization continues to be a significant concern in a field that requires common analysis procedures. AFM-based mechanical measurements involve applying a controlled force to the sample and measure the resulting deformation in the so-called force-distance curves. These may include simple approach and retract or oscillatory cycles at various frequencies (microrheology). To extract quantitative parameters, such as the elastic modulus, from these measurements, AFM measurements are processed using data analysis software. Although open tools exist and allow obtaining the mechanical properties of the sample, most of them only include standard elastic models and do not allow the processing of microrheology data. In this work, we have developed an open-source software package (called PyFMLab, as of python force microscopy laboratory) capable of determining the viscoelastic properties of samples from both conventional force-distance curves and microrheology measurements., Methods: PyFMLab has been written in Python, which provides an accessible syntax and sufficient computational efficiency. The software features were divided into separate, self-contained libraries to enhance code organization and modularity and to improve readability, maintainability, testability, and reusability. To validate PyFMLab, two AFM datasets, one composed of simple force curves and another including oscillatory measurements, were collected on HeLa cells., Results: The viscoelastic parameters obtained on the two datasets analysed using PyFMLab were validated against data processing proprietary software and against validated MATLAB routines developed before obtaining equivalent results., Conclusions: Its open-source nature and versatility makes PyFMLab an open-source solution that paves the way for standardized viscoelastic characterization of biological samples from both force-distance curves and microrheology measurements., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 López-Alonso J et al.)

The plasticity and growth of plant cell walls (CWs) remain poorly understood at the molecular level. In this work, we used atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4-day-old Arabidopsis thaliana wild-type and almt1-mutant seedlings grown under Fe or Al stresses. Elastic parameters were deduced from force-distance curve measurements using the trimechanic-3PCS framework. The presence of single metal species Fe 2+ or Al 3+ at 10 µM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root-extension arrest concomitant with significant increase of CW stiffness. Raising the concentration of either Fe 2+ or Al 3+ to 20 µM, no root-extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 µM, root-extension arrest was not observed in the almt1 mutant, which substantially abolishes the ability to exude malate. Our results indicate that the combination of Fe 2+ and Al 3+ with exuded malate is crucial for both CW stiffening and root-extension arrest. However, stiffness increase induced by single Fe 2+ or Al 3+ is not sufficient for arresting root growth in our experimental conditions., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo-electron microscopy structure of the helical NP-RNA complex (8.7 to 5.3 Å). The helical RNP-like structure reveals a parallel double-stranded conformation, allowing the visualization of NP-NP and NP-RNA interactions. The RNA, located at the interface of neighboring NP protomers, interacts with conserved residues previously described as essential for the NP-RNA interaction. The NP undergoes conformational changes to enable RNA binding and helix formation. Together, our findings provide relevant insights for understanding the mechanism for influenza genome encapsidation.

Cry11Aa and Cyt1Aa are two pesticidal toxins produced by Bacillus thuringiensis subsp. israelensis. To improve our understanding of the nature of their oligomers in the toxic actions and synergistic effects, we performed the atomic force microscopy to probe the surfaces of their natively grown crystals, and used the L-weight filter to enhance the structural features. By L-weight filtering, molecular sizes of the Cry11Aa and Cyt1Aa monomers obtained are in excellent agreement with the three-dimensional structures determined by x-ray crystallography. Moreover, our results show that the layered feature of a structural element distinguishes the topographic characteristics of Cry11Aa and Cyt1Aa crystals, suggesting that the Cry11Aa toxin has a better chance than Cyt1Aa for multimerization and therefore cooperativeness of the toxic actions., (© 2023 The Authors. Journal of Molecular Recognition published by John Wiley & Sons Ltd.)

