Every refuge has its price: Ostreobium as a model for understanding how algae can live in rock and stay in business.

Ostreobium is a siphonous green alga in the Bryopsidales (Chlorophyta) that burrows into calcium carbonate (CaCO3) substrates. In this habitat, it lives under environmental conditions unusual for an alga (i.e., low light and low oxygen) and it is a major agent of carbonate reef bioerosion. In coral skeletons, Ostreobium can form conspicuous green bands recognizable by the naked eye and it is thought to contribute to the coral's nutritional needs. With coral reefs in global decline, there is a renewed focus on understanding Ostreobium biology and its roles in the coral holobiont. This review summarizes knowledge on Ostreobium's morphological structure, biodiversity and evolution, photosynthesis, mechanism of bioerosion and its role as a member of the coral holobiont. We discuss the resources available to study Ostreobium biology, lay out some of the uncharted territories in Ostreobium biology and offer perspectives for future research.

Clamping enables enhanced electromechanical responses in antiferroelectric thin films.

Thin-film materials with large electromechanical responses are fundamental enablers of next-generation micro-/nano-electromechanical applications. Conventional electromechanical materials (for example, ferroelectrics and relaxors), however, exhibit severely degraded responses when scaled down to submicrometre-thick films due to substrate constraints (clamping). This limitation is overcome, and substantial electromechanical responses in antiferroelectric thin films are achieved through an unconventional coupling of the field-induced antiferroelectric-to-ferroelectric phase transition and the substrate constraints. A detilting of the oxygen octahedra and lattice-volume expansion in all dimensions are observed commensurate with the phase transition using operando electron microscopy, such that the in-plane clamping further enhances the out-of-plane expansion, as rationalized using first-principles calculations. In turn, a non-traditional thickness scaling is realized wherein an electromechanical strain (1.7%) is produced from a model antiferroelectric PbZrO3 film that is just 100 nm thick. The high performance and understanding of the mechanism provide a promising pathway to develop high-performance micro-/nano-electromechanical systems.

ΔNp63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes.

SignificanceΔNp63 is a master regulator of skin homeostasis since it finely controls keratinocyte differentiation and proliferation. Here, we provide cellular and molecular evidence demonstrating the functional role of a ΔNp63 interactor, the R-loop-resolving enzyme Senataxin (SETX), in fine-tuning keratinocyte differentiation. We found that SETX physically binds the p63 DNA-binding motif present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3' ends and thus allowing efficient transcriptional termination and gene expression. These molecular events translate into the inability of SETX-depleted keratinocytes to undergo the correct epidermal differentiation program. Remarkably, SETX is dysregulated in cutaneous squamous cell carcinoma, suggesting its potential involvement in the pathogenesis of skin disorders.

Strongly Bound Excitons and Anisotropic Linear Absorption in Monolayer Graphullerene.

Graphullerene is a novel two-dimensional carbon allotrope with unique optoelectronic properties. Despite significant experimental characterization and prior density functional theory calculations, unanswered questions remain as to the nature, energy, and intensity of the electronic and optical excitations. Here, we present first-principles calculations of the quasiparticle band structure, neutral excitations, and absorption spectra of monolayer graphullerene and bulk graphullerite, employing the GW-Bethe-Salpeter equation (GW-BSE) approach. We show that strongly bound excitons dominate the absorption spectrum of monolayer graphullerene with binding energies up to 0.8 eV, while graphullerite exhibits less pronounced excitonic effects. Our calculations also reveal a strong linear polarization anisotropy, reflecting the in-plane structural anisotropy from intermolecular coupling between neighboring C60 units. We further show that the presence of Mg atoms, crucial to the synthesis process, induces structural modifications and polarizability effects, resulting in a ∼1 eV quasiparticle gap renormalization and a reduction in the exciton binding energy to ∼0.6 eV.

Synthetic DNA-based Swimmers Driven by Enzyme Catalysis.

Here, we report DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA-enzyme swimmers) that self-propel by converting the enzymatic substrate to the product in solution. The DNA-enzyme swimmers are obtained from tubular DNA structures that self-assemble spontaneously by the hybridization of DNA tiles. We functionalize these DNA structures with two different enzymes, urease and catalase, and show that they exhibit concentration-dependent movement and enhanced diffusion upon addition of the enzymatic substrate (i.e., urea and H2O2). To demonstrate the programmability of such DNA-based swimmers, we also engineer DNA strands that displace the enzyme from the DNA scaffold, thus acting as molecular "brakes" on the DNA swimmers. These results serve as a first proof of principle for the development of synthetic DNA-based enzyme-powered swimmers that can self-propel in fluids.

RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Blood-brain barrier permeability is associated with different neuroinflammatory profiles in Alzheimer's disease.

INTRODUCTION: Inflammation is an important player in Alzheimer's disease (AD), whose effects can be influenced by the blood-brain barrier (BBB). Here, we investigated the relationship between BBB permeability, indicated by cerebrospinal fluid (CSF)/plasma albumin quotient (Qalb), and CSF indexes of neuroinflammation in a cohort of biologically defined AD patients. METHODS: Fifty-nine consecutive patients with mild cognitive impairment (MCI) or early AD (Mini-Mental State Examination [MMSE] >22) underwent CSF analysis for inflammatory cytokines (interleukin [IL]-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, Il-10, IL-12, IL-13, IL-17, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], granulocyte-monocyte colony-stimulating factor [GM-CSF], granulocyte colony-stimulating factor [G-CSF]). Using backward stepwise linear regression analysis, we explored the potential influence of each cytokine CSF level on Qalb considering age, sex, and apolipoprotein E (APOE) as covariates. RESULTS: Higher levels of IL-4 (ß = 0.356, 0.005) and IL-8 (ß = 0.249, 0.05) were associated with higher Qalb values, while macrophage inflammatory protein-1α (MIP-1ß) (ß = -0.274; p = 0.032) and TNF-α (ß = -0.248; p = 0.031) showed a significant negative association with BBB permeability. Age was also positively associated with Qalb (ß = 0.283; p = 0.016). CONCLUSIONS: Despite the overall integrity of the BBB, its permeability could either influence or be influenced by central neuroinflammation, reflected by CSF cytokine levels. This is in line with previous studies that showed that patients with a more intact barrier are those with more prominent neurodegeneration. Our findings suggest that different neuroinflammatory profiles can be associated with different levels of BBB permeability in AD.

Terminal crystalline solid solutions, solubility enhancements and T-X phase diagram of salicylic acid - 4-hydroxybenzoic acid.

The binary T-X phase diagram of salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA) has been constructed from 20 °C to melting, revealing a partially miscible system with an eutectic composition of 27.3 mol% 4HBA in SA. Terminal crystalline solid solutions were obtained at the extremes of the phase diagram with solid-state miscibility limits below 0.4% at 20 °C. The limited phase boundaries could be captured experimentally by both DSC analyses at around melting temperature and solid-liquid equilibria studies at 20 °C in two solvent systems. The NRTL model was applied to regress phase boundaries and generate the final binary T-X phase diagram. The NRTL model was also used to regress solubility data, and reproduce the ternary SA/4HBA/solvent phase diagram at 20 °C and 1 atm. 4HBA was obtained as two crystal forms, viz. anhydrate and monohydrate. It is shown how the monohydrate of 4HBA is less miscible with SA in the solid state than the anhydrous form of 4HBA. As compared to pure SA and 4HBA, the crystalline solid solutions exhibited significant changes in physical properties that are relevant for organic and pharmaceutical materials in the context of impurity effects. A lattice incorporation of just 0.2 mol% 4HBA in SA caused a 10% reduction in melting enthalpy and a 66% solubility increase in 40 wt% MeOH in H2O. The reasons for this thermodynamic effect are discussed.

Complex oiling-out behavior of procaine with stable and metastable liquid phases.

During the crystallization of a solute from solvent(s), spontaneous liquid-liquid phase separation (LLPS) might occur, under certain conditions. This phenomenon, colloquially referred to as "oiling-out" in the pharmaceutical industry, often leads to undesired outcomes, including undesired particle properties, encrustation, ineffective impurity rejection, and excessively long process time. Therefore, it is critical to understand the thermodynamic driving force and phase boundaries of this phenomenon, such that rational strategies can be developed to avoid oiling-out or minimize its negative impact. In this study, we systematically evaluated the oiling-out behavior of procaine, a low melting point drug, in the solvent systems heptane, and ethanol-heptane as a function of temperature and solvent composition. In the procaine-heptane binary system, we observed a region where the LLPS is metastable with respect to crystallization, which is most commonly observed in the crystallization of modern active pharmaceutical ingredients (APIs); however, we also identified a region of the phase diagram where the LLPS is stable with respect to crystallization, and therefore will persist indefinitely. In the procaine-ethanol-heptane ternary system we identified five different regions, including a homogeneous liquid (L) region, two solid-liquid (SLI and SLII) regions, a liquid-liquid (LILII) region, and a solid-liquid-liquid (SLILII) region. The binary and ternary phase diagrams were also predicted using a state-of-the-art thermodynamic model: the SAFT-γ-Mie equation of state, and the results were compared with experimental data. Our findings highlight the complexity of oiling-out behavior. This work also represents a combined modeling and experimental platform to identify phase boundaries that will enable rational selection of strategies to crystallize active pharmaceutical ingredients with oiling-out risks.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

Enzyme-Linked DNA Displacement (ELIDIS) Assay for Ultrasensitive Electrochemical Detection of Antibodies.

Here we report the development of a method for the electrochemical ultrasensitive detection of antibodies that couples the programmability and versatility of DNA-based systems with the sensitivity provided by enzymatic amplification. The platform, termed Enzyme-Linked DNA Displacement (ELIDIS), is based on the use of antigen-DNA conjugates that, upon the bivalent binding of a specific target antibody, induce the release of an enzyme-DNA hybrid strand from a preformed duplex. Such enzyme-DNA hybrid strand can then be electrochemically detected with a disposable electrode with high sensitivity. We applied ELIDIS to demonstrate the sensitive (limit of detection in the picomolar range), specific and multiplexed detection of five different antibodies including three clinically relevant ones. ELIDIS is also rapid (it only requires two reaction steps), works well in complex media (serum) and is cost-effective. A direct comparison with a commercial ELISA kit for the detection of Cetuximab demonstrates the promising features of ELIDIS as a point-of-care platform for antibodies detection.

Synthetic Genes For Dynamic Regulation Of DNA-Based Receptors.

We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA-based receptors: a triplex-forming DNA complex, an ATP-binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling.

Timed Pulses in DNA Strand Displacement Reactions.

Inspired by naturally occurring regulatory mechanisms that allow complex temporal pulse features with programmable delays, we demonstrate here a strategy to achieve temporally programmed pulse output signals in DNA-based strand displacement reactions (SDRs). To achieve this, we rationally designed input strands that, once bound to their target duplex, can be gradually degraded, resulting in a pulse output signal. We also designed blocker strands that suppress strand displacement and determine the time at which the pulse reaction is generated. We show that by controlling the degradation rate of blocker and input strands, we can finely control the delayed pulse output over a range of 10 h. We also prove that it is possible to orthogonally delay two different pulse reactions in the same solution by taking advantage of the specificity of the degradation reactions for the input and blocker strands. Finally, we show here two possible applications of such delayed pulse SDRs: the time-programmed pulse decoration of DNA nanostructures and the sequentially appearing and self-erasing formation of DNA-based patterns.

Fine-scale mapping of physicochemical and microbial landscapes of the coral skeleton.

The coral skeleton harbours a diverse community of bacteria and microeukaryotes exposed to light, O2 and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O2 and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef-building corals Porites lutea and Paragoniastrea benhami. The physicochemical environment was more stable in the deep skeleton, and the diversity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O2 habitat, whereas pH varied from pH 6-9 with depth. Physicochemical gradients of O2 and pH of the coral skeleton explained the ß-diversity of the bacterial communities, and skeletal layers that showed O2 peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.

Protein-Templated Reactions Using DNA-Antibody Conjugates.

DNA-templated chemical reactions have found wide applications in drug discovery, programmed multistep synthesis, nucleic acid detection, and targeted drug delivery. The control of these reactions has, however, been limited to nucleic acid hybridization as a means to direct the proximity between reactants. In this work a system capable of translating protein-protein binding events into a DNA-templated reaction which leads to the covalent formation of a product is introduced. Protein-templated reactions by employing two DNA-antibody conjugates that are both able to recognize the same target protein and to colocalize a pair of reactant DNA strands able to undergo a click reaction are achieved. Two individual systems, each responsive to human serum albumin (HSA) and human IgG, are engineered and it is demonstrated that, while no reaction occurs in the absence of proteins, both protein-templated reactions can occur simultaneously in the same solution without any inter-system crosstalk.

Using Spectroscopy to Guide the Adaptation of Aptamers into Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.

Autonomous and Programmable Reorganization of DNA-Based Polymers Using Redox Chemistry.

We demonstrate here a strategy that allows the programmable and autonomous reorganization of self-assembled DNA polymers using redox chemistry. We have rationally designed different DNA monomers (tiles) that can co-assemble into tubular structures. The tiles can be orthogonally activated/deactivated with disulfide-linked DNA fuel strands that are degraded over time upon reduction because of the presence of a reducing agent in the system. The concentration of the disulfide fuels determines the activation kinetics of each DNA tile, which controls the degree of order/disorder in the formed co-polymer. The disulfide-reduction pathway can be employed together with enzymatic fuel-degradation pathways providing an additional level of control in the re-organization of DNA structures. Taking advantage of the different pH-sensitivities of disulfide-thiol and enzymatic reactions, we show that we can control the order in DNA-based co-polymers as a function of pH.

Rational design to control the trade-off between receptor affinity and cooperativity.

Cooperativity enhances the responsiveness of biomolecular receptors to small changes in the concentration of their target ligand, albeit with a concomitant reduction in affinity. The binding midpoint of a two-site receptor with a Hill coefficient of 1.9, for example, must be at least 19 times higher than the dissociation constant of the higher affinity of its two binding sites. This trade-off can be overcome, however, by the extra binding energy provided by the addition of more binding sites, which can be used to achieve highly cooperative receptors that still retain high affinity. Exploring this experimentally, we have employed an "intrinsic disorder" mechanism to design two cooperative, three-binding-site receptors starting from a single-site-and thus noncooperative-doxorubicin-binding aptamer. The first receptor follows a binding energy landscape that partitions the energy provided by the additional binding event to favor affinity, achieving a Hill coefficient of 1.9 but affinity within a factor of 2 of the parent aptamer. The binding energy landscape of the second receptor, in contrast, partitions more of this energy toward cooperativity, achieving a Hill coefficient of 2.3, but at the cost of 4-fold poorer affinity than that of the parent aptamer. The switch between these two behaviors is driven primarily by the affinity of the receptors' second binding event, which serves as an allosteric "gatekeeper" defining the extent to which the system is weighted toward higher cooperativity or higher affinity.

Electrochemical Cell-Free Biosensors for Antibody Detection.

We report here the development of an electrochemical cell-free biosensor for antibody detection directly in complex sample matrices with high sensitivity and specificity that is particularly suitable for point-of-care applications. The approach is based on the use of programmable antigen-conjugated gene circuits that, upon recognition of a specific target antibody, trigger the cell-free transcription of an RNA sequence that can be consequently detected using a redox-modified probe strand immobilized to a disposable electrode. The platform couples the features of high sensitivity and specificity of cell-free systems and the strength of cost-effectiveness and possible miniaturization provided by the electrochemical detection. We demonstrate the sensitive, specific, selective, and multiplexed detection of three different antibodies, including the clinically-relevant Anti-HA antibody.

Decorated DNA-Based Scaffolds as Lateral Flow Biosensors.

Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have rationally re-engineered tile-based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof-of-principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti-Dig, Anti-DNP and Anti-MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA-based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity.