METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes.

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

Simultaneous super-linear excitation-emission and emission depletion allows imaging of upconversion nanoparticles with higher sub-diffraction resolution.

Upconversion nanoparticles (UCNPs) are becoming increasingly popular as biological markers as they offer photo-stable imaging in the near-infrared (NIR) biological transparency window. Imaging at NIR wavelengths benefits from low auto-fluorescence background and minimal photo-damage. However, as the diffraction limit increases with the wavelength, the imaging resolution deteriorates. To address this limitation, recently two independent approaches have been proposed for imaging UCNPs with sub-diffraction resolution, namely stimulated emission-depletion (STED) microscopy and super linear excitation-emission (uSEE) microscopy. Both methods are very sensitive to the UCNP composition and the imaging conditions, i.e. to the excitation and depletion power. Here, we demonstrate that the imaging conditions can be chosen in a way that activates both super-resolution regimes simultaneously when imaging NaYF4:Yb,Tm UCNPs. The combined uSEE-STED mode benefits from the advantages of both techniques, allowing for imaging with lateral resolution about six times better than the diffraction limit due to STED and simultaneous improvement of the axial resolution about twice over the diffraction limit due to uSEE. Conveniently, at certain imaging conditions, the uSEE-STED modality can achieve better resolution at four times lower laser power compared to STED mode, making the method appealing for biological applications. We illustrate this by imaging UCNPs functionalized by colominic acid in fixed neuronal phenotype cells.

Eph-ephrin signaling modulated by polymerization and condensation of receptors.

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization-condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.

Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand-Receptor Interactions That Tune EphB2 Receptor Clustering.

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate-with molecular sensitivity-the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems.

Exploration of sequence space as the basis of viral RNA genome segmentation.

The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

Viral genome segmentation can result from a trade-off between genetic content and particle stability.

The evolutionary benefit of viral genome segmentation is a classical, yet unsolved question in evolutionary biology and RNA genetics. Theoretical studies anticipated that replication of shorter RNA segments could provide a replicative advantage over standard size genomes. However, this question has remained elusive to experimentalists because of the lack of a proper viral model system. Here we present a study with a stable segmented bipartite RNA virus and its ancestor non-segmented counterpart, in an identical genomic nucleotide sequence context. Results of RNA replication, protein expression, competition experiments, and inactivation of infectious particles point to a non-replicative trait, the particle stability, as the main driver of fitness gain of segmented genomes. Accordingly, measurements of the volume occupation of the genome inside viral capsids indicate that packaging shorter genomes involves a relaxation of the packaging density that is energetically favourable. The empirical observations are used to design a computational model that predicts the existence of a critical multiplicity of infection for domination of segmented over standard types. Our experiments suggest that viral segmented genomes may have arisen as a molecular solution for the trade-off between genome length and particle stability. Genome segmentation allows maximizing the genetic content without the detrimental effect in stability derived from incresing genome length.

Competition-colonization dynamics in an RNA virus.

During replication, RNA viruses rapidly generate diverse mutant progeny which differ in their ability to kill host cells. We report that the progeny of a single RNA viral genome diversified during hundreds of passages in cell culture and self-organized into two genetically distinct subpopulations that exhibited the competition-colonization dynamics previously recognized in many classical ecological systems. Viral colonizers alone were more efficient in killing cells than competitors in culture. In cells coinfected with both competitors and colonizers, viral interference resulted in reduced cell killing, and competitors replaced colonizers. Mathematical modeling of this coinfection dynamics predicted selection to be density dependent, which was confirmed experimentally. Thus, as is known for other ecological systems, biodiversity and even cell killing of virus populations can be shaped by a tradeoff between competition and colonization. Our results suggest a model for the evolution of virulence in viruses based on internal interactions within mutant spectra of viral quasispecies.

The evolution of virulence in RNA viruses under a competition-colonization trade-off.

RNA viruses exist in large intra-host populations which display great genotypic and phenotypic diversity. We analyze a model of viral competition between two viruses infecting a constantly replenished cell pool. We assume a trade-off between the ability of the virus to colonize new cells (cell killing rate or virulence) and its local competitiveness (replicative success within coinfected cells). We characterize the conditions that allow for viral spread by means of the basic reproductive number and show that a local coexistence equilibrium exists, which is asymptotically stable. At this equilibrium, the less virulent competitor has a reproductive advantage over the more virulent colonizer reflected by a larger equilibrium population size of the competitor. The equilibria at which one virus outcompetes the other one are unstable, i.e., a second virus is always able to permanently invade. We generalize the two-virus model to multiple viral strains, each displaying a different virulence. To account for the large phenotypic diversity in viral populations, we consider a continuous spectrum of virulences and present a continuum limit of this multiple viral strains model that describes the time evolution of an initial continuous distribution of virulence without mutations. We provide a proof of the existence of solutions of the model equations, analytically assess the properties of stationary solutions, and present numerical approximations of solutions for different initial distributions. Our simulations suggest that initial continuous distributions of virulence evolve toward a distribution that is extremely skewed in favor of competitors. At equilibrium, only the least virulent part of the population survives. The discrepancy of this finding in the continuum limit with the two-virus model is attributed to the skewed equilibrium subpopulation sizes and to the transition to a continuum. Consequently, in viral quasispecies with high virulence diversity, the model predicts collective virulence attenuation. This result may contribute to understanding virulence attenuation, which has been reported in several experimental studies.

Embryo implantation in the laboratory: an update on current techniques.

BACKGROUND: The embryo implantation process is crucial for the correct establishment and progress of pregnancy. During implantation, the blastocyst trophectoderm cells attach to the epithelium of the endometrium, triggering intense cell-to-cell crosstalk that leads to trophoblast outgrowth, invasion of the endometrial tissue, and formation of the placenta. However, this process, which is vital for embryo and foetal development in utero, is still elusive to experimentation because of its inaccessibility. Experimental implantation is cumbersome and impractical in adult animal models and is inconceivable in humans. OBJECTIVE AND RATIONALE: A number of custom experimental solutions have been proposed to recreate different stages of the implantation process in vitro, by combining a human embryo (or a human embryo surrogate) and endometrial cells (or a surrogate for the endometrial tissue). In vitro models allow rapid high-throughput interrogation of embryos and cells, and efficient screening of molecules, such as cytokines, drugs, or transcription factors, that control embryo implantation and the receptivity of the endometrium. However, the broad selection of available in vitro systems makes it complicated to decide which system best fits the needs of a specific experiment or scientific question. To orient the reader, this review will explore the experimental options proposed in the literature, and classify them into amenable categories based on the embryo/cell pairs employed.The goal is to give an overview of the tools available to study the complex process of human embryo implantation, and explain the differences between them, including the advantages and disadvantages of each system. SEARCH METHODS: We performed a comprehensive review of the literature to come up with different categories that mimic the different stages of embryo implantation in vitro, ranging from initial blastocyst apposition to later stages of trophoblast invasion or gastrulation. We will also review recent breakthrough advances on stem cells and organoids, assembling embryo-like structures and endometrial tissues. OUTCOMES: We highlight the most relevant systems and describe the most significant experiments. We focus on in vitro systems that have contributed to the study of human reproduction by discovering molecules that control implantation, including hormones, signalling molecules, transcription factors and cytokines. WIDER IMPLICATIONS: The momentum of this field is growing thanks to the use of stem cells to build embryo-like structures and endometrial tissues, and the use of bioengineering to extend the life of embryos in culture. We propose to merge bioengineering methods derived from the fields of stem cells and reproduction to develop new systems covering a wider window of the implantation process.

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells.

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

BACKGROUND: The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. RESULTS: Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. CONCLUSION: Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes.

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.

Competition-colonization trade-off promotes coexistence of low-virulence viral strains.

RNA viruses exist as genetically diverse populations displaying a range of virulence degrees. The evolution of virulence in viral populations is, however, poorly understood. On the basis of the experimental observation of an RNA virus clone in cell culture diversifying into two subpopulations of different virulence, we study the dynamics of mutating virus populations with varying virulence. We introduce a competition-colonization trade-off into standard mathematical models of intra-host viral infection. Colonizers are fast-spreading virulent strains, whereas the competitors are less-virulent variants but more successful within co-infected cells. We observe a two-step dynamics of the population. Early in the infection, the population is dominated by colonizers, which later are outcompeted by competitors. Our simulations suggest the existence of steady state in which all virulence classes coexist but are dominated by the most competitive ones. This equilibrium implies collective virulence attenuation in the population, in contrast to previous models predicting evolution of the population towards increased virulence.

Quasispecies as a matter of fact: viruses and beyond.

We review the origins of the quasispecies concept and its relevance for RNA virus evolution, viral pathogenesis and antiviral treatment strategies. We emphasize a critical point of quasispecies that refers to genome collectivities as the unit of selection, and establish parallels between RNA viruses and some cellular systems such as bacteria and tumor cells. We refer also to tantalizing new observations that suggest quasispecies behavior in prions, perhaps as a result of the same quantum-mechanical indeterminations that underlie protein conformation and error-prone replication in genetic systems. If substantiated, these observations with prions could lead to new research on the structure-function relationship of non-nucleic acid biological molecules.

Models of RNA virus evolution and their roles in vaccine design.

Viruses are fast evolving pathogens that continuously adapt to the highly variable environments they live and reproduce in. Strategies devoted to inhibit virus replication and to control their spread among hosts need to cope with these extremely heterogeneous populations and with their potential to avoid medical interventions. Computational techniques such as phylogenetic methods have broadened our picture of viral evolution both in time and space, and mathematical modeling has contributed substantially to our progress in unraveling the dynamics of virus replication, fitness, and virulence. Integration of multiple computational and mathematical approaches with experimental data can help to predict the behavior of viral pathogens and to anticipate their escape dynamics. This piece of information plays a critical role in some aspects of vaccine development, such as viral strain selection for vaccinations or rational attenuation of viruses. Here we review several aspects of viral evolution that can be addressed quantitatively, and we discuss computational methods that have the potential to improve vaccine design.

Competition-colonization dynamics: An ecology approach to quasispecies dynamics and virulence evolution in RNA viruses.

A single and purified clone of foot-and-mouth disease virus diversified in cell culture into two subpopulations that were genetically distinct. The subpopulation with higher virulence was a minority and was suppressed by the dominant but less virulent one. These two populations follow the competitioncolonization dynamics described in ecology. Virulent viruses can be regarded as colonizers because they killed the cells faster and they spread faster. The attenuated subpopulation resembles competitors because of its higher replication efficiency in coinfected cells. Our results suggest a new model for the evolution of virulence which is based on interactions between components of the quasispecies. Competition between viral mutants takes place at two levels, intracellular competition and competition for new cells. The two strategies are subjected to densitydependent selection.

New vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines.

BACKGROUND: New vaccine designs are needed to control diseases associated with antigenically variable RNA viruses. Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that inflicts severe economic losses. Although the current whole-virus chemically inactivated vaccine has proven effective, it has led to new outbreaks of FMD because of incomplete inactivation of the virus or the escape of infectious virus from vaccine production premises. We have previously shown that serial passages of FMD virus (FMDV) C-S8c1 at high multiplicity of infection in cell culture resulted in virus populations consisting of defective genomes that are infectious by complementation (termed C-S8p260). PRINCIPAL FINDING: Here we evaluate the immunogenicity of C-S8p260, first in a mouse model system to establish a proof of principle, and second, in swine, the natural host of FMDV C-S8c1. Mice were completely protected against a lethal challenge with FMDV C-S8c1, after vaccination with a single dose of C-S8p260. Pigs immunized with different C-S8p260 doses and challenged with FMDV C-S8c1 either did not develop any clinical signs or showed delayed and mild disease symptoms. C-S8p260 induced high titers of both FMDV-specific, neutralizing antibodies and activated FMDV-specific T cells in swine, that correlated with solid protection against FMDV. CONCLUSIONS: The defective virus-based vaccine did not produce detectable levels of transmissible FMDV. Therefore, a segmented, replication-competent form of a virus, such as FMDV C-S8p260, can provide the basis of a new generation of attenuated antiviral vaccines with two safety barriers. The design can be extended to any viral pathogen that encodes trans-acting gene products, allowing complementation between replication-competent, defective forms.