Hepatomegaly and Splenomegaly: An Approach to the Diagnosis of Lysosomal Storage Diseases.

Clinical findings of hepatomegaly and splenomegaly, the abnormal enlargement of the liver and spleen, respectively, should prompt a broad differential diagnosis that includes metabolic, congestive, neoplastic, infectious, toxic, and inflammatory conditions. Among the metabolic diseases, lysosomal storage diseases (LSDs) are a group of rare and ultrarare conditions with a collective incidence of 1 in 5000 live births. LSDs are caused by genetic variants affecting the lysosomal enzymes, transporters, or integral membrane proteins. As a result, abnormal metabolites accumulate in the organelle, leading to dysfunction. Therapeutic advances, including early diagnosis and disease-targeted management, have improved the life expectancy and quality of life of people affected by certain LSDs. To access these new interventions, LSDs must be considered in patients presenting with hepatomegaly and splenomegaly throughout the lifespan. This review article navigates the diagnostic approach for individuals with hepatosplenomegaly particularly focusing on LSDs. We provide hints in the history, physical exam, laboratories, and imaging that may identify LSDs. Additionally, we discuss molecular testing, arguably the preferred confirmatory test (over biopsy), accompanied by enzymatic testing when feasible.

Cardiac sodium channel inhibition by lamotrigine: In vitro characterization and clinical implications.

Lamotrigine, approved for use as an antiseizure medication as well as the treatment of bipolar disorder, inhibits sodium channels in the brain to reduce repetitive neuronal firing and pathological release of glutamate. The shared homology of sodium channels and lack of selectivity associated with channel blocking agents can cause slowing of cardiac conduction and increased proarrhythmic potential. The Vaughan-Williams classification system differentiates sodium channel blockers using biophysical properties of binding. As such, Class Ib inhibitors, including mexiletine, do not slow cardiac conduction as measured by the electrocardiogram, at therapeutically relevant exposure. Our goal was to characterize the biophysical properties of NaV 1.5 block and to support the observed clinical safety of lamotrigine. We used HEK-293 cells stably expressing the hNaV 1.5 channel and voltage clamp electrophysiology to quantify the potency (half-maximal inhibitory concentration) against peak and late channel current, on-/off-rate binding kinetics, voltage-dependence, and tonic block of the cardiac sodium channel by lamotrigine; and compared to clinically relevant Class Ia (quinidine), Ib (mexiletine), and Ic (flecainide) inhibitors. Lamotrigine blocked peak and late NaV 1.5 current at therapeutically relevant exposure, with rapid kinetics and biophysical properties similar to the class Ib inhibitor mexiletine. However, no clinically meaningful prolongation in QRS or PR interval was observed in healthy subjects in a new analysis of a previously reported thorough QT clinical trial (SCA104648). In conclusion, the weak NaV 1.5 block and rapid kinetics do not translate into clinically relevant conduction slowing at therapeutic exposure and support the clinical safety of lamotrigine in patients suffering from epilepsy and bipolar disorder.

Discrete Open-Shell Tris(bipyridinium radical cationic) Inclusion Complexes in the Solid State.

The solid-state properties of organic radicals depend on radical-radical interactions that are influenced by the superstructure of the crystalline phase. Here, we report the synthesis and characterization of a substituted tetracationic cyclophane, cyclobis(paraquat-p-1,4-dimethoxyphenylene), which associates in its bisradical dicationic redox state with the methyl viologen radical cation (MV•+) to give a 1:1 inclusion complex. The (super)structures of the reduced cyclophane and this 1:1 complex in the solid state deviate from the analogous (super)structures observed for the reduced state of cyclobis(paraquat-p-phenylene) and that of its trisradical tricationic complex. Titration experiments reveal that the methoxy substituents on the p-phenylene linkers do not influence binding of the cyclophane toward small neutral guests-such as dimethoxybenzene and tetrathiafulvalene-whereas binding of larger radical cationic guests such as MV•+ by the reduced cyclophane decreases 10-fold. X-ray diffraction analysis reveals that the solid-state superstructure of the 1:1 complex constitutes a discrete entity with weak intermolecular orbital overlap between neighboring complexes. Transient nutation EPR experiments and DFT calculations confirm that the complex has a doublet spin configuration in the ground state as a result of the strong orbital overlap, while the quartet-state spin configuration is higher in energy and inaccessible at ambient temperature. Superconducting quantum interference device (SQUID) measurements reveal that the trisradical tricationic complexes interact antiferromagnetically and form a one-dimensional Heisenberg antiferromagnetic chain along the a-axis of the crystal. These results offer insights into the design and synthesis of organic magnetic materials based on host-guest complexes.

Precise Tuning of Cortical Contractility Regulates Cell Shape during Cytokinesis.

The mechanical properties of the actin cortex regulate shape changes during cell division, cell migration, and tissue morphogenesis. We show that modulation of myosin II (MII) filament composition allows tuning of surface tension at the cortex to maintain cell shape during cytokinesis. Our results reveal that MIIA generates cortex tension, while MIIB acts as a stabilizing motor and its inclusion in MII hetero-filaments reduces cortex tension. Tension generation by MIIA drives faster cleavage furrow ingression and bleb formation. We also show distinct roles for the motor and tail domains of MIIB in maintaining cytokinetic fidelity. Maintenance of cortical stability by the motor domain of MIIB safeguards against shape instability-induced chromosome missegregation, while its tail domain mediates cortical localization at the terminal stages of cytokinesis to mediate cell abscission. Because most non-muscle contractile systems are cortical, this tuning mechanism will likely be applicable to numerous processes driven by myosin-II contractility.

Organic Counteranion Co-assembly Strategy for the Formation of γ-Cyclodextrin-Containing Hybrid Frameworks.

A class of γ-cyclodextrin-containing hybrid frameworks (CD-HFs) has been synthesized, employing γ-cyclodextrin (γ-CD) as the primary building blocks, along with 4-methoxysalicylate (4-MS-) anions as the secondary building blocks. CD-HFs are constructed through the synergistic exploitation of coordinative, electrostatic, and dispersive forces. The syntheses have been carried out using an organic counteranion co-assembly strategy, which allows for the introduction of 4-MS-, in place of inorganic OH-, into the cationic γ-CD-containing metal-organic frameworks (CD-MOFs). Although the packing arrangement of the γ-CD tori in the solid-state superstructure of CD-HFs is identical to that of the previously reported CD-MOFs, CD-HFs crystallize with lower symmetry and in the cuboid space group P43212-when compared to CD-MOF-1, which has the cubic unit cell of I432 space group-on account of the chiral packing of the 4-MS- anions in the CD-HF superstructures. Importantly, CD-HFs have ultramicroporous apertures associated with the pore channels, a significant deviation from CD-MOF-1, as a consequence of the contribution from the 4-MS- anions, which serve as supramolecular baffles. In gas adsorption-desorption experiments, CD-HF-1 exhibits a Brunauer-Emmett-Teller (BET) surface area of 306 m2 g-1 for CO2 at 195 K, yet does not uptake N2 at 77 K, confirming the difference in porosity between CD-HF-1 and CD-MOF-1. Furthermore, the 4-MS- anions in CD-HF-1 can be exchanged with OH- anions, leading to an irreversible single-crystal to single-crystal transformation, with rearrangement of coordinated metal ions. Reversible transformations were also observed in CD-MOF-1 when OH- ions were exchanged for 4-MS- anions, with the space group changing from I432 to R32. This organic counteranion co-assembly strategy opens up new routes for the construction of hybrid frameworks, which are inaccessible by existing de novo MOF assembly methodologies.

Characterization of Spatially Graded Biomechanical Scaffolds.

Advances in fabrication have allowed tissue engineers to better mimic complex structures and tissue interfaces by designing nanofibrous scaffolds with spatially graded material properties. However, the nonuniform properties that grant the desired biomechanical function also make these constructs difficult to characterize. In light of this, we developed a novel procedure to create graded nanofibrous scaffolds and determine the spatial distribution of their material properties. Multilayered nanofiber constructs were synthesized, controlling spatial gradation of the stiffness to mimic the soft tissue gradients found in tendon or ligament tissue. Constructs were characterized using uniaxial tension testing with digital image correlation (DIC) to measure the displacements throughout the sample, in a noncontacting fashion, as it deformed. Noise was removed from the displacement data using principal component analysis (PCA), and the final denoised field served as the input to an inverse elasticity problem whose solution determines the spatial distribution of the Young's modulus throughout the material, up to a multiplicative factor. Our approach was able to construct, characterize, and determine the spatially varying moduli, in four electrospun scaffolds, highlighting its great promise for analyzing tissues and engineered constructs with spatial gradations in modulus, such as those at the interfaces between two disparate tissues (e.g., myotendinous junction, tendon- and ligament-to-bone entheses).

Cyclotris(paraquat-p-phenylenes).

Reported here is the synthesis, solid-state characterization, and redox properties of new triangular, threefold symmetric, viologen-containing macrocycles. Cyclotris(paraquat-p-phenylene) (CTPQT6+ ) and cyclotris(paraquat-p-1,4-dimethoxyphenylene) (MCTPQT6+ ) were prepared and their X-ray single-crystal (super)structures reveal intricate three-dimensional packing. MCTPQT6+ results in nanometer-sized channels, in contrast with its parent counterpart CTPQT6+ which crystallizes as a couple of polymorphs in the form of intercalated assemblies. In the solid state, MCTPQT3(.+) exhibits stacks between the 1,4-dimethoxyphenylene and bipyridinium radical cations, providing new opportunities for the manipulation and control of the recognition motif associated with viologen radical cations. These redox-active cyclophanes demonstrate that geometry-matching and weak intermolecular interactions are of paramount importance in dictating the formation of their intricate solid-state superstructures.

A Supramolecular Approach for Modulated Photoprotection, Lysosomal Delivery, and Photodynamic Activity of a Photosensitizer.

Prompted by a knowledge of the photoprotective mechanism operating in photosystem supercomplexes and bacterial antenna complexes by pigment binding proteins, we have appealed to a boxlike synthetic receptor (ExBox·4Cl) that binds a photosensitizer, 5,15-diphenylporphyrin (DPP), to provide photoprotection by regulating light energy. The hydrophilic ExBox4+ renders DPP soluble in water and modulates the phototoxicity of DPP by trapping it in its cavity and releasing it when required. While trapping removes access to the DPP triplet state, a pH-dependent release of diprotonated DPP (DPPH22+) restores the triplet deactivation pathway, thereby activating its ability to generate reactive oxygen species. We have employed the ExBox4+-bound DPP complex (ExBox4+⊃DPP) for the safe delivery of DPP into the lysosomes of cancer cells, imaging the cells by utilizing the fluorescence of the released DPPH22+ and regulating photodynamic therapy to kill cancer cells with high efficiency.

Anion carriers as potential treatments for cystic fibrosis: transport in cystic fibrosis cells, and additivity to channel-targeting drugs.

Defective anion transport is a hallmark of the genetic disease cystic fibrosis (CF). One approach to restore anion transport to CF cells utilises alternative pathways for transmembrane anion transport, including artificial anion carriers (anionophores). Here, we screened 22 anionophores for biological activity using fluorescence emission from the halide-sensitive yellow fluorescent protein. Three compounds possessed anion transport activity similar to or greater than that of a bis-(p-nitrophenyl)ureidodecalin previously shown to have promising biological activity. Anion transport by these anionophores was concentration-dependent and persistent. All four anionophores mediated anion transport in CF cells, and their activity was additive to rescue of the predominant disease-causing variant F508del-CFTR using the clinically-licensed drugs lumacaftor and ivacaftor. Toxicity was variable but minimal at the lower end. The results provide further evidence that anionophores, by themselves or together with other treatments that restore anion transport, offer a potential therapeutic strategy for CF.

Nanopore Detection of Single-Molecule Binding within a Metallosupramolecular Cage.