Stiffness plays a central action in plant cell extension. Here, we present a protocol to detect changes in stiffness on the external epidermal cell wall of living plant roots using atomic force microscopy (AFM). We provide generalized instructions for collecting force-distance curves and analysis of stiffness using contact-based mechanical model. With this protocol, and some initial training in AFM, a user is able to perform indentation experiments on 4- and 5-day-old Arabidopsis thaliana and determine stiffness properties. For complete details on the use and execution of this protocol, please refer to Godon et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Measuring the structural stiffness aims to reveal the impact of nanostructured components or various physiological circumstances on the elastic response of material to an external indentation. With a pyramidal tip at a nano-scale, we employed the atomic force microscopy (AFM) to indent the surfaces of two compositions of polyacrylamide gels with different softness and seedling roots of Arabidopsis thaliana . We found that the stiffness-depth curve derived from the measured force exhibits a heterogeneous character in elasticity. According to the tendency of stiffness-depth curve, we decomposed the responding force into depth-impact ( F C ), Hookean ( F H ) and tip-shape ( F S ) components, called trimechanic, where F S and its gradient should be offset at the surface or subsurfaces of the indented material. Thereby, trimechnic theory allows us to observe how the three restoring nanomechanics change with varied depth. Their strengths are represented by the respective spring constants ( k C , k H , k S ) of three parallel-connected spring (3PCS) analogs to differentiate restoring nanomechansims of indented materials. The effective Young's modulus Ê and the total stiffness k T (= k H + k S ) globally unambiguously distinguish the softness between the two gel categories. Data fluctuations were observed in the elasticity parameters of individual samples, reflecting nanostructural variations in the gel matrix. Similar tendencies were found in the results from growing plant roots, though the data fluctuations are expectedly much more dramatic. The zone-wise representation of stiffness by the trimechanic-3PCS framework demonstrates a stiffness measure that reflects beneath nanostructures encountered by deepened depth. The trimechanic-3PCS framework can apply any mechanical model of power-law based force-depth relationship and is compatible with thin layer corrections. It provides a new paradigm for analyzing restoring nanomechanics of soft biomaterials in response to indenting forces.

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control., (© 2022. The Author(s).)

Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.5 Å and further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade-unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS-8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature-dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The proton pump transmembrane protein bacteriorhodopsin was successfully incorporated into planar floating lipid bilayers in gel and fluid phases, by applying a detergent-mediated incorporation method. The method was optimized on single supported bilayers by using quartz crystal microbalance, atomic force and fluorescence microscopy techniques. Neutron and X-ray reflectometry were used on both single and floating bilayers with the aim of determining the structure and composition of this membrane-protein system before and after protein reconstitution at sub-nanometer resolution. Lipid bilayer integrity and protein activity were preserved upon the reconstitution process. Reversible structural modifications of the membrane, induced by the bacteriorhodopsin functional activity triggered by visible light, were observed and characterized at the nanoscale., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Deinococcus radiodurans protein HU (DrHU) was shown to be critical for nucleoid activities, yet its functional and structural properties remain largely unexplored. We have applied atomic force microscopy (AFM) imaging to study DrHU binding to pUC19-DNA in vitro and analyzed the topographic structures formed at the nanoscale. At the single-molecule level, AFM imaging allows visualization of super-helical turns on naked DNA surfaces and characterization of free DrHU molecules observed as homodimers. When enhancing the molecular surface structures of AFM images by the Laplacian weight filter, the distribution of bound DrHUs was visibly varied as a function of the DrHU/DNA molar ratio. At a low molar ratio, DrHU binding was found to reduce the volume of condensed DNA configuration by about 50%. We also show that DrHU is capable of bridging distinct DNA segments. Moreover, at a low molar ratio, the binding orientation of individual DrHU dimers could be perceived on partially "open" DNA configuration. At a high molar ratio, DrHU stiffened the DNA molecule and enlarged the spread of the open DNA configuration. Furthermore, a lattice-like pattern could be seen on the surface of DrHU-DNA complex, indicating that DrHU multimerization had occurred leading to the formation of a higher order architecture. Together, our results show that the functional plasticity of DrHU in mediating DNA organization is subject to both the conformational dynamics of DNA molecules and protein abundance.

Bacteria employ several mechanisms, and most notably secretion systems, to translocate effectors from the cytoplasm to the extracellular environment or the cell surface. Pseudomonas aeruginosa widely employs secretion machineries such as the Type III Secretion System to support virulence and cytotoxicity. However, recently identified P. aeruginosa strains that do not express the Type III Secretion System have been shown to express ExlA, an exolysin translocated through a two-partner secretion system, and are the causative agents of severe lung hemorrhage. Sequence predictions of ExlA indicate filamentous hemagglutinin (FHA-2) domains as the prevalent features, followed by a C-terminal domain with no known homologs. In this work, we have addressed the mechanism employed by ExlA to target membrane bilayers by using NMR, small-angle X-ray scattering, atomic force microscopy, and cellular infection techniques. We show that the C-terminal domain of ExlA displays a "molten globule-like" fold that punctures small holes into membranes composed of negatively charged lipids, while other domains could play a lesser role in target recognition. In addition, epithelial cells infected with P. aeruginosa strains expressing different ExlA variants allow localization of the toxin to lipid rafts. ExlA homologs have been identified in numerous bacterial strains, indicating that lipid bilayer destruction is an effective strategy employed by bacteria to establish interactions with multiple hosts., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution. It is thus paramount to develop novel approaches for biochemical and biophysical characterization of these challenging targets. Here, we present a protocol that integrates non-denaturing lncRNA purification with in-solution hydrodynamic analysis and single-particle atomic force microscopy (AFM) imaging to produce highly homogeneous lncRNA preparations and visualize their 3D topology at ~15-Å resolution. Our protocol is suitable for imaging lncRNAs in biologically active conformations and for measuring structural defects of functionally inactive mutants that have been identified by cell-based functional assays. Once optimized for the specific target lncRNA of choice, our protocol leads from cloning to AFM imaging within 3-4 weeks and can be implemented using state-of-the-art biochemical and biophysical instrumentation by trained researchers familiar with RNA handling and supported by AFM and small-angle X-ray scattering (SAXS) experts.

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

Hb Dompierre [β29(B11)Gly→Arg, HBB : c.88G>C] is a rare β-globin gene variant that was previously described in the heterozygous state in a 24-year-old female patient. It is defined in the HbVar database as being clinically and biologically asymptomatic. A few years after the first description, we had an opportunity of reassessing the index case because she presented with splenomegaly and clinical and biological manifestations of hemolysis. After ruling out the most common causes of hemolysis, further analyses on the variant hemoglobin (Hb) using brilliant cresyl blue staining, indicated that it showed mild instability, which may explain the clinical and biological manifestations. A structural bioinformatic analysis on the Hb variant suggested that the amino acid replacement may be deleterious to the integrity of the Hb. This report confirms the importance of completely characterizing all new Hb variants in order to guide the patients' clinical management and follow-up, as well as to provide the probands and their family members with appropriate genetic counseling.

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with two catalytic A subunits and two carrier-protective B subunits in a heterotetramer (FXIII-A 2 B 2 ). FXIII-A 2 and -B 2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B subunits, activated FXIII (FXIIIa) covalently cross links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A 2 B 2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A 2 B 2 complex. We also used molecular dynamics simulations in combination with isothermal titration calorimetry to characterize FXIII-A 2 B 2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A 2 B 2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly or activity to reduce bleeding and thrombotic complications., Competing Interests: Authors declare no conflict of interest.

Long non-coding RNAs (lncRNAs) are key regulatory molecules, but unlike with other RNAs, the direct link between their tertiary structure motifs and their function has proven elusive. Here we report structural and functional studies of human maternally expressed gene 3 (MEG3), a tumor suppressor lncRNA that modulates the p53 response. We found that, in an evolutionary conserved region of MEG3, two distal motifs interact by base complementarity to form alternative, mutually exclusive pseudoknot structures ("kissing loops"). Mutations that disrupt these interactions impair MEG3-dependent p53 stimulation in vivo and disrupt MEG3 folding in vitro. These findings provide mechanistic insights into regulation of the p53 pathway by MEG3 and reveal how conserved motifs of tertiary structure can regulate lncRNA biological function., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death 1,2 ; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo , in vitro , and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro . These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.

