Quantitative Magnetic Resonance Imaging (qMRI) of the Small Bowel in Crohn's Disease: State-of-the-Art and Future Directions.

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract in which repeated episodes of acute inflammation may lead to long-term bowel damage. Cross-sectional imaging is used in conjunction with endoscopy to diagnose and monitor disease and detect complications. Magnetic resonance imaging (MRI) has demonstrable utility in evaluating inflammatory activity. However, subjective interpretation of conventional MR sequences is limited in its ability to fully phenotype the underlying histopathological processes in chronic disease. In particular, conventional MRI can be confounded by the presence of mural fibrosis and muscle hypertrophy, which can mask or sometimes mimic inflammation. Quantitative MRI (qMRI) methods provide a means to better differentiate mural inflammation from fibrosis and improve quantification of these processes. qMRI may also provide more objective measures of disease activity and enable better tailoring of treatment. Here, we review quantitative MRI methods for imaging the small bowel in CD and consider the path to their clinical translation. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Deep exome sequencing identifies enrichment of deleterious mosaic variants in neurodevelopmental disorder genes and mitochondrial tRNA regions in bipolar disorder.

Bipolar disorder (BD) is a global medical issue, afflicting around 1% of the population with manic and depressive episodes. Despite various genetic studies, the genetic architecture and pathogenesis of BD have not been fully resolved. Besides germline variants, postzygotic mosaic variants are proposed as new candidate mechanisms contributing to BD. Here, we performed extensive deep exome sequencing (DES, ~300×) and validation experiments to investigate the roles of mosaic variants in BD with 235 BD cases (194 probands of trios and 41 single cases) and 39 controls. We found an enrichment of developmental disorder (DD) genes in the genes hit by deleterious mosaic variants in BD (P = 0.000552), including a ClinVar-registered pathogenic variant in ARID2. An enrichment of deleterious mosaic variants was also observed for autism spectrum disorder (ASD) genes (P = 0.000428). The proteins coded by the DD/ASD genes with non-synonymous mosaic variants in BD form more protein-protein interaction than expected, suggesting molecular mechanisms shared with DD/ASD but restricted to a subset of cells in BD. We also found significant enrichment of mitochondrial heteroplasmic variants, another class of mosaic variants, in mitochondrial tRNA genes in BD (P = 0.0102). Among them, recurrent m.3243 A > G variants known as causal for mitochondrial diseases were found in two unrelated BD probands with allele fractions of 5-12%, lower than in mitochondrial diseases. Despite the limitation of using peripheral tissues, our DES investigation supports the possible contribution of deleterious mosaic variants in the nuclear genome responsible for severer phenotypes, such as DD/ASD, to the risk of BD and further demonstrates that the same paradigm can be applied to the mitochondrial genome. These results, as well as the enrichment of heteroplasmic mitochondrial tRNA variants in BD, add a new piece to the understanding of the genetic architecture of BD and provide general insights into the pathological roles of mosaic variants in human diseases.

Subphthalocyanine-flipper dyads for selective membrane staining.

The design, synthesis and evaluation of a subphthalocyanine-flipper (SubPc-Flipper) amphiphilic dyad is reported. This dyad combines two fluorophores that function in the visible region (420-800 nm) for the simultaneous sensing of both ordered and disordered lipidic membranes. The flipper probes part of the dyad possesses mechanosensitivity, long fluorescence lifetimes (τ = 3.5-5 ns) and selective staining of ordered membranes. On the other hand, subphthalocyanines (SubPc) are short-lifetime (τ = 1-2.5 ns) fluorophores that are insensitive to membrane tension. As a result of a Förster Resonance Energy Transfer (FRET) process, the dyad not only retains the mechanosensitivity of flippers but also demonstrates high selectivity and emission in different kinds of lipidic membranes. The dyad exhibits high emission and sensitivity to membrane tension (Δτ = 3.5 ns) when tested in giant unilamellar vesicles (GUVs) with different membrane orders. Overall, the results of this study represent a significant advancement in the applications of flippers and dyads in mechanobiology.

Multiple-Community-Based Epidemiological Study of Stuttering among 3-Year-Old Children in Japan.

INTRODUCTION: Many epidemiological studies of the disorder of stuttering have been conducted during the 20th century, continuing during the current one. Unfortunately, only a few were carried out in Japan. This study aimed at assessing (1) the incidence and prevalence of stuttering in 3-year-old children in multiple Japanese communities and (2) factors associated with the onset of stuttering among these children. METHODS: A questionnaire aimed at screening for the presence of stuttering was employed for 2,055 children aged 3 years, who underwent a standard nationwide health checkup. Positive responses were confirmed in several ways: (1) direct interviews and assessment of the child's speech, (2) confirmatory questionnaire, or (3) telephone interviews by licensed Speech Language Hearing Therapists. RESULTS: Approximately 6.5% of the children were found to exhibit stuttering at the time of their health checkup. This figure rose to 8.9% after including individuals who previously, but not currently, were reported to have exhibited stuttering. Among the putative risk factors, higher stuttering odds (odds ratio, OR = 3.27) were detected in those with a family history of stuttering, those whose guardians had concerns about their child's development (OR = 1.75), and those with diagnosed diseases or disabilities (OR = 2.13). DISCUSSION/CONCLUSIONS: It was concluded that, in Japan, both the risk of stuttering incidence (8.9%) in children up to, and including, the age of 3 years, as well as its prevalence (6.5%) in this population, was similar to those reported by recent studies in other countries. Additionally, our findings also confirmed that an increased risk for stuttering at age 3 is associated with a family history of stuttering.

Pnictogen-Bonding Enzymes.

The objective of this study was to create artificial enzymes that capitalize on pnictogen bonding, a s-hole interaction that is essentially absent in biocatalysis.  For this purpose, stibine catalysts were equipped with a biotin derivative and combined with streptavidin mutants to identify an efficient transfer hydrogenation catalyst for the reduction of a fluorogenic quinoline substrate.  Increased catalytic activity from wild-type streptavidin to the best mutants coincides with the depth of the s hole on the Sb(V) center, and the emergence of saturation kinetic behavior.  Michaelis-Menten analysis reveals transition-state recognition in the low micromolar range, more than three orders of magnitude stronger than the millimolar substrate recognition.  Carboxylates preferred by the best mutants contribute to transition-state recognition by hydrogen-bonded ion pairing and anion-π interactions with the emerging pyridinium product.  The emergence of challenging stereoselectivity in aqueous systems further emphasizes compatibility of pnictogen bonding with higher order systems catalysis.

Core-Alkynylated Fluorescent Flippers: Altered Ultrafast Photophysics to Track Thick Membranes.

Fluorescent flippers have been introduced as small-molecule probes to image membrane tension in living systems. This study describes the design, synthesis, spectroscopic and imaging properties of flippers that are elongated by one and two alkynes inserted between the push and the pull dithienothiophene domains. The resulting mechanophores combine characteristics of flippers, reporting on physical compression in the ground state, and molecular rotors, reporting on torsional motion in the excited state, to take their photophysics to new level of sophistication. Intensity ratios in broadened excitation bands from differently twisted conformers of core-alkynylated flippers thus report on mechanical compression. Lifetime boosts from ultrafast excited-state planarization and lifetime drops from competitive intersystem crossing into triplet states report on viscosity. In standard lipid bilayer membranes, core-alkynylated flippers are too long for one leaflet and tilt or extend into disordered interleaflet space, which preserves rotor-like torsional disorder and thus weak, blue-shifted fluorescence. Flipper-like planarization occurs only in highly ordered membranes of matching leaflet thickness, where they light up and selectively report on these thick membranes with red-shifted, sharpened excitation maxima, high intensity and long lifetime.

Dynamic Phosphorus: Thiolate Exchange Cascades with Higher Phosphorothioates.

In this study, we introduce phosphorus, a pnictogen, as an exchange center for dynamic covalent chemistry. Cascade exchange of neutral phosphorotri- and -tetrathioates with thiolates is demonstrated in organic solvents, aqueous micellar systems, and in living cells. Exchange rates increase with the pH value, electrophilicity of the exchange center, and nucleophilicity of the exchangers. Molecular walking of the dynamic phosphorus center along Hammett gradients is simulated by the sequential addition of thiolate exchangers. Compared to phosphorotrithioates, tetrathioates are better electrophiles with higher exchange rates. Dynamic phosphorotri- and -tetrathioates are non-toxic to HeLa Kyoto cells and participate in the dynamic networks that account for thiol-mediated uptake into living cells.

Anion-(π)n -π Catalytic Micelles.

Anion-π catalysis operates by stabilizing anionic transition states on π-acidic aromatic surfaces. In anion-(π)n -π catalysis, π stacks add polarizability to strengthen interactions. In search of synthetic methods to extend π stacks beyond the limits of foldamers, the self-assembly of micelles from amphiphilic naphthalenediimides (NDIs) is introduced. To interface substrates and catalysts, charge-transfer complexes with dialkoxynaphthalenes (DANs), a classic in supramolecular chemistry, are installed. In π-stacked micelles, the rates of bioinspired ether cyclizations exceed rates on monomers in organic solvents by far. This is particularly impressive considering that anion-π catalysis in water has been elusive so far. Increasing rates with increasing π acidity of the micelles evince operational anion-(π)n -π catalysis. At maximal π acidity, autocatalytic behavior emerges. Dependence on position and order in confined micellar space promises access to emergent properties. Anion-(π)n -π catalytic micelles in water thus expand supramolecular systems catalysis accessible with anion-π interactions with an inspiring topic of general interest and great perspectives.

Fluorescent Flippers: Small-Molecule Probes to Image Membrane Tension in Living Systems.

Flipper probes have been introduced as small molecule fluorophores to image physical forces, that is, membrane tension in living systems. Their emergence over one decade is described, from evolution in design and synthesis to spectroscopic properties. Responsiveness to physical compression in equilibrium at the ground state is identified as the ideal origin of mechanosensitivity to image membrane tension in living cells. A rich collection of flippers is described to deliver and release in any subcellular membrane of interest in a leaflet-specific manner. Chalcogen-bonding cascade switching and dynamic covalent flippers are developed for super-resolution imaging and dual-sensing of membrane compression and hydration. Availability and broad use in the community validate flipper probes as a fine example of the power of translational supramolecular chemistry, moving from fundamental principles to success on the market.

Anion-π catalysis on carbon allotropes.

Anion-π catalysis, introduced in 2013, stands for the stabilization of anionic transition states on π-acidic aromatic surfaces. Anion-π catalysis on carbon allotropes is particularly attractive because high polarizability promises access to really strong anion-π interactions. With these expectations, anion-π catalysis on fullerenes has been introduced in 2017, followed by carbon nanotubes in 2019. Consistent with expectations from theory, anion-π catalysis on carbon allotropes generally increases with polarizability. Realized examples reach from enolate addition chemistry to asymmetric Diels-Alder reactions and autocatalytic ether cyclizations. Currently, anion-π catalysis on carbon allotropes gains momentum because the combination with electric-field-assisted catalysis promises transformative impact on organic synthesis.

Safety and efficacy of hydrothermal duodenal mucosal resurfacing in patients with type 2 diabetes: the randomised, double-blind, sham-controlled, multicentre REVITA-2 feasibility trial.

OBJECTIVE: Hydrothermal duodenal mucosal resurfacing (DMR) is a safe, outpatient endoscopic procedure. REVITA-2, a double-blind, superiority randomised controlled trial, investigates safety and efficacy of DMR using the single catheter Revita system (Revita DMR (catheter and system)), on glycaemic control and liver fat content in type 2 diabetes (T2D). DESIGN: Eligible patients (haemoglobin A1c (HbA1c) 59-86 mmol/mol, body mass index≥24 and ≤40 kg/m2, fasting insulin >48.6 pmol/L, ≥1 oral antidiabetic medication) enrolled in Europe and Brazil. Primary endpoints were safety, change from baseline in HbA1c at 24 weeks, and liver MRI proton-density fat fraction (MRI-PDFF) at 12 weeks. RESULTS: Overall mITT (DMR n=56; sham n=52), 24 weeks post DMR, median (IQR) HbA1c change was -10.4 (18.6) mmol/mol in DMR group versus -7.1 (16.4) mmol/mol in sham group (p=0.147). In patients with baseline liver MRI-PDFF >5% (DMR n=48; sham n=43), 12-week post-DMR liver-fat change was -5.4 (5.6)% in DMR group versus -2.9 (6.2)% in sham group (p=0.096). Results from prespecified interaction testing and clinical parameter assessment showed heterogeneity between European (DMR n=39; sham n=37) and Brazilian (DMR n=17; sham n=16) populations (p=0.063); therefore, results were stratified by region. In European mITT, 24 weeks post DMR, median (IQR) HbA1c change was -6.6 mmol/mol (17.5 mmol/mol) versus -3.3 mmol/mol (10.9 mmol/mol) post-sham (p=0.033); 12-week post-DMR liver-fat change was -5.4% (6.1%) versus -2.2% (4.3%) post-sham (p=0.035). Brazilian mITT results trended towards DMR benefit in HbA1c, but not liver fat, in context of a large sham effect. In overall PP, patients with high baseline fasting plasma glucose ((FPG)≥10 mmol/L) had significantly greater reductions in HbA1c post-DMR versus sham (p=0.002). Most adverse events were mild and transient. CONCLUSIONS: DMR is safe and exerts beneficial disease-modifying metabolic effects in T2D with or without non-alcoholic liver disease, particularly in patients with high FPG. TRIAL REGISTRATION NUMBER: NCT02879383.

The Dynamic Range of Acidity: Tracking Rules for the Unidirectional Penetration of Cellular Compartments.

Labeled ammonium cations with pKa ∼7.4 accumulate in acidic organelles because they can be neutralized transiently to cross the membrane at cytosolic pH 7.2 but not at their internal pH<5.5. Retention in early endosomes with less acidic internal pH was achieved recently using weaker acids of up to pKa 9.8. We report here that primary ammonium cations with higher pKa 10.6, label early endosomes more efficiently. This maximized early endosome tracking coincides with increasing labeling of Golgi networks with similarly weak internal acidity. Guanidinium cations with pKa 13.5 cannot cross the plasma membrane in monomeric form and label the plasma membrane with selectivity for vesicles embarking into endocytosis. Self-assembled into micelles, guanidinium cations enter cells like arginine-rich cell-penetrating peptides and, driven by their membrane potential, penetrate mitochondria unidirectionally despite their high inner pH. The resulting tracking rules with an approximated dynamic range of pKa change ∼3.5 are expected to be generally valid, thus enabling the design of chemistry tools for biology research in the broadest sense. From a practical point of view, most relevant are two complementary fluorescent flipper probes that can be used to image the mechanics at the very beginning of endocytosis.

An Orthogonal Dynamic Covalent Chemistry Tool for Ring-Opening Polymerization of Cyclic Oligochalcogenides on Detachable Helical Peptide Templates.

A model system is introduced as a general tool to elaborate on orthogonal templation of dynamic covalent ring-opening polymerization (ODC-TROP). The tool consists of 310 helical peptides as unprecedented templates and semicarbazones as orthogonal dynamic covalent linkers. With difficult-to-control 1,2-dithiolanes, ODC-TROP on the level of short model oligomers occurs with high templation efficiency, increasing and diminishing upon helix stabilization and denaturation, respectively. Further, an anti-templated conjugate with mispositioned monomers gave reduced templation upon helix twisting. Even with the "unpolymerizable" 1,2-diselenolanes, initial studies already afford mild templation efficiency. These proof-of-principle results promise that the here introduced tool, recyclable and enabling late-stage side chain modification, will be useful to realize ODC-TROP of intractable or unknown cyclic dynamic covalent monomers for dynamer materials as well as cellular uptake and signaling applications.

Dynamic Covalent Michael Acceptors to Penetrate Cells: Thiol-Mediated Uptake with Tetrel-Centered Exchange Cascades, Assisted by Halogen-Bonding Switches.

Chalcogen-centered cascade exchange chemistry is increasingly understood to account for thiol-mediated uptake, that is, the ability of reversibly thiol-reactive agents to penetrate cells. Here, reversible Michael acceptors are shown to enable and inhibit thiol-mediated uptake, including the cytosolic delivery of proteins. Dynamic cyano-cinnamate dimers rival the best chalcogen-centered inhibitors. Patterns generated in inhibition heatmaps reveal contributions from halogen-bonding switches that occur independent from the thyroid transporter MCT8. The uniqueness of these patterns supports that the entry of tetrel-centered exchangers into cells differs from chalcogen-centered systems. These results expand the chemical space of thiol-mediated uptake and support the existence of a universal exchange network to bring matter into cells, abiding to be decoded for drug delivery and drug discovery in the broadest sense.

Photocleavable Fluorescent Membrane Tension Probes: Fast Release with Spatiotemporal Control in Inner Leaflets of Plasma Membrane, Nuclear Envelope, and Secretory Pathway.

Mechanosensitive flipper probes are attracting interest as fluorescent reporters of membrane order and tension in biological systems. We introduce PhotoFlippers, which contain a photocleavable linker and an ultralong tether between mechanophore and various targeting motifs. Upon irradiation, the original probe is released and labels the most ordered membrane that is accessible by intermembrane transfer. Spatiotemporal control from photocleavable flippers is essential to access open, dynamic or elusive membrane motifs without chemical or physical interference. For instance, fast release with light is shown to place the original small-molecule probes into the innermost leaflet of the nuclear envelope to image changes in membrane tension, at specific points in time of membrane trafficking along the secretory pathway, or in the inner leaflet of the plasma membrane to explore membrane asymmetry. These results identify PhotoFlippers as useful chemistry tools to enable research in biology.

Oligomers of Cyclic Oligochalcogenides for Enhanced Cellular Uptake.

Monomeric cyclic oligochalcogenides (COCs) are emerging as attractive transporters to deliver substrates of interest into the cytosol through thiol-mediated uptake. The objective of this study was to explore COC oligomers. We report a systematic evaluation of monomers, dimers, and trimers of asparagusic, lipoic, and diselenolipoic acid as well as their supramolecular monomers, dimers, trimers, and tetramers. COC dimers were more than twice as active as the monomers on both the covalent and noncovalent levels, whereas COC trimers were not much better than dimers. These trends might suggest that thiol-mediated uptake of COCs is synergistic over both short and long distances, that is, it involves more than two COCs and more than one membrane protein, although other interpretations cannot be excluded at this level of complexity. These results thus provide attractive perspectives for structural evolution as well as imminent use in practice. Moreover, they validate automated HC-CAPA as an invaluable method to collect comprehensive data on cytosolic delivery within a reasonable time at a level of confidence that is otherwise inconceivable.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Fluorescent Membrane Tension Probes for Early Endosomes.

Fluorescent flipper probes have been introduced recently to image membrane tension in live cells, and strategies to target these probes to specific membranes are emerging. In this context, early endosome (EE) targeting without the use of protein engineering is especially appealing because it translates into a fascinating transport problem. Weakly basic probes, commonly used to track the inside of acidic late endosomes and lysosomes, are poorly retained in EE because they are sufficiently neutralized in weakly acidic EE, thus able to diffuse out. Here, we disclose a rational strategy to target EE using a substituted benzylamine with a higher pKa value as a head group of the flipper probe. The resulting EE flippers are validated for preserved mechanosensitivity, ready for use in biology, particularly to elucidate the mechanics of endocytosis.

Oligonucleotide Phosphorothioates Enter Cells by Thiol-Mediated Uptake.

Oligonucleotide phosphorothioates (OPS) are DNA or RNA mimics where one phosphate oxygen is replaced by a sulfur atom. They have been shown to enter mammalian cells much more efficiently than non-modified DNA. Thus, solving one of the key challenges with oligonucleotide technology, OPS became very useful in practice, with several FDA-approved drugs on the market or in late clinical trials. However, the mechanism accounting for this facile cellular uptake is unknown. Here, we show that OPS enter cells by thiol-mediated uptake. The transient adaptive network produced by dynamic covalent pseudo-disulfide exchange is characterized in action. Inhibitors with nanomolar efficiency are provided, together with activators that reduce endosomal capture for efficient delivery of OPS into the cytosol, the site of action.

Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks.

In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin-biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin-biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin-biotin biotechnology in cellular uptake.