E‐cadherin mediates ultraviolet radiation‐ and calcium‐induced melanin transfer in human skin cells

Skin pigmentation is directed by epidermal melanin units, characterized by long‐lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X‐mediated control of MC filopodia. Both MC and KC express Ca²⁺‐dependent E‐cadherins. These homophilic adhesion contacts induce transient increases in intra‐KC Ca²⁺, while ultraviolet radiation (UVR) raises intra‐MC Ca²⁺ via calcium‐selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca²⁺ triggers melanin transfer remains unclear. Here we evaluated the role of E‐cadherin in UVR‐mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia‐associated E‐cadherin, Cdc42, VASP and β‐catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E‐cadherin revealed that this cadherin is essential for UVR‐induced MC filopodia formation and melanin transfer. Moreover, Ca²⁺ induced a dose‐dependent increase in filopodia formation and melanin transfer, as well as increased β‐catenin, Cdc42, Myosin X and E‐cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E‐cadherin, which are upregulated by intracellular (UVR‐stimulated) and extracellular Ca²⁺ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca²⁺ signalling influences human pigmentation.     

Disrupting 5-HT2A Receptor/PDZ Protein Interactions Reduces Hyperalgesia and Enhances SSRI Efficacy in Neuropathic Pain

Antidepressants are one of the first-line treatments for neuropathic pain. Despite the influence of serotonin (5-hydroxytryptamine, 5-HT) in pain modulation, selective serotonin reuptake inhibitors (SSRIs) are less effective than tricyclic antidepressants. Here, we show, in diabetic neuropathic rats, an alteration of the antihyperalgesic effect induced by stimulation of 5-HT_2A receptors, which are known to mediate SSRI-induced analgesia. 5-HT_2A receptor density was not changed in the spinal cord of diabetic rats, whereas postsynaptic density protein-95 (PSD-95), one of the PSD-95/disc large suppressor/zonula occludens-1 (PDZ) domain containing proteins interacting with these receptors, was upregulated. Intrathecal injection of a cell-penetrating peptidyl mimetic of the 5-HT_2A receptor C-terminus, which disrupts 5-HT_2A receptor–PDZ protein interactions, induced an antihyperalgesic effect in diabetic rats, which results from activation of 5-HT_2A receptors by endogenous 5-HT. The peptide also enhanced antihyperalgesia induced by the SSRI fluoxetine. Its effects likely resulted from an increase in receptor responsiveness, because it revealed functional 5-HT_2A receptor-operated Ca²⁺ responses in neurons, an effect mimicked by knockdown of PSD-95. Hence, 5-HT_2A receptor/PDZ protein interactions might contribute to the resistance to SSRI-induced analgesia in painful diabetic neuropathy. Disruption of these interactions might be a valuable strategy to design novel treatments for neuropathic pain and to increase the effectiveness of SSRIs.     

Hepatic Stellate Cells and Liver Retinoid Content in Alcoholic Liver Disease in Humans

Body retinoids are stored in the lipid droplets of hepatic stellate (Ito) cells. In chronic liver disease, the stellate cells differentiate into myo-fibroblast-like cells, a process whereby they lose their retinoid-con-taining lipid droplets. We studied the relation between liver retinoid content, the number of lipid droplets per stellate cell, and the number of stellate cells per mm² in human alcoholic liver disease. Semithin sections of liver biopsies from normal subjects and patients with early (steatosis, inflammation, and mild fibrosis) and late (cirrhosis and cirrhosis with acute alcoholic hepatitis) alcoholic liver disease were morphometrically evaluated. Liver retinoid content was determined by HPLC. In normal patients, liver retinoid content was 901 ± 213 lU/g of liver (mean ± SEM). There was a decrease in liver retinoid content in early alcoholic liver disease (409 ± 50 IU/g) and a further reduction in cirrhosis (153 ± 50 IU/g). In patients with acute alcoholic hepatitis, retinoid content was strikingly low (5.2 ± 1.8 IU/g). There was a progressive decrease in the number of stellate cells per mm² associated with progressive liver damage. We found a fair correlation between the number of stellate cells per mm² and liver retinoid content in all patient groups (overall correlation: 0.71). In normal subjects, the mean number of lipid droplets per stellate cell was 7.4 ± 0.7. In patients with early alcoholic liver disease and in patients with alcoholic cirrhosis, this value was increased to 13.6 ± 0.8 and 10.4 ± 2.0, respectively. In patients with acute alcoholic hepatitis, only a few lipid droplets were present (4.2 ± 0.5). There was a good correlation between liver retinoid content and mean number of lipid droplets in normal patients (r= 0.58). In alcoholic cirrhosis, however, correlation was poor (r= 0.34). In early alcoholic liver disease, the correlation was absent (r= 0.004). In conclusion, the major finding of our study is that the correlation between the mean number of lipid droplets per stellate cell and liver retinoid content varies according to the hepatic pathology considered. Marked lipid droplet accumulation occurs in stellate cells in early alcoholic liver disease and, to a lesser extent, in alcoholic cirrhosis, but there is no correlation between the mean number of lipid droplets per stellate cell and liver retinoid content. Therefore, not retinoids but probably lipids are responsible for the accumulation of lipid droplets. We also find that there is a fair correlation between the number of stellate cells per mm² and liver retinoid content in all patient groups. Finally, we confirm the decrease in hepatic retinoid content that occurs in alcoholic liver disease in humans, even at the early stages of the disease.     

Further delineation of the KBG syndrome caused by ANKRD11 aberrations

Loss-of-function variants in ANKRD11 were identified as the cause of KBG syndrome, an autosomal dominant syndrome with specific dental, neurobehavioural, craniofacial and skeletal anomalies. We present the largest cohort of KBG syndrome cases confirmed by ANKRD11 variants reported so far, consisting of 20 patients from 13 families. Sixteen patients were molecularly diagnosed by Sanger sequencing of ANKRD11, one familial case and three sporadic patients were diagnosed through whole-exome sequencing and one patient was identified through genomewide array analysis. All patients were evaluated by a clinical geneticist. Detailed orofacial phenotyping, including orthodontic evaluation, intra-oral photographs and orthopantomograms, was performed in 10 patients and revealed besides the hallmark feature of macrodontia of central upper incisors, several additional dental anomalies as oligodontia, talon cusps and macrodontia of other teeth. Three-dimensional (3D) stereophotogrammetry was performed in 14 patients and 3D analysis of patients compared with controls showed consistent facial dysmorphisms comprising a bulbous nasal tip, upturned nose with a broad base and a round or triangular face. Many patients exhibited neurobehavioural problems, such as autism spectrum disorder or hyperactivity. One-third of patients presented with (conductive) hearing loss. Congenital heart defects, velopharyngeal insufficiency and hip anomalies were less frequent. On the basis of our observations, we recommend cardiac assessment in children and regular hearing tests in all individuals with a molecular diagnosis of KBG syndrome. As ANKRD11 is a relatively common gene in which sequence variants have been identified in individuals with neurodevelopmental disorders, it seems an important contributor to the aetiology of both sporadic and familial cases.     

Perylene bisbenzimidazole nonlinear dielectric material for energy storage

A novel perylene bisbenzimidazole comprising both donor and acceptor functional groups was designed, synthesized, and characterized. This structure exhibits potentially useful physical properties, including a nonlinear dielectric response to an increasing electric field. This material can be used in energy storage devices as the dielectric part of a capacitor. Energy storage devices based on film capacitors are targeting applications in a wide range of industrial, residential and transportation systems.

We synthesized and characterized an organic molecule which can serve as a unit for high-density energy storage.

Capacitors are among the simplest devices that store electrical energy and feature high charge–discharge rates, high power density, and a wide range of operating temperatures.¹ As a result, they have found use in many electronic devices and in numerous industrial applications. However, while capacitors feature high power density, they do not compete with the high gravimetric energy density of batteries, precluding their use as long-term energy storage devices. This disadvantage originates from the low polarizability of conventional dielectric materials found in modern industrial capacitors. We are developing new dielectric materials with high polarizability, high resistivity, and high breakdown voltage. These materials will widen the scope of capacitor applications to a variety of energy storage devices including transportation, industry, and residential applications.For traditional materials, dielectric polarizability is a constant value with stored energy sharing a linear relationship to the dielectric permittivity of the material. At high electric fields, however, many materials exhibit a nonlinear dielectric constant.² Commonly, these materials consist of highly polarizable molecules with electron-donating and/or electron-withdrawing groups on opposite sides of a large conjugated core. These groups can increase polarizability and further impart directionality to the delocalized electrons when an electric field is applied.Here we present the general structure for a non-linear dielectric chromophore, that we named dielectrophore, that would bring the combination of required properties into capacitors: high permittivity, resistivity, and breakdown strength, as well as good processability and mechanical flexibility.³ The main three components that build these properties are: the polyaromatic core, allowing π–π stacking within the material as well as high polarizability, donor and acceptor groups at each end of the conjugated core increasing the first hyperpolarizability, and insulating subunits that prevent current leakage within the capacitor (Fig. 1). In such structures, the cores are predominantly planar polycyclic molecular system which forms column-like supramolecular stacks by π–π interactions.Open in a separate windowFig. 1General representation of an ideal dielectrophore, such as 15.Perylene-3,4,9,10-tetracarboxylic acid diimides (PDIs) are among the most extensively used cores for studies of π–π stacking and columnar liquid crystalline structures. It is noteworthy that PDI-based molecules have been widely used as colorants and dyes, as well as chromophores for optoelectronic applications such as photovoltaics and organic field effect transistors. This is due to the combination of their optoelectronic properties, such as large extinction coefficients, high fluorescent quantum yields, strong electron-accepting ability (N-type semiconductors), and high thermal and chemical stability.⁴ Most of these properties originate from the delocalization of the π electrons of the PDI''s. In addition, the modular structure and straightforward synthesis of PDI-based molecules streamline the modification of their structures, which helps to bring all the required properties of our dielectrophores.Our theoretical studies demonstrated that linear polarizability of the PDIs can be strongly increased by transforming the diimide functions into the more conjugated benzimidazole derivatives and adding donor (NEt₂) and acceptor groups (NO₂) at the opposite sides of a molecule.⁵ Considering the predicted design, we propose molecule 15 as our target molecule (Fig. 1). Long alkyl chains are added via phenyl linkers at both sides of the molecule and bring significant impact into the resistivity of the potential capacitor and help with processability during the synthetic manipulations and film coating.One of the main advantages of 15 is the modularity of its synthesis, which can be performed as a step-wise addition of two substituted o-phenylenediamines to 13 (Scheme 1). 13 was synthesized according to a modified procedure reported by Xiao and co-workers (outlined in Scheme 1).⁶ In our synthesis, 3 is accessed through a 2-step derivatization of catechol (1) through an alkylation followed by a C–H activated borylation.⁷ The electron-acceptor diamine (7) was synthesized from 2,5-dinitroaniline (4) via bromination with NBS, followed by Suzuki coupling with 6, and further reduction of one of the nitro groups with ammonium sulfide. The electron donating diamine (12) was synthesized from 3-bromo-2,5-difluoronitrobenzene (8) via nucleophilic aromatic substitution at room temperature, followed by Suzuki coupling with 3 to isolate 10. A nucleophilic aromatic substitution with benzylamine at elevated temperature yields 11 and a deprotection/reduction using Pd(OH)₂/C and H₂ at 55 psi afforded 12. 14 was obtained in two steps by stirring diamine 7 with 13 in molten imidazole at 140 °C for 12 hours then further treating the product with an excess of para-toluenesulfonic acid in toluene at 100 °C to afford 14 in 37% overall yield (Scheme 1).Open in a separate windowScheme 1Synthesis of 15.15 was made through the condensation of mono-anhydride 14 with diamine 12 with the presence of zinc acetate in quinoline at 140 °C for 12 hours. Upon precipitation into methanol, 15 was isolated as a dark purple solid. It is important to mention that reactions of 13 with 7 and then 14 with 12 are likely not regioselective, even though steric factors favour the formation of the isