Reactive mixing enables enzymatic depolymerization of recalcitrant or unsortable polyester wastes.

Enzyme-catalyzed depolymerization allows efficient recycling of poly(ethylene terephthalate) (PET) bottles, which are easy to sort and made of slowly crystallizing PET. However, because crystalline phases are recalcitrant to enzymatic hydrolysis, this technology fails for rapidly crystallizing polyester wastes such as poly(butylene terephthalate) (PBT), unsortable mixed polyesters, or heterogeneous formulated PET waste streams. We show that melt transesterification and vitrimerization of mixtures of rapidly crystallizing polyester wastes, leveraging catalysts already present, produce copolyesters that crystallize slowly and are readily depolymerized.

Optimised nanobody-based quenchbodies for enhanced protein detection.

Quenchbodies, antibodies labelled with fluorophores that increase in intensity upon antigen binding, offer great promise for biosensor development. Nanobody-based quenchbodies are particularly attractive due to their small size, ease of expression, high stability, rapid evolvability, and amenability to protein engineering. However, existing designs for protein detection show limited dynamic range, with fluorescence increases of only 1.

Degradation of Reporter Molecules Imposes a Fundamental Limit of Detection on CRISPR Diagnostics.

The sensitivity of CRISPR-Cas systems used for molecular diagnostics remains a major bottleneck in the adoption of this technology. The vast majority of CRISPR-based assays use dually labeled, single-stranded reporters and fluorescence signal readouts to infer enzymatic activity and the presence (or absence) of target nucleic acid. The limit of detection of such assays is set by the kinetics of the Cas enzymes and a slow yet measurable increase in the fluorescence signal.

Global regulators enable bacterial adaptation to a phenotypic trade-off.

Cellular fitness depends on multiple phenotypes that must be balanced during evolutionary adaptation. For instance, coordinating growth and motility is critical for microbial colonization and cancer invasiveness. In bacteria, these phenotypes are controlled by local regulators that target single operons, as well as by global regulators that impact hundreds of genes.

Small-molecule autocatalysis drives compartment growth, competition and reproduction.

Sustained autocatalysis coupled to compartment growth and division is a key step in the origin of life, but an experimental demonstration of this phenomenon in an artificial system has previously proven elusive. We show that autocatalytic reactions within compartments-when autocatalysis, and reactant and solvent exchange outpace product exchange-drive osmosis and diffusion, resulting in compartment growth. We demonstrate, using the formose reaction compartmentalized in aqueous droplets in an emulsion, that compartment volume can more than double.

Monocyte Trajectories Endotypes Are Associated With Worsening in Septic Patients.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The immune system plays a key role in sepsis onset and remains dysregulated over time in a heterogeneous manner. Here, we decipher the heterogeneity of the first week evolution of the monocyte HLA-DR (mHLA-DR) surface protein expression in septic patients, a key molecule for adaptive immunity onset.

Herpes DNAemia and TTV Viraemia in Intensive Care Unit Critically Ill Patients: A Single-Centre Prospective Longitudinal Study.

Introduction: We analysed blood DNAemia of TTV and four herpesviruses (CMV, EBV, HHV6, and HSV-1) in the REAnimation Low Immune Status Marker (REALISM) cohort of critically ill patients who had presented with either sepsis, burns, severe trauma, or major surgery. The aim was to identify common features related to virus and injury-associated pathologies and specific features linking one or several viruses to a particular pathological context.

Methods: Overall and individual viral DNAemia were measured over a month using quantitative PCR assays from the 377 patients in the REALISM cohort.

The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands.

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands.

Immune Profiling Demonstrates a Common Immune Signature of Delayed Acquired Immunodeficiency in Patients With Various Etiologies of Severe Injury.

Objectives: The host response plays a central role in the pathophysiology of sepsis and severe injuries. So far, no study has comprehensively described the overtime changes of the injury-induced immune profile in a large cohort of critically ill patients with different etiologies.

Design: Prospective observational cohort study.

RNA diversification by a self-reproducing ribozyme revealed by deep sequencing and kinetic modelling.

We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by a kinetic model. This shows that key mechanisms can be integrated and controlled toward Darwinian evolution in RNA reaction networks.

Darwinian properties and their trade-offs in autocatalytic RNA reaction networks.

Discovering autocatalytic chemistries that can evolve is a major goal in systems chemistry and a critical step towards understanding the origin of life. Autocatalytic networks have been discovered in various chemistries, but we lack a general understanding of how network topology controls the Darwinian properties of variation, differential reproduction, and heredity, which are mediated by the chemical composition. Using barcoded sequencing and droplet microfluidics, we establish a landscape of thousands of networks of RNAs that catalyze their own formation from fragments, and derive relationships between network topology and chemical composition.

Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassay.

Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the anti-bacterial antibody repertoire, we developed an in-droplet bioassay with single-antibody resolution. The assay not only allowed us to identify whether the secreted antibodies recognized a bacterial surface antigen, but also to estimate the apparent dissociation constant (K) of the interaction and the density of the recognized epitope on the bacteria.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.

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 Impact of Anticoagulation on Trauma Outcomes : An National Trauma Data Bank Study.

Background: Increased prevalence of patients on anticoagulants and the advent of new therapies raise concern over how these patients fare if they sustain a traumatic injury. We investigated the role of prehospitalization anticoagulation therapy in trauma-related mortality and postacute disposition.

Methods: A retrospective analysis was performed on patients who sustained traumatic injury identified in the 2017 National Trauma Data Bank (NTDB).

The Quantitative Assessment of the Secreted IgG Repertoire after Recall to Evaluate the Quality of Immunizations.

One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme.

Author Correction: High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic cost of rapid adaptation of single yeast cells.

Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated.

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes. 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 bioassays, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription.

High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer.

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution.

Coupled catabolism and anabolism in autocatalytic RNA sets.

The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway.

Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.

Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.