Guest encapsulation is a fundamental property of coordination cages. However, there is a paucity of methods capable of quantifying the dynamics of guest binding processes. Here, we demonstrate nanopore detection of single-molecule binding within metallosupramolecular cages. Real-time monitoring of the ion current flowing through a transmembrane α-hemolysin nanopore resolved the binding of different guests to both cage enantiomers. This enabled the single-molecule kinetics of guest binding to be quantified, whereas the ordering and durations of events were consistent with a guest-exchange mechanism that does not involve ligand dissociation. In addition to providing a new approach for single-molecule interrogation of dynamic supramolecular processes, this work also establishes that cage complexes which are too large to enter the nanopore can be exploited for detecting small molecules, thus constituting a new class of molecular adapter.

The Role of Terminal Functionality in the Membrane and Antibacterial Activity of Peptaibol-Mimetic Aib Foldamers.

Peptaibols are peptide antibiotics that typically feature an N-terminal acetyl cap, a C-terminal aminoalcohol, and a high proportion of α-aminoisobutyric acid (Aib) residues. To establish how each feature might affect the membrane-activity of peptaibols, biomimetic Aib foldamers with different lengths and terminal groups were synthesised. Vesicle assays showed that long foldamers (eleven Aib residues) with hydrophobic termini had the highest ionophoric activity. C-terminal acids or primary amides inhibited activity, while replacement of an N-terminal acetyl with an azide group made little difference. Crystallography showed that N3 Aib11 CH2 OTIPS folded into a 310 helix 2.91 nm long, which is close to the bilayer hydrophobic width. Planar bilayer conductance assays showed discrete ion channels only for N-acetylated foldamers. However long foldamers with hydrophobic termini had the highest antibacterial activity, indicating that ionophoric activity in vesicles was a better indicator of antibacterial activity than the observation of discrete ion channels.

Discrimination of supramolecular chirality using a protein nanopore.

Supramolecular chirality may emerge from self-assembly processes to yield architectures that differ only in the topological arrangement of their constituent parts. Since the properties of the resulting enantiomeric assemblies are identical, purification and characterisation can be challenging. Here, we have examined the hypothesis that the intrinsic chirality of a protein nanopore can be exploited to detect supramolecular chirality. Transient blockages in the ion current flowing through a single membrane-spanning α-haemolysin nanopore were shown to discriminate between M4L6 tetrahedral coordination cages of opposing chiralities. The single-molecule nature of the approach facilitated direct access to the rates of association and dissociation with the nanopore, which allowed the concentrations of the enantiomeric supramolecular assemblies to be determined in situ. Thus, we have established that a protein nanopore can be used to discriminate the chiral topologies of supramolecular assemblies, even when they are too large to fully enter the nanopore.

Survey of Physicians' Understanding of Specific Risks Associated with Retigabine.

BACKGROUND: Following reports of discoloration, including retinal pigmentation, in addition to known significant risks of urinary retention, central nervous system effects, and QTc prolongation, the retigabine indication was restricted to adjunctive treatment of partial onset seizures where other appropriate drug combinations have proved inadequate or have not been tolerated. OBJECTIVE: To ascertain the effectiveness of educational initiatives as reflected in physicians' understanding of retigabine-associated risks, management, and patient selection. METHODOLOGY: An online, cross-sectional survey, designated a post-authorization safety study (24/9/2014-30/1/2015), recruited retigabine prescribers (RP) and retigabine non-prescribers (RNP) in seven countries, who had been sent a retigabine Dear Health Care Professional letter (June 2013). Questions tested understanding of the significant risks associated with retigabine. RESULTS: 414/467 participants completed all questions (RP, n = 141; RNP, n = 273) and were included in the analysis. 74.2 % of these participants (RP, 77.3 %; RNP, 72.5 %) correctly identified the label indication. 81.9 % of participants (RP, 86.5 %; RNP, 79.5 %) recognized that specific retigabine-associated risks included pigment changes of ocular tissues, including the retina. 81.6 % of participants (RP, 87.2 %; RNP, 78.8 %) recognized that a comprehensive ophthalmologic examination is required. 99.8 % of participants (RP, 100.0 %; RNP, 99.6 %) acknowledged the requirement for action in case of retinal pigmentation or vision changes. RP and RNP results were similar to the overall participants' analysis, with a trend toward stronger understanding among RP. CONCLUSION: Most participants recognized the appropriate population for retigabine treatment and the requirement to monitor for adverse events including retinal pigmentation and vision changes. Understanding was satisfactory among RNP but stronger among RP.

The influence of specimen thickness and alignment on the material and failure properties of electrospun polycaprolactone nanofiber mats.

Electrospinning is a versatile fabrication technique that has been recently expanded to create nanofibrous structures that mimic ECM topography. Like many materials, electrospun constructs are typically characterized on a smaller scale, and scaled up for various applications. This established practice is based on the assumption that material properties, such as toughness, failure stress and strain, are intrinsic to the material, and thus will not be influenced by specimen geometry. However, we hypothesized that the material and failure properties of electrospun nanofiber mats vary with specimen thickness. To test this, we mechanically characterized polycaprolactone (PCL) nanofiber mats of three different thicknesses in response to constant rate elongation to failure. To identify if any observed thickness-dependence could be attributed to fiber alignment, such as the effects of fiber reorientation during elongation, these tests were performed in mats with either random or aligned nanofiber orientation. Contrary to our hypothesis, the failure strain was conserved across the different thicknesses, indicating similar maximal elongation for specimens of different thickness. However, in both the aligned and randomly oriented groups, the ultimate tensile stress, short-range modulus, yield modulus, and toughness all decreased with increasing mat thickness, thereby indicating that these are not intrinsic material properties. These findings have important implications in engineered scaffolds for fibrous and soft tissue applications (e.g., tendon, ligament, muscle, and skin), where such oversights could result in unwanted laxity or reduced resistance to failure. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2794-2800, 2016.

Sequential gelation of tyramine-substituted hyaluronic acid hydrogels enhances mechanical integrity and cell viability.

Tyramine-substituted hyaluronic acid (HA-Tyr) hydrogels formed by the oxidative coupling reaction of hydrogen peroxide (H2O2) and horseradish peroxidase (HRP) have been used for cellular encapsulation and protein delivery. Crosslinking density and gelation time can be tuned by altering the H2O2 and HRP concentrations. Previous studies using HA-Tyr constructs report significant mechanical degradation after 21 days of culture. In this work, exogenous supplementation of HRP after initial gelation resulted in superior mechanical properties in acellular hydrogels and improved viability and proliferation in cell-laden constructs. Swelling of the acellular hydrogels was prevented in the samples receiving exogenous HRP. Monolayer studies showed no negative effect of relevant HRP concentrations on the viability of human adipose-derived stem cells (hASCs) and improved the viability of hASCs cultured with HRP and H2O2 compared to H2O2 alone. Taken together, this study demonstrates that HA-Tyr hydrogel properties could be modified by exogenous supplementation of HRP to tune scaffold degradation and improve cell viability by mitigating the negative effects of oxidative stress.

Health-related quality of life in Parkinson's: impact of 'off' time and stated treatment preferences.

PURPOSE: Long-term levodopa therapy and related fluctuating plasma concentrations are associated with between-dose periods of 'off time' resulting in substantial variation in symptoms and functioning throughout the day in people with Parkinson's (PwP). METHODS: PwP across UK, France, Spain and Italy completed an online survey to explore: the impact of 'off time' on (1) health-related quality of life (HRQL) and (2) on functioning and ability to undertake usual activities; (3) the value of 'off time' relative to other factors associated with Parkinson's through a stated preference discrete choice experiment (SPDCE). RESULTS: In total, 305 PwP completed the online survey. Overall mean HRQL (utility) score was significantly lower for 'off time' (0.37) than for 'on time' (0.60). All attributes within the SPDCE were significant predictors of treatment choice, although increased duration of 'on time' (per hour per day: odds ratio (OR) = 1.40) and predictability of 'off time' to within 30 min (OR = 1.42) were valued most highly. CONCLUSIONS: 'On time' and predictability of 'off time' are highly valued by PwP. Due to substantial diurnal variation of Parkinson's symptoms, standard patient-reported outcome (PRO) assessments may not adequately capture the impact of 'off time' on HRQL and participation in daily activities.

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia.

Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.

A flexible solution to anion transport: powerful anionophores based on a cyclohexane scaffold.

Transmembrane anion carriers (anionophores) have potential for biological activity, including the treatment of channelopathies such as cystic fibrosis. A new family of anionophores has been synthesized, in which three thiourea groups are mounted on a cyclohexane-based scaffold. Though conceptually related to earlier polycyclic systems, these molecules are simpler and far more accessible. Preorganization is somewhat reduced compared to earlier systems, and anion affinities are correspondingly lower. However, transport activities set new records. This surprising performance suggests a role for controlled flexibility in the design of transmembrane anion carriers.

Hybrid hyaluronic acid hydrogel/poly(ɛ-caprolactone) scaffold provides mechanically favorable platform for cartilage tissue engineering studies.

Hybrid scaffolds for cartilage tissue engineering provide the potential for high stiffness properties in tension and compression while exhibiting the viscoelastic response found in hydrogels and native cartilage tissue. We investigate the impact of a hybrid scaffold fabricated from a hyaluronic acid (HA)-based hydrogel combined with porous poly(ε-caprolactone) (PCL) material formed by a particulate leaching method to study dedifferentiated chondrocyte response. The material properties of the hybrid scaffold showed mean Young's moduli in tension which were similar to human articular cartilage but not statistically different between the hybrid and porous PCL scaffolds at 2.02 and 2.07 MPa, respectively. For both the hybrid and porous PCL control scaffolds in compression at low loading frequencies (<1 Hz) and 10% strain peak amplitude the Young's moduli are not statistically distinct. However, at frequencies in the range of normal human gait from 1 to2 Hz, hybrid scaffolds exhibit significantly (p < 0.01) increased loss moduli indicating additional contribution of the viscous phase to stiffness. Dedifferentiated chondrocytes seeded onto the scaffolds exhibited a rounded morphology in hybrid scaffolds however ECM protein expression levels of collagen type I, collagen type II, and aggrecan are not different from the PCL control scaffolds. These results provide a model platform to investigate cell response to mechanical and chemical cues in a hybrid scaffold system with mechanical properties similar to human cartilage that does not contribute to differentiation in order to identify the appropriate design and development parameters to promote formation of extracellular matrix and investigate chondrocyte scaffold interactions.

Resolution of sleepiness and fatigue: a comparison of bupropion and selective serotonin reuptake inhibitors in subjects with major depressive disorder achieving remission at doses approved in the European Union.

Unlike selective serotonin reuptake inhibitors (SSRIs), bupropion may be classified as a dual noradrenaline and dopamine reuptake inhibitor, a difference with potential implications for the treatment of residual sleepiness and fatigue in major depressive disorder (MDD). Post-hoc analysis of subjects with remitted MDD was performed on data pooled from six double-blind, randomized trials comparing the European Union (EU)-approved dose of ≤300 mg/day bupropion with SSRIs (sertraline, paroxetine or escitalopram) for the resolution of sleepiness and fatigue. Hypersomnia score was defined as the sum of scores of the Hamilton Depression Rating Scale (HDRS) items 22, 23, and 24; fatigue score as HDRS item 13 score; and remission as HDRS-17≤7. Similar proportions of bupropion- and SSRI-treated subjects achieved remission at study endpoint (169/343, 49.3% vs 324/656, 49.4%; last observation carried forward (LOCF), p=0.45). Fewer bupropion-treated remitters had residual symptoms of sleepiness (32/169, 18.9% vs 104/324, 32.1%; p<0.01) and fatigue (33/169, 19.5% vs 98/324, 30.2%; p<0.05). Bupropion-treated remitters also showed greater improvement (mean change from baseline) in sleepiness (p<0.05) and fatigue scores (p<0.01) at endpoint: benefits were evident from week 2 for sleepiness (p<0.01) and week 4 for fatigue (p<0.01). Bupropion treatment at the EU-approved dose of ≤300 mg/day may offer advantages over SSRIs in the resolution of sleepiness and fatigue in remitted MDD patients.