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.

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.

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. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.

mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.

mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.

Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.

To divide in a tissue, both normal and cancer cells become spherical and mechanically stiffen as they enter mitosis. We investigated the effect of oncogene activation on this process in normal epithelial cells. We found that short-term induction of oncogenic RasV12 activates downstream mitogen-activated protein kinase (MEK-ERK) signaling to alter cell mechanics and enhance mitotic rounding, so that RasV12-expressing cells are softer in interphase but stiffen more upon entry into mitosis. These RasV12-dependent changes allow cells to round up and divide faithfully when confined underneath a stiff hydrogel, conditions in which normal cells and cells with reduced levels of Ras-ERK signaling suffer multiple spindle assembly and chromosome segregation errors. Thus, by promoting cell rounding and stiffening in mitosis, oncogenic RasV12 enables cells to proliferate under conditions of mechanical confinement like those experienced by cells in crowded tumors.

The human body is a crowded place. This crowding is even more acute when the regulation of cell growth and proliferation fails during the formation of a tumor. Dealing with the lack of space in crowded environments presents cells with a challenge. This is especially true for immune cells, whose task is to patrol tissues, causing them to experience both acute and sustained deformation as they move. Although changes in tissue crowding and associated cell shape alterations have been known by pathologists to be key diagnostic traits of late-stage tumors since the 19th century, the impact of these changes on the biology of cancer and immune cells remains unclear. Moreover, it is not known whether cells can detect and adaptively respond to deformations in densely packed spaces.