Nanoparticles are defined as elementary particles with a size between 1 and 100 nm for at least 50% (in number). They can be made from natural materials, or manufactured. Due to their small sizes, novel toxicological issues are raised and thus determining the accurate size of these nanoparticles is a major challenge. In this study, we performed an intercomparison experiment with the goal to measure sizes of several nanoparticles, in a first step, calibrated beads and monodispersed SiO₂ Ludox®, and, in a second step, nanoparticles (NPs) of toxicological interest, such as Silver NM-300 K and PVP-coated Ag NPs, Titanium dioxide A12, P25(Degussa), and E171(A), using commonly available laboratory techniques such as transmission electron microscopy, scanning electron microscopy, small-angle X-ray scattering, dynamic light scattering, wet scanning transmission electron microscopy (and its dry state, STEM) and atomic force microscopy. With monomodal distributed NPs (polystyrene beads and SiO₂ Ludox®), all tested techniques provide a global size value amplitude within 25% from each other, whereas on multimodal distributed NPs (Ag and TiO₂) the inter-technique variation in size values reaches 300%. Our results highlight several pitfalls of NP size measurements such as operational aspects, which are unexpected consequences in the choice of experimental protocols. It reinforces the idea that averaging the NP size from different biophysical techniques (and experimental protocols) is more robust than focusing on repetitions of a single technique. Besides, when characterizing a heterogeneous NP in size, a size distribution is more informative than a simple average value. This work emphasizes the need for nanotoxicologists (and regulatory agencies) to test a large panel of different techniques before making a choice for the most appropriate technique(s)/protocol(s) to characterize a peculiar NP.

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Environmental cues profoundly modulate cell proliferation and cell elongation to inform and direct plant growth and development. External phosphate (Pi) limitation inhibits primary root growth in many plant species. However, the underlying Pi sensory mechanisms are unknown. Here we genetically uncouple two Pi sensing pathways in the root apex of Arabidopsis thaliana. First, the rapid inhibition of cell elongation in the transition zone is controlled by transcription factor STOP1, by its direct target, ALMT1, encoding a malate channel, and by ferroxidase LPR1, which together mediate Fe and peroxidase-dependent cell wall stiffening. Second, during the subsequent slow inhibition of cell proliferation in the apical meristem, which is mediated by LPR1-dependent, but largely STOP1-ALMT1-independent, Fe and callose accumulate in the stem cell niche, leading to meristem reduction. Our work uncovers STOP1 and ALMT1 as a signalling pathway of low Pi availability and exuded malate as an unexpected apoplastic inhibitor of root cell wall expansion.

A recurrent interrogation when imaging soft biomolecules using atomic force microscopy (AFM) is the putative deformation of molecules leading to a bias in recording true topographical surfaces. Deformation of biomolecules comes from three sources: sample instability, adsorption to the imaging substrate, and crushing under tip pressure. To disentangle these causes, we measured the maximum height of a well-known biomolecule, the tobacco mosaic virus (TMV), under eight different experimental conditions positing that the maximum height value is a specific indicator of sample deformations. Six basic AFM experimental factors were tested: imaging in air (AIR) versus in liquid (LIQ), imaging with flat minerals (MICA) versus flat organic surfaces (self-assembled monolayers, SAM), and imaging forces with oscillating tapping mode (TAP) versus PeakForce tapping (PFT). The results show that the most critical parameter in accurately measuring the height of TMV in air is the substrate. In a liquid environment, regardless of the substrate, the most critical parameter is the imaging mode. Most importantly, the expected TMV height values were obtained with both imaging with the PeakForce tapping mode either in liquid or in air at the condition of using self-assembled monolayers as substrate. This study unambiguously explains previous poor results of imaging biomolecules on mica in air and suggests alternative methodologies for depositing soft biomolecules on well organized self-assembled monolayers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage., Results: By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (β-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius., Conclusions: These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.

Purpose: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here., Methods: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline., Results: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 μg/mL), (super)FVa did not (6.7 μg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 μg/mL) or saline (5.6 μg/mL)., Conclusion: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.

Image visibility is a central issue in analyzing all kinds of microscopic images. An increase of intensity contrast helps to raise the image visibility, thereby to reveal fine image features. Accordingly, a proper evaluation of results with current imaging parameters can be used for feedback on future imaging experiments. In this work, we have applied the Laplacian function of image intensity as either an additive component (Laplacian mask) or a multiplying factor (Laplacian weight) for enhancing image contrast of high-resolution AFM images of two molecular systems, an unknown protein imaged in air, provided by AFM COST Action TD1002 (http://www.afm4nanomedbio.eu/), and tobacco mosaic virus (TMV) particles imaged in liquid. Based on both visual inspection and quantitative representation of contrast measurements, we found that the Laplacian weight is more effective than the Laplacian mask for the unknown protein, whereas for the TMV system the strengthened Laplacian mask is superior to the Laplacian weight. The present results indicate that a mathematical function, as exemplified by the Laplacian function, may yield varied processing effects with different operations. To interpret the diversity of molecular structure and topology in images, an explicit expression for processing procedures should be included in scientific reports alongside instrumental setups., (Copyright © 2015 John Wiley & Sons, Ltd.)

Protein conformational variability (or dynamics) for large macromolecules and its implication for their biological function attracts more and more attention. Collective motions of domains increase the ability of a protein to bind to partner molecules. Using atomic force microscopy (AFM) topographic images, it is possible to take snapshots of large multi-component macromolecules at the single molecule level and to reconstruct complete molecular conformations. Here, we report the application of a reconstruction protocol, named AFM-assembly, to characterise the conformational variability of the two C domains of human coagulation factor Va (FVa). Using AFM topographic surfaces obtained in liquid environment, it is shown that the angle between C1 and C2 domains of FVa can vary between 40° and 166°. Such dynamical variation in C1 and C2 domain arrangement may have important implications regarding the binding of FVa to phospholipid membranes.

Tobacco mosaic virus particles can be rapidly assembled into 3D-domains by capillary flow-driven alignment at the triple contact-line of an evaporating droplet. Virus particles of ∼150 Å diameter can be resolved within individual domains at the outer rim of the "coffee-ring" type residue by atomic force microscopy. The crystalline domains can also be probed by X-ray microdiffraction techniques. Both techniques reveal that the rod-like virus particles are oriented parallel to the rim. We further demonstrate the feasibility of collection of hk0 reflection intensities in GISAXS geometry and show it allows calculating a low-resolution electron density projection along the rod axis.

The sodium/iodide symporter (NIS or SLC5A5) is an intrinsic membrane protein implicated in iodide uptake into thyroid follicular cells. It plays a crucial role in iodine metabolism and thyroid regulation and its function is widely exploited in the diagnosis and treatment of benign and malignant thyroid diseases. A great effort is currently being made to develop a NIS-based gene therapy also allowing the radiotreatment of nonthyroidal tumors. NIS is also expressed in other tissues, such as salivary gland, stomach and mammary gland during lactation, where its physiological role remains unclear. The molecular identity of the thyroid iodide transporter was elucidated approximately fifteen years ago. It belongs to the superfamily of sodium/solute symporters, SSS (and to the human transporter family, SLC5), and is composed of 13 transmembrane helices and 643 amino acid residues in humans. Knowledge concerning NIS structure/function relationship has been obtained by taking advantage of the high resolution structure of one member of the SSS family, the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT), and from studies of gene mutations leading to congenital iodine transport defects (ITD). This review will summarize current knowledge regarding the molecular characterization of NIS., (© 2013.)

Unlabelled: Proteins can adopt a variety of conformations. We present a simple server for scoring the agreement between 3D atomic structures and experimental envelopes obtained by atomic force microscopy. Three different structures of immunoglobulins (IgG) or blood coagulation factor V activated were tested and their agreement with several topographical surfaces was computed. This approach can be used to test structural variability within a family of proteins., Availability and Implementation: DockAFM is available at http://biodev.cea.fr/dockafm.

In this work, we propose "single-image analysis", as opposed to multi-image averaging, for extracting valuable information from AFM images of single bio-particles. This approach allows us to study molecular systems imaged by AFM under general circumstances without restrictions on their structural forms. As feature exhibition is a resolution correlation, we have performed AFM imaging on surfaces of tobacco mosaic virus (TMV) to demonstrate variations of structural patterns with probing resolution. Two AFM images were acquired with the same tip at different probing resolutions in terms of pixel width, i.e., 1.95 and 0.49 nm per pixel. For assessment, we have constructed an in silico topograph based on the three-dimensional crystal structure of TMV as a reference. The prominent artifacts observed in the AFM-determined shape of TMV were attributed to tip convolutions. The width of TMV rod was systematically overestimated by ~10 nm at both probing resolutions of AFM. Nevertheless, the effects of tip convolution were less severe in vertical orientation so that the estimated height of TMV by AFM imaging was in close agreement with the in silico X-ray topograph. Using dedicated image processing algorithms, we found that at low resolution (i.e., 1.95 nm per pixel), the extracted surface features of TMV can be interpreted as a partial or full helical repeat (three complete turns with ~7.0 nm in length), while individual protein subunits (~2.5 nm) were perceivable only at high resolution. The present study shows that the scales of revealed structural features in AFM images are subject to both probing resolution and processing algorithms for image analysis.

Piezoelectric quartz tuning fork has drawn the attention of many researchers for the development of new atomic force microscopy (AFM) self-sensing probes. However, only few works have been done for soft biological materials imaging in air or aqueous conditions. The aim of this work was to demonstrate the efficiency of the AFM tuning fork probe to perform high-resolution imaging of proteins and to study the specific interaction between a ligand and its receptor in aqueous media. Thus, a new kind of self-sensing AFM sensor was introduced to realize imaging and biochemical specific recognition spectroscopy of glucose oxidase enzyme using a new chemical functionalization procedure of the metallic tips based on the electrochemical reduction of diazonium salt. This scanning probe as well as the functionalization strategy proved to be efficient respectively for the topography and force spectroscopy of soft biological materials in buffer conditions., (Copyright © 2013 John Wiley & Sons, Ltd.)

Inherited factor VII (FVII) deficiency is one of the commonest rare bleeding disorders. It is characterized by a wide molecular and clinical heterogeneity and an autosomal recessive pattern of inheritance. Factor VII-deficient patients are still scarcely explored in Pakistan although rare bleeding disorders became quite common as a result of traditional consanguineous marriages. The aim of the study was to give a first insight of F7 gene mutations in Pakistani population. Ten unrelated FVII-deficient patients living in Pakistan were investigated (median FVII:C = 2%; range = 2-37%). A clinical questionnaire was filled out for each patient and direct sequencing was performed on the coding regions, intron/exon boundaries and 5' and 3' untranslated regions of the F7 gene. Nine different mutations (eight missense mutations and one located within the F7 promoter) were identified on the F7 gene. Five of them were novel (p.Cys82Tyr, p.Cys322Ser, p.Leu357Phe, p.Thr410Ala, c-57C>T, the last being predicted to alter the binding site of transcription factor HNF-4). Half of the patients had single mutations in Cys residues involved in disulfide bridges. The p.Cys82Arg mutation was the most frequent in our series. Six of seven patients with FVII:C levels below 10% were homozygous in connection with the high percentage of consanguinity in our series. In addition, we graded the 10 patients according to three previously published classifications for rare bleeding disorders. The use of the bleeding score proposed by Tosetto and co-workers in 2006 appears to well qualify the bleeding tendency in our series., (© 2013 John Wiley & Sons Ltd.)

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen-binding fragments and one crystallizable fragment using a surface-constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X-ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X-ray structure consequence of the antibody flexibility. We conclude that high-resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica., (Copyright © 2013 John Wiley & Sons, Ltd.)

We applied the signed distance function (SDF) for representing the depths of atoms in a macromolecule. The calculations of SDF values were performed on grid points in a rectangular box that accommodates the macromolecule. The depth for an atom inside the molecule was then obtained as a result of tri-linear interpolation of SDF values at the nearest grid points surrounding the atom. For testing the performance of present program Adepth, we have constructed an artificial molecule whose atomic depths are known as the gold standard for accuracy assessments. On average, our results showed that Adepth reached an accuracy of 1.6% at 0.5 Å of grid spacing, whereas the current reference server DEPTH reached 7.5%. The Adepth program provides both depth and height representations; it is capable of computing iso-surfaces for atomic depths and presenting graphical view of macromolecular shape at some distance away from the surface. Web interface is available at http://biodev.cea.fr/adepth.

The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

GTPases are molecular switches that regulate a wide-range of cellular processes. The GPN-loop GTPase (GPN) is a sub-family of P-loop NTPase that evolved from a single gene copy in archaea to triplicate paralog genes in eukaryotes, each having a non-redundant essential function in cell. In Saccharomyces cerevisiae, yGPN1 and yGPN2 are involved in sister chromatid cohesion mechanism, whereas nothing is known regarding yGPN3 function. Previous high-throughput experiments suggested that GPN paralogs interaction may occur. In this work, GPN|GPN contact was analyzed in details using TAP-Tag approach, yeast two-hybrid assay, in silico energy computation and site-directed mutagenesis of a conserved Glu residue located at the center of the interaction interface. It is demonstrated that this residue is essential for cell viability. A chromatid cohesion assay revealed that, like yGPN1 and yGPN2, yGPN3 also plays a role in sister chromatid cohesion. These results suggest that all three GPN proteins act at the molecular level in sister chromatid cohesion mechanism as a GPN|GPN complex reminiscent of the homodimeric structure of PAB0955, an archaeal member of GPN-loop GTPase.

Over 30 thromboembolic events have been reported in factor VII (FVII) deficiency either associated with previously asymptomatic forms or bleeding diathesis. Whether this coexistence is fortuitous or not is still a mater of debate. Nevertheless, it is well admitted that (i) thrombotic events occurring in FVII-deficient patients with any apparent triggering factors are very rare, (ii) surgical procedures, replacement therapy (especially containing activated factors) but also the presence of an antiphospholipid syndrome are frequently associated with these particular thrombotic events, (iii) in the same way, R304Q and A294V FVII variants appear to be more prevalent than other FVII equally frequent mutations and finally (iv) low FVII coagulant activity levels do not protect against thrombosis. Therefore, peri-operative thrombotic prophylaxis should be relevant for these particular FVII-deficient patients. However, safety, treatment modalities and specific indications of such an antithrombotic prophylaxis remain to be established., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Coagulation factor VIII (FVIII) is a multidomain glycoprotein in which the FVIII A2 domain is a key structural element. We aimed at identifying residues within FVIII A2 domain that are crucial for the maintenance of the cofactor function. A high number (n=206) of mutants were generated by substituting original residues with alanine. The mutants were expressed in COS-1 cells and their antigen levels and procoagulant activities were measured. The residues were classified in three categories: those with a non-detrimental alteration of their activities (activity >50 % of control FVIII; n=98), those with a moderate alteration (15 %

Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The factor VIII (FVIII) is a cofactor of the coagulation cascade. The FVIII C2 domain is a critical domain that participates in the interactions with the von Willebrand factor and the phospholipidic surfaces. To assess the importance of each residue of this domain in the maintenance of the structure and the function of FVIII, a number (n=139) of mutants were generated by substituting the original residues, from Ser2173 to Gly2325, by an alanine. Mutants were built within a complete B domain-deleted FVIII and expressed in COS-1 cells. Mutant antigen levels and procoagulant activities were measured. Two in silico analyses, a sliding average procedure and an analysis of the mutation energy cost were conducted in parallel on the FVIII structure. Both results were in agreement with the functional data, and illustrated the benefit of using such strategies prior to targeting specific residues in the aim of generating active recombinant molecules. The functional assays identify the residues that are important to maintaining the structure of the C2 domain, mainly those forming β-sheet, and those that can afford substitution, establishing a detailed functional relation with the available crystallographic data. This study provided a comprehensive functional mapping of the FVIII C2 domain and discussed the implication of specific residues in respect to the maintenance in the activity and structure stability, the efficiency in secretion, the binding to phospholipids and the formation of epitope.