Site-specific R-loops induce CGG repeat contraction and fragile X gene reactivation.

Here, we describe an approach to correct the genetic defect in fragile X syndrome (FXS) via recruitment of endogenous repair mechanisms. A leading cause of autism spectrum disorders, FXS results from epigenetic silencing of FMR1 due to a congenital trinucleotide (CGG) repeat expansion. By investigating conditions favorable to FMR1 reactivation, we find MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We trace the mechanism to DNA demethylation and site-specific R-loops, which are necessary and sufficient for repeat contraction. A positive feedback cycle comprising demethylation, de novo FMR1 transcription, and R-loop formation results in the recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction is specific to FMR1 and restores the production of FMRP protein. Our study therefore identifies a potential method of treating FXS in the future.

Jpx RNA regulates CTCF anchor site selection and formation of chromosome loops.

Chromosome loops shift dynamically during development, homeostasis, and disease. CCCTC-binding factor (CTCF) is known to anchor loops and construct 3D genomes, but how anchor sites are selected is not yet understood. Here, we unveil Jpx RNA as a determinant of anchor selectivity. Jpx RNA targets thousands of genomic sites, preferentially binding promoters of active genes. Depleting Jpx RNA causes ectopic CTCF binding, massive shifts in chromosome looping, and downregulation of >700 Jpx target genes. Without Jpx, thousands of lost loops are replaced by de novo loops anchored by ectopic CTCF sites. Although Jpx controls CTCF binding on a genome-wide basis, it acts selectively at the subset of developmentally sensitive CTCF sites. Specifically, Jpx targets low-affinity CTCF motifs and displaces CTCF protein through competitive inhibition. We conclude that Jpx acts as a CTCF release factor and shapes the 3D genome by regulating anchor site usage.

A disproportionate impact of G9a methyltransferase deficiency on the X chromosome.

G9a is a histone methyltransferase responsible for the dimethylation of histone H3 at lysine 9 (H3K9me2). G9a plays key roles in transcriptional silencing of developmentally regulated genes, but its role in X-chromosome inactivation (XCI) has been under debate. Here, we uncover a female-specific function of G9a and demonstrate that deleting G9a has a disproportionate impact on the X chromosome relative to the rest of the genome. G9a deficiency causes a failure of XCI and female-specific hypersensitivity to drug inhibition of H3K9me2. We show that G9a interacts with Tsix and Xist RNAs, and that competitive inhibition of the G9a-RNA interaction recapitulates the XCI defect. During XCI, Xist recruits G9a to silence X-linked genes on the future inactive X. In parallel on the future Xa, Tsix recruits G9a to silence Xist in cis Thus, RNA tethers G9a for allele-specific targeting of the H3K9me2 modification and the G9a-RNA interaction is essential for XCI.

Targeting Xist with compounds that disrupt RNA structure and X inactivation.

Although more than 98% of the human genome is non-coding1, nearly all of the drugs on the market target one of about 700 disease-related proteins. The historical reluctance to invest in non-coding RNA stems partly from requirements for drug targets to adopt a single stable conformation2. Most RNAs can adopt several conformations of similar stabilities. RNA structures also remain challenging to determine3. Nonetheless, an increasing number of diseases are now being attributed to non-coding RNA4 and the ability to target them would vastly expand the chemical space for drug development. Here we devise a screening strategy and identify small molecules that bind the non-coding RNA prototype Xist5. The X1 compound has drug-like properties and binds specifically the RepA motif6 of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that RepA can adopt multiple conformations but favours one structure in solution. X1 binding reduces the conformational space of RepA, displaces cognate interacting protein factors (PRC2 and SPEN), suppresses histone H3K27 trimethylation, and blocks initiation of X-chromosome inactivation. X1 inhibits cell differentiation and growth in a female-specific manner. Thus, RNA can be systematically targeted by drug-like compounds that disrupt RNA structure and epigenetic function.

Droplet motion driven by humidity gradients during evaporation and condensation.

The motion of droplets on solid surfaces in response to an external gradient is a fundamental problem with a broad range of applications, including water harvesting, heat exchange, mixing and printing. Here we study the motion of droplets driven by a humidity gradient, i.e. a variation in concentration of their own vapour in the surrounding gas phase. Using lattice-Boltzmann simulations of a diffuse-interface hydrodynamic model to account for the liquid and gas phases, we demonstrate that the droplet migrates towards the region of higher vapour concentration. This effect holds in situations where the ambient gradient drives either the evaporation or the condensation of the droplet, or both simultaneously. We identify two main mechanisms responsible for the observed motion: a difference in surface wettability, which we measure in terms of the Young stress, and a variation in surface tension, which drives a Marangoni flow. Our results are relevant in advancing our knowledge of the interplay between gas and liquid phases out of thermodynamic equilibrium, as well as for applications involving the control of droplet motion.

Biomarkers in Thyroid Cancer: Emerging Opportunities from Non-Coding RNAs and Mitochondrial Space.

Thyroid cancer diagnosis primarily relies on imaging techniques and cytological analyses. In cases where the diagnosis is uncertain, the quantification of molecular markers has been incorporated after cytological examination. This approach helps physicians to make surgical decisions, estimate cancer aggressiveness, and monitor the response to treatments. Despite the availability of commercial molecular tests, their widespread use has been hindered in our experience due to cost constraints and variability between them. Thus, numerous groups are currently evaluating new molecular markers that ultimately will lead to improved diagnostic certainty, as well as better classification of prognosis and recurrence. In this review, we start reviewing the current preoperative testing methodologies, followed by a comprehensive review of emerging molecular markers. We focus on micro RNAs, long non-coding RNAs, and mitochondrial (mt) signatures, including mtDNA genes and circulating cell-free mtDNA. We envision that a robust set of molecular markers will complement the national and international clinical guides for proper assessment of the disease.

Selective Concurrence of the Long Non-Coding RNA MALAT1 and the Polycomb Repressive Complex 2 to Promoter Regions of Active Genes in MCF7 Breast Cancer Cells.

In cancer cells, the long non-coding RNA (lncRNA) MALAT1 has arisen as a key partner for the Polycomb Repressive Complex 2 (PRC2), an epigenetic modifier. However, it is unknown whether this partnership occurs genome-wide at the chromatin level, as most of the studies focus on single genes that are usually repressed. Due to the genomic binding properties of both macromolecules, we wondered whether there are binding sites shared by PRC2 and MALAT1. Using public genome-binding datasets for PRC2 and MALAT1 derived from independent ChIP- and CHART-seq experiments performed with the breast cancer cell line MCF7, we searched for regions containing PRC2 and MALAT1 overlapping peaks. Peak calls for each molecule were performed using MACS2 and then overlapping peaks were identified by bedtools intersect. Using this approach, we identified 1293 genomic sites where PRC2 and MALAT1 concur. Interestingly, 54.75% of those sites are within gene promoter regions (<3000 bases from the TSS). These analyses were also linked with the transcription profiles of MCF7 cells, obtained from public RNA-seq data. Hence, it is suggested that MALAT1 and PRC2 can concomitantly bind to promoters of actively-transcribed genes in MCF7 cells. Gene ontology analyses revealed an enrichment of genes related to categories including cancer malignancy and epigenetic regulation. Thus, by re-visiting occupancy and transcriptomic data, we identified a key gene subset controlled by the collaboration of MALAT1 and PRC2.

Cassie's Law Reformulated: Composite Surfaces from Superspreading to Superhydrophobic.

In 1948, Cassie provided an equation describing the wetting of a smooth, heterogeneous surface. He proposed that the cosine of the contact angle, θc, for a droplet on a composite surface could be predicted from a weighted average using the fractional surface areas, fi, of the cosines of contact angles of a droplet on the individual component surfaces, i.e., cos θc = f1 cos θ1 + f2 cos θ2. This was a generalization of an earlier equation for porous materials, which has recently proven fundamental to underpinning the theoretical description of wetting of superhydrophobic and superoleophobic surfaces. However, there has been little attention paid to what happens when a liquid exhibits complete wetting on one of the surface components. Here, we show that Cassie's equation can be reformulated using spreading coefficients. This reformulated equation is capable of describing composite surfaces where the individual surface components have negative (droplet state/partial wetting) or positive (film-forming/complete wetting) spreading coefficients. The original Cassie equation is then a special case when the combination of interfacial tensions results in a droplet state on the composite surface for which a contact angle can be defined. In the case of a composite surface created from a partial wetting (droplet state) surface and a complete wetting (film-forming) surface, there is a threshold surface area fraction at which a liquid on the composite surface transitions from a droplet to a film state. The applicability of this equation is demonstrated from literature data including data on mixed self-assembled monolayers on copper, silver, and gold surfaces that was regarded as definitive in establishing the validity of the Cassie equation. Finally, we discuss the implications of these ideas for super-liquid repellent surfaces.

Droplet Self-Propulsion on Slippery Liquid-Infused Surfaces with Dual-Lubricant Wedge-Shaped Wettability Patterns.

Young's equation is fundamental to the concept of the wettability of a solid surface. It defines the contact angle for a droplet on a solid surface through a local equilibrium at the three-phase contact line. Recently, the concept of a liquid Young's law contact angle has been developed to describe the wettability of slippery liquid-infused porous surfaces (SLIPS) by droplets of an immiscible liquid. In this work, we present a new method to fabricate biphilic SLIP surfaces and show how the wettability of the composite SLIPS can be exploited with a macroscopic wedge-shaped pattern of two distinct lubricant liquids. In particular, we report the development of composite liquid surfaces on silicon substrates based on lithographically patterning a Teflon AF1600 coating and a superhydrophobic coating (Glaco Mirror Coat Zero), where the latter selectively dewets from the former. This creates a patterned base surface with preferential wetting to matched liquids: the fluoropolymer PTFE with a perfluorinated oil Krytox and the hydrophobic silica-based GLACO with olive oil (or other mineral oils or silicone oil). This allows us to successively imbibe our patterned solid substrates with two distinct oils and produce a composite liquid lubricant surface with the oils segregated as thin films into separate domains defined by the patterning. We illustrate that macroscopic wedge-shaped patterned SLIP surfaces enable low-friction droplet self-propulsion. Finally, we formulate an analytical model that captures the dependence of the droplet motion as a function of the wettability of the two liquid lubricant domains and the opening angle of the wedge. This allows us to derive scaling relationships between various physical and geometrical parameters. This work introduces a new approach to creating patterned liquid lubricant surfaces, demonstrates long-distance droplet self-propulsion on such surfaces, and sheds light on the interactions between liquid droplets and liquid surfaces.

Suppression of crystallization in saline drop evaporation on pinning-free surfaces.

For sessile droplets of pure liquid on a surface, evaporation depends on surface wettability, the surrounding environment, contact angle hysteresis, and surface roughness. For non-pure liquids, the evaporation characteristics are further complicated by the constituents and impurities within the droplet. For saline solutions, this complication takes the form of a modified partial vapor pressure/water activity caused by the increasing salt concentration as the aqueous solvent evaporates. It is generally thought that droplets on surfaces will crystallize when the saturation concentration is reached, i.e., 26.3% for NaCl in water. This crystallization is initiated by contact with the surface and is thus due to surface roughness and heterogeneities. Recently, smooth, low contact angle hysteresis surfaces have been created by molecular grafting of polymer chains. In this work, we hypothesize that by using these very smooth surfaces to evaporate saline droplets, we can suppress the crystallization caused by the surface interactions and thus achieve constant volume droplets above the saturation concentration. In our experiments, we used several different surfaces to examine the possibility of crystallization suppression. We show that on polymer grafted surfaces, i.e., Slippery Omniphobic Covalently Attached Liquid-like (SOCAL) and polyethyleneglycol(PEGylated) surfaces, we can achieve stable droplets as low as 55% relative humidity at 25 °C with high reproducibility using NaCl in water solutions. We also show that it is possible to achieve stable droplets above the saturation concentration on other surfaces, including superhydrophobic surfaces. We present an analytical model, based on water activity, which accurately describes the final stable volume as a function of the initial salt concentration. These findings are important for heat and mass transfer in relatively low humidity environments.

The Liquid Young's Law on SLIPS: Liquid-Liquid Interfacial Tensions and Zisman Plots.

Slippery liquid-infused porous surfaces (SLIPS) are an innovation that reduces droplet-solid contact line pinning and interfacial friction. Recently, it has been shown that a liquid analogue of Young's law can be deduced for the apparent contact angle of a sessile droplet on SLIPS despite there never being contact by the droplet with the underlying solid. Since contact angles on solids are used to characterize solid-liquid interfacial interactions and the wetting of a solid by a liquid, it is our hypothesis that liquid-liquid interactions and the wetting of a liquid surface by a liquid can be characterized by apparent contact angles on SLIPS. Here, we first present a theory for deducing liquid-liquid interfacial tensions from apparent contact angles. This theory is valid irrespective of whether or not a film of the infusing liquid cloaks the droplet-vapor interface. We show experimentally that liquid-liquid interfacial tensions deduced from apparent contact angles of droplets on SLIPS are in excellent agreement with values from the traditional pendant drop technique. We then consider whether the Zisman method for characterizing the wettability of a solid surface can be applied to liquid surfaces created using SLIPS. We report apparent contact angles for a homologous series of alkanes on Krytox-infused SLIPS and for water-IPA mixtures on both the Krytox-infused SLIPS and a silicone oil-infused SLIPS. The alkanes on the Krytox-infused SLIPS follow a linear relationship in the liquid form of the Zisman plot provided that the effective droplet-vapor interfacial tension is used. All three systems follow a linear relationship on a modified Zisman plot. We interpret these results using the concept of the critical surface tension (CST) for the wettability of a solid surface introduced by Zisman. In our liquid surface case, the obtained critical surface tensions were found to be lower than the infusing liquid-vapor surface tensions.

Friction Coefficients for Droplets on Solids: The Liquid-Solid Amontons' Laws.

The empirical laws of dry friction between two solid bodies date back to the work of Amontons in 1699 and are pre-dated by the work of Leonardo da Vinci. Fundamental to those laws are the concepts of static and kinetic coefficients of friction relating the pinning and sliding friction forces along a surface to the normal load force. For liquids on solid surfaces, contact lines also experience pinning and the language of friction is used when droplets are in motion. However, it is only recently that the concept of coefficients of friction has been defined in this context and that droplet friction has been discussed as having a static and a kinetic regime. Here, we use surface free energy considerations to show that the frictional force per unit length of a contact line is directly proportional to the normal component of the surface tension force. We define coefficients of friction for both contact lines and droplets and provide a droplet analogy of Amontons' first and second laws but with the normal load force of a solid replaced by the normal surface tension force of a liquid. In the static regime, the coefficient of static friction, defined by the maximum pinning force of a droplet, is proportional to the contact angle hysteresis, whereas in the kinetic regime, the coefficient of kinetic friction is proportional to the difference in dynamic advancing and receding contact angles. We show the consistency between the droplet form of Amontons' first and second laws and an equation derived by Furmidge. We use these liquid-solid Amontons' laws to describe literature data and report friction coefficients for various liquid-solid systems. The conceptual framework reported here should provide insight into the design of superhydrophobic, slippery liquid-infused porous surfaces (SLIPS) and other surfaces designed to control droplet motion.

Self-Assembled, Hierarchical Structured Surfaces for Applications in (Super)hydrophobic Antiviral Coatings.

A versatile method for the creation of multitier hierarchical structured surfaces is reported, which optimizes both antiviral and hydrophobic (easy-clean) properties. The methodology exploits the availability of surface-active chemical groups while also manipulating both the surface micro- and nanostructure to control the way the surface coating interacts with virus particles within a liquid droplet. This methodology has significant advantages over single-tier structured surfaces, including the ability to overcome the droplet-pinning effect and in delivering surfaces with high static contact angles (>130°) and good antiviral efficacy (log kill >2). In addition, the methodology highlights a valuable approach for the creation of mechanically robust, nanostructured surfaces which can be prepared by spray application using nonspecialized equipment.

Epigenetic silencing of the osteoblast-lineage gene program during hippocampal maturation.

Accumulating evidence indicates that epigenetic control of gene expression plays a significant role during cell lineage commitment and subsequent cell fate maintenance. Here, we assess epigenetic mechanisms operating in the rat brain that mediate silencing of genes that are expressed during early and late stages of osteogenesis. We report that repression of the osteoblast master regulator Sp7 in embryonic (E18) hippocampus is mainly mediated through the Polycomb complex PRC2 and its enzymatic product H3K27me3. During early postnatal (P10), juvenile (P30), and adult (P90) hippocampal stages, the repressive H3K27me3 mark is progressively replaced by nucleosome enrichment and increased CpG DNA methylation at the Sp7 gene promoter. In contrast, silencing of the late bone phenotypic Bglap gene in the hippocampus is PRC2-independent and accompanied by strong CpG methylation from E18 through postnatal and adult stages. Forced ectopic expression of the primary master regulator of osteogenesis Runx2 in embryonic hippocampal neurons activates the expression of its downstream target Sp7 gene. Moreover, transcriptomic analyses show that several genes associated with the mesenchymal-osteogenic lineages are transcriptionally activated in these hippocampal cells that express Runx2 and Sp7. This effect is accompanied by a loss in neuronal properties, including a significant reduction in secondary processes at the dendritic arbor and reduced expression of critical postsynaptic genes like PSD95. Together, our results reveal a developmental progression in epigenetic control mechanisms that repress the expression of the osteogenic program in hippocampal neurons at embryonic, postnatal, and adult stages.

Lattice Boltzmann Simulations of Multiphase Dielectric Fluids.

The dynamic effect of an electric field on dielectric liquids is called liquid dielectrophoresis. It is widely used in several industrial and scientific applications, including inkjet printing, microfabrication, and optical devices. Numerical simulations of liquid-dielectrophoresis are necessary to understand the fundamental physics of the phenomenon, but also to explore situations that might be difficult or expensive to implement experimentally. However, such modeling is challenging, as one needs to solve the electrostatic and fluid dynamics equations simultaneously. Here, we formulate a new lattice-Boltzmann method capable of modeling the dynamics of immiscible dielectric fluids coupled with electric fields within a single framework, thus eliminating the need of using separate algorithms to solve the electrostatic and fluid dynamics equations. We validate the numerical method by comparing it with analytical solutions and previously reported experimental results. Beyond the benchmarking of the method, we study the spreading of a droplet using a dielectrowetting setup and quantify the mechanism driving the variation of the apparent contact angle of the droplet with the applied voltage. Our method provides a useful tool to study liquid-dielectrophoresis and can be used to model dielectric fluids in general, such as liquid-liquid and liquid-gas systems.

Contact-Angle Hysteresis and Contact-Line Friction on Slippery Liquid-like Surfaces.

Contact-line pinning and dynamic friction are fundamental forces that oppose the motion of droplets on solid surfaces. Everyday experience suggests that if a solid surface offers low contact-line pinning, it will also impart a relatively low dynamic friction to a moving droplet. Examples of such surfaces are superhydrophobic, slippery porous liquid-infused, and lubricant-impregnated surfaces. Here, however, we show that slippery omniphobic covalently attached liquid-like (SOCAL) surfaces have a remarkable combination of contact-angle hysteresis and contact-line friction properties, which lead to very low droplet pinning but high dynamic friction against the motion of droplets. We present experiments of the response of water droplets to changes in volume at controlled temperature and humidity conditions, which we separately compare to the predictions of a hydrodynamic model and a contact-line model based on molecular kinetic theory. Our results show that SOCAL surfaces offer very low contact-angle hysteresis, between 1 and 3°, but an unexpectedly high dynamic friction controlled by the contact line, where the typical relaxation time scale is on the order of seconds, 4 orders of magnitude larger than the prediction of the classical hydrodynamic model. Our results highlight the remarkable wettability of SOCAL surfaces and their potential application as low-pinning, slow droplet shedding surfaces.

Downregulation of the Polycomb-Associated Methyltransferase Ezh2 during Maturation of Hippocampal Neurons Is Mediated by MicroRNAs Let-7 and miR-124.

Ezh2 is a catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates epigenetic gene silencing through depositing the mark histone H3 lysine 27 trimethylation (H3K27me3) at target genomic sequences. Previous studies have demonstrated that Enhancer of Zeste Homolog 2 (Ezh2) was differentially expressed during maturation of hippocampal neurons; in immature neurons, Ezh2 was abundantly expressed, whereas in mature neurons the expression Ezh2 was significantly reduced. Here, we report that Ezh2 is downregulated by microRNAs (miRs) that are expressed during the hippocampal maturation process. We show that, in mature hippocampal neurons, lethal-7 (let-7) and microRNA-124 (miR-124) are robustly expressed and can target cognate motifs at the 3'-UTR of the Ezh2 gene sequence to downregulate Ezh2 expression. Together, these data demonstrate that the PRC2 repressive activity during hippocampal maturation is controlled through a post-transcriptional mechanism that mediates Ezh2 downregulation in mature neurons.

Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts.

Expression of Runx2/p57 is a hallmark of the osteoblast-lineage identity. Although several regulators that control the expression of Runx2/p57 during osteoblast-lineage commitment have been identified, the epigenetic mechanisms that sustain this expression in differentiated osteoblasts remain to be completely determined. Here, we assess epigenetic mechanisms associated with Runx2/p57 gene transcription in differentiating MC3T3 mouse osteoblasts. Our results show that an enrichment of activating histone marks at the Runx2/p57 P1 promoter is accompanied by the simultaneous interaction of Wdr5 and Utx proteins, both are components of COMPASS complexes. Knockdown of Wdr5 and Utx expression confirms the activating role of both proteins at the Runx2-P1 promoter. Other chromatin modifiers that were previously described to regulate Runx2/p57 transcription in mesenchymal precursor cells (Ezh2, Prmt5, and Jarid1b proteins) were not found to contribute to Runx2/p57 transcription in full-committed osteoblasts. We also determined the presence of additional components of COMPASS complexes at the Runx2/p57 promoter, evidencing that the Mll2/COMPASS- and Mll3/COMPASS-like complexes bind to the P1 promoter in osteoblastic cells expressing Runx2/p57 to modulate the H3K4me1 to H3K4me3 transition.

Wnt/ß-catenin signaling enhances transcription of the CX43 gene in murine Sertoli cells.

Sertoli cells provide the nutritional and metabolic support for germ cells. Wnt/ß-catenin signaling is important for the development of the seminiferous epithelium during embryonic age, although after birth this pathway is downregulated. Cx43 gene codes for a protein that is critical during testicular development. The Cx43 promoter contains TCF/ß-catenin binding elements (TBEs) that contribute CX43 expression in different cell types and which may also be regulating the expression of this gene in Sertoli cells. In this study, we demonstrate that 42GPA9 Sertoli cells respond to treatments that result in accumulation of ß-catenin within the nucleus and in upregulation of CX43 gene transcription. ß-Catenin binds to TBEs located both upstream and downstream of the transcriptional start site (TSS). Luciferase reporter experiments revealed that TBEs located upstream of the TSS are necessary for ß-catenin-mediated upregulation. Our results also indicate that the Wnt/ß-catenin-dependent upregulation of the Cx43 gene in Sertoli cells is accompanied by changes in epigenetic parameters that may be directly contributing to generating a chromatin environment that facilitates the establishment of the transcriptional machinery at this promoter.

Lattice-Boltzmann Simulations of Electrowetting Phenomena.

When a voltage difference is applied between a conducting liquid and a conducting (solid) electrode, the liquid is observed to spread on the solid. This phenomenon, generally referred to as electrowetting, underpins a number of interfacial phenomena of interest in applications that range from droplet microfluidics to optics. Here, we present a lattice-Boltzmann method that can simulate the coupled hydrodynamics and electrostatics equations of motion of a two-phase fluid as a means to model the electrowetting phenomena. Our method has the advantage of modeling the electrostatic fields within the lattice-Boltzmann algorithm itself, eliminating the need for a hybrid method. We validate our method by reproducing the static equilibrium configuration of a droplet subject to an applied voltage and show that the apparent contact angle of the drop depends on the voltage following the Young-Lippmann equation up to contact angles of ≈50°. At higher voltages, we observe a saturation of the contact angle caused by the competition between electric and capillary stresses, similar to previous experimental observations. We also study the stability of a dielectric film trapped between a conducting fluid and a solid electrode and find a good agreement with analytical predictions based on lubrication theory. Finally, we investigate the film dynamics at long times and report observations of film breakup and entrapment similar to previously reported experimental results.