We report that user-defined DNA nanostructures, such as two-dimensional (2D) origamis and nanogrids, undergo a rapid higher-order folding transition, referred to as supra-folding, into three-dimensional (3D) compact structures (origamis) or well-defined μm-long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer-by-layer deposition of polyelectrolytes. Once supra-folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA-polyelectrolyte complexation. Orthogonal to DNA base-pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site-specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami-tethered proteins.

Oxazepam, one of the most frequently prescribed anxiolytic drugs, is not completely removed from wastewater with conventional treatment processes. It can thus be found at trace levels in environmental water, with human urine constituting the major source of contamination. This study focused on the development and characterization of molecularly imprinted polymers (MIPs) for the selective solid-phase extraction of oxazepam at trace levels from environmental water and human urine samples. Two MIPs were synthesized, and their selectivity in pure organic and aqueous media were assayed. After optimizing the extraction procedure adapted to a large sample volume to reach a high enrichment factor, the most promising MIP was applied to the selective extraction of oxazepam from environmental water. Extraction recoveries of 83 ± 12, 92 ± 4 and 89 ± 10% were obtained using the MIP for tap, mineral and river water, respectively, while a recovery close to 40% was obtained on the corresponding non-imprinted polymer (NIP). Thanks to the high enrichment factors, a limit of quantification (LOQ) of 4.5 ng L-1 was obtained for river water. A selective extraction procedure was also developed for urine samples and gave rise to extraction recoveries close to 95% for the MIP and only 23% for the NIP. Using the MIP, a LOQ of 357 ng L-1 was obtained for oxazepam in urine. The use of the MIP also helped to limit the matrix effects encountered for the quantification of oxazepam in environmental samples and in human urine samples after extraction on an Oasis HLB sorbent.

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGβ glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGβ glycoforms, which allowed for the first time the detection of 33 hCGβ glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

Sulfur mustard is a highly reactive chemical warfare agent that causes severe damages to the victims exposed by alkylating multiple biomolecules such as proteins. Resulting alkylated products can be used as biomarkers of exposure to this chemical agent. A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was thus developed to detect alkylated peptides after the tryptic digestion of hemoglobin (50 mg.mL−1) incubated with sulfur mustard at different concentrations (0.25, 0.5, 1, 10 and 100 µg.mL−1). Five new alkylation sites were accurately identified on the protein (α-His72, α-His87, α-His89, β-His2 and β-Val98) and fifteen adducted peptides were detected, among which eight of them resulted from the alkylation of four peptides, each presenting two potential sites of adduction that could be discriminated by the method specificity. Similarly, it was possible to discriminate the three potential adduction sites of the peptide α-T9. Moreover, the method allowed the quantification of all the alkylated peptides with a satisfying repeatability, with RSD ranging from 0.5 to 9.3% for an exposure of hemoglobin to sulfur mustard at 100 µg.mL−1. The analysis of hemoglobin incubated with different concentrations of sulfur mustard levels led to a linear response for all the alkylated peptides with the studied concentrations (0.25, 0.5, 1, 10 and 100 µg.mL−1). A variation of the alkylation rate was also observed between the different peptides studied, with a preferential adduction of sulfur mustard on the histidine residues but also on the N-terminal valine residues of both globin chains and on the Val98 residue of globin β. Furthermore, the presented method proved to be sensitive, with a theoretical possibility to detect alkylated peptides resulting from in vitro incubation of hemoglobin in deionized water with sulfur mustard at 2.63 ng.mL−1. After further development, this method could potentially be used for the analysis of blood samples in vivo exposed to sulfur mustard.

The outbreak of Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has significantly affected the dental care sector. Dental professionals are at high risk of being infected, and therefore transmitting SARS-CoV-2, due to the nature of their profession, with close proximity to the patient’s oropharyngeal and nasal regions and the use of aerosol-generating procedures. The aim of this article is to provide an update on different issues regarding SARS-CoV-2 and COVID-19 that may be relevant for dentists. Members of the French National College of Oral Biology Lecturers (“Collège National des EnseignantS en Biologie Orale”; CNESBO-COVID19 Task Force) answered seventy-two questions related to various topics, including epidemiology, virology, immunology, diagnosis and testing, SARS-CoV-2 transmission and oral cavity, COVID-19 clinical presentation, current treatment options, vaccine strategies, as well as infection prevention and control in dental practice. The questions were selected based on their relevance for dental practitioners. Authors independently extracted and gathered scientific data related to COVID-19, SARS-CoV-2 and the specific topics using scientific databases. With this review, the dental practitioners will have a general overview of the COVID-19 pandemic and its impact on their practice. View Full-Text

Molecularly imprinted polymers are highly selective and cost-effective materials, which have attracted significant interest in various areas such as sample pretreatment and chromatographic and electrophoretic separations. This review aims to present the state of the art concerning the miniaturization of these materials in order to meet the societal demand for reliable, fast, cheap, and solvent/sample saving analyses. The polymerization route specificities for the production of miniaturized molecularly imprinted polymers in capillaries or chip channels, such as open tubular, packed particles, magnetic nanoparticles, and in situ imprinted monoliths, are investigated. Their performances as selective supports in solid phase extraction and as stationary phases in electrochromatography and liquid chromatography, as well as their possible perspectives are discussed.

The use of protein templates for the controlled synthesis of inorganic nanostructures has gained considerable attention in multidisciplinary fields, including electronics, optics, energy, sensing, and biomedicine, owing to their biocompatibility and structural programmability. The possible synergistic combination of protein scaffolds (and other biomolecules/biopolymers) with metal nanoclusters (MNCs) has created a new class of highly photoluminescent nanoprobes and nanodevices. For the first time, we will discuss the different types of protein templates used for MNC preparation with an emphasis on their optoelectronic properties for application. In particular, applications of protein-coated MNCs for chemosensing or biosensing of cancer biomarkers, neurotransmitters, pathogenic microorganisms, biomolecules, pharmaceutical compounds, and immunoassays are discussed in detail herein. Fluorescence-based and multimodal molecular imaging, both in vitro and in vivo based on functional proteins are also covered. Furthermore, we discuss the burgeoning growth of protein-coated MNCs (e.g., gold (Au) and silver (Ag) NCs) to develop synergistic nanotherapeutics with potential biomedical applications in chemotherapy, radiotherapy, photodynamic therapy (PDT), photothermal therapy (PTT), and antibacterial activity, as well as MNC-containing nanocomposites for enhanced bioimaging and controlled drug release. Overall, the proposed review highlights the recent progress, technical challenges and new horizons in this field, and summarizes our understanding of how MNC properties interact with the biological function of protein scaffolds to develop synergistic nanotherapeutics towards clinical translation.

Chemotherapeutic drugs cause harmful side effects in cancer patients due to their low specificity, calling for the development of more effective strategies for their dosage and administration. In this work, a smart drug nanocarrier was synthesized through the covalent functionalization of superparamagnetic iron oxide nanoparticles with a triblock copolymer, which includes a dual alkoxysilane array, ((3-aminopropyl)triethoxysilane and (trimethoxysilyl)propyl methacrylate), and the pH-responsive poly(4-vinylpyridine). The synthetic conditions were optimized through structural and physicochemical characterization after every functionalization step. Afterward, the systematic loading, capture, and release of the anticancer drug doxorubicin (Dox) were demonstrated at relevant pH values using a specially designed square wave voltammetry technique. This strategy revealed that the P4VP polymeric chains underwent reversible hydrophobic to hydrophilic transitions in acidic media, triggering a molecular distention driven by the induced intermolecular electro-repulsive forces. Thereafter, the Dox solution can easily penetrate the polymeric layer at pH values below 5.62 (the pKa of poly(4-vinylpyridine)), allowing a loading of 61.9 ± 5.4 mg g−1 in the nanocomplex. After deprotonation in a pH 7.4 buffer solution, the polymer chains underwent intermolecular interactions again, capturing the drug molecules. Subsequently, 93.5 ± 3.5% of the payload was released upon suspension of the nanocomplex in pH 4.0 media, which is significantly more acidic than healthy tissues. Since the magnetic properties of the MNPs were practically unaffected by the surface modification, this nanocomplex offers a versatile strategy for the pH-selective and magnetically-guided release of drugs.

We report here an impedimetric genosensor for the quantification of microRNA-21 using [Fe(CN)6]3−/4− as redox probe to transduce the hybridization event. The biosensing platform was built at a thiolated-gold electrode by covalent bond of reduced graphene oxide (RGO) modified with chitosan (CHIT) and further covalent attachment of the aminated DNA probe. GO was used to provide the carboxylic groups for the covalent attachment of CHIT and, once reduced, to improve the electroactivity of the resulting platform, while CHIT served as a bridge between the thiol and the aminated probe DNA. The proposed bioanalytical platform allows the label-free, non-amplified, simple and fast biosensing of microRNA-21, with a linear range between 1.0 × 10−12 M and 1.0 × 10−8 M, a sensitivity of (134 ± 4) ΩM−1 (r2 = 0.996), a detection limit of 300 fM, and a reproducibility of 5.9% for 1.0 × 10−12 M miRNA-21 and 2.2% for 1.0 × 10−9 M miRNA-21. The genosensor was successfully used for the quantification of microRNA-21 in enriched human blood serum, urine and saliva samples.

AISI 430 stainless steel is an attractive material to be used in the healthcare industry, particularly as a sensor due to its low cost, corrosion resistance, as well as being Ni-free. AISI 430 was evaluated in an artificial sweat solution with the presence of Escherichia coli, and Staphylococcus aureus. Surface microbial analyses did not reveal colonization of bacteria on metallic surfaces, even when bacteria adhesion was investigated in a Müeller-Hinton solution. However, by electrochemical techniques, the AISI 430 surfaces demonstrated clear signs of corrosion mainly in a sterile medium after two weeks of exposure.

Phase separation of nucleic acids and proteins is a ubiquitous phenomenon regulating subcellular compartment structure and function. While complex coacervation of flexible single-stranded nucleic acids is broadly investigated, coacervation of double-stranded DNA (dsDNA) is less studied because of its propensity to generate solid precipitates. Here, we reverse this perspective by showing that short dsDNA and poly-l-lysine coacervates can escape precipitation while displaying a surprisingly complex phase diagram, including the full set of liquid crystal (LC) mesophases observed to date in bulk dsDNA. Short dsDNA supramolecular aggregation and packing in the dense coacervate phase are the main parameters regulating the global LC-coacervate phase behavior. LC-coacervate structure was characterized upon variations in temperature and monovalent salt, DNA, and peptide concentrations, which allow continuous reversible transitions between all accessible phases. A deeper understanding of LC-coacervates can gain insights to decipher structures and phase transition mechanisms within biomolecular condensates, to design stimuli-responsive multiphase synthetic compartments with different degrees of order and to exploit self-assembly driven cooperative prebiotic evolution of nucleic acids and peptides.

Concentrated solutions of blunt-ended DNA oligomer duplexes self-assemble in living polymers and order into lyotropic nematic liquid crystal phase. Using the optical torque provided by three distinct illumination geometries, we induce independent splay, twist, and bend deformations of the DNA nematic and measure the corresponding elastic coefficients K1, K2, and K3, and viscosities ηsplay, ηtwist, and ηbend. We find the viscoelasticity of the system to be remarkably soft, as the viscoelastic coefficients are smaller than in other lyotropic liquid crystals. We find K1 > K3 > K2, in agreement with the elasticity of the nematic phase of flexible polymers, and ηbend > ηsplay > ηtwist a behavior that is nonconventional in the context of chromonic, polymeric, and thermotropic liquid crystals, indicating a possible role of the weakness and reversibility of the DNA aggregates.

The onset of liquid crystal (LC) phases in concentrated aqueous solutions of DNA oligomers crucially depends on the end-to-end interaction between the DNA duplexes, which can be provided by the aromatic stacking of the terminal base-pairs or by the pairing of complementary dangling-ends. Here we investigated the LC behavior of three blunt-end 12-base-long DNA duplexes synthesized with hydroxyl, phosphate and triphosphate 5’-termini. We experimentally characterized the concentration-temperature phase diagrams and we quantitatively estimated the end-to-end stacking free energy, by comparing the empirical data with the predictions of coarse-grained linear aggregation models. The preservation of LC ordering, even in presence of the bulky and highly charged triphosphate group, indicates that attractive stacking interactions are still present and capable of induce linear aggregation of the DNA duplexes. This finding strengthens the potential role of chromonic like self-assembly for the prebiotic formation of linear polymeric nucleic acids.

Liquid–liquid phase separation (LLPS) phenomena are ubiquitous in biological systems, as various cellular LLPS structures control important biological processes. Due to their ease of in vitro assembly into membraneless compartments and their presence within modern cells, LLPS systems have been postulated to be one potential form that the first cells on Earth took on. Recently, liquid crystal (LC)-coacervate droplets assembled from aqueous solutions of short double-stranded DNA (s-dsDNA) and poly-L-lysine (PLL) have been reported. Such LC-coacervates conjugate the advantages of an associative LLPS with the relevant long-range ordering and fluidity properties typical of LC, which reflect and propagate the physico-chemical properties of their molecular constituents. Here, we investigate the structure, assembly, and function of DNA LC-coacervates in the context of prebiotic molecular evolution and the emergence of functional protocells on early Earth. We observe through polarization microscopy that LC-coacervate systems can be dynamically assembled and disassembled based on prebiotically available environmental factors including temperature, salinity, and dehydration/rehydration cycles. Based on these observations, we discuss how LC-coacervates can in principle provide selective pressures effecting and sustaining chemical evolution within partially ordered compartments. Finally, we speculate about the potential for LC-coacervates to perform various biologically relevant properties, such as segregation and concentration of biomolecules, catalysis, and scaffolding, potentially providing additional structural complexity, such as linearization of nucleic acids and peptides within the LC ordered matrix, that could have promoted more efficient polymerization. While there are still a number of remaining open questions regarding coacervates, as protocell models, including how modern biologies acquired such membraneless organelles, further elucidation of the structure and function of different LLPS systems in the context of origins of life and prebiotic chemistry could provide new insights for understanding new pathways of molecular evolution possibly leading to the emergence of the first cells on Earth

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

Can prelife proceed without cell division? A recently proposed mechanism suggests that transient compartmentalization could have preceded cell division in prebiotic scenarios. Here, we study transient compartmentalization dynamics in the presence of mutations and noise in replication, as both can be detrimental the survival of compartments. Our study comprises situations where compartments contain uncoupled autocatalytic reactions feeding on a common resource, and systems based on RNA molecules copied by replicases, following a recent experimental study. Using the theory of branching processes, we show analytically that two regimes are possible. In the diffusion-limited regime, replication is asynchronous which leads to a large variability in the composition of compartments. In contrast, in a replication-limited regime, the growth is synchronous and thus the compositional variability is low. Typically, simple autocatalysts are in the former regime, while polymeric replicators can access the latter. For deterministic growth dynamics, we introduce mutations that turn functional replicators into parasites. We derive the phase boundary separating coexistence or parasite dominance as a function of relative growth, inoculation size and mutation rate. We show that transient compartmentalization allows coexistence beyond the classical error threshold, above which the parasite dominates. Our findings invite to revisit major prebiotic transitions, notably the transitions towards cooperation, complex polymers and cell division.

The limits of evolution have long fascinated biologists. However, the causes of evolutionary constraint have remained elusive due to a poor mechanistic understanding of studied phenotypes. Recently, a range of innovative approaches have leveraged mechanistic information on regulatory networks and cellular biology. These methods combine systems biology models with population and single-cell quantification and with new genetic tools, and they have been applied to a range of complex cellular functions and engineered networks. In this article, we review these developments, which are revealing the mechanistic causes of epistasis at different levels of biological organization-in molecular recognition, within a single regulatory network, and between different networks-providing first indications of predictable features of evolutionary constraint.

We establish, using single-cell analysis of metabolism and division in a droplet microfluidic system, that yeast can adapt, resuming division, extremely rapidly to an unforeseen environmental challenge, and that adaptation is an active process, requiring the consumption of a characteristic amount energy. The adapted state is stable over at least several days, showing that this is a genuine adaptation process. The adaptation rate (10−3 cells per hour) is orders of magnitude higher than expected based on known mutation rates, suggesting an epigenetic origin, and the tight energetic coupling implies that there is active exploration of different states, and fixation of the solution(s) that allow adaptation.

Our ability to predict the impact of mutations on traits relevant for disease and evolution remains severely limited by the dependence of their effects on the genetic background and environment. Even when molecular interactions between genes are known, it is unclear how these translate to organism-level interactions between alleles. We therefore characterized the interplay of genetic and environmental dependencies in determining fitness by quantifying ~4000 fitness interactions between expression variants of two metabolic genes, starting from various environmentally modulated expression levels. We detect a remarkable variety of interactions dependent on initial expression levels and demonstrate that they can be quantitatively explained by a mechanistic model accounting for catabolic flux, metabolite toxicity, and expression costs. Complex fitness interactions between mutations can therefore be predicted simply from their simultaneous impact on a few connected molecular phenotypes.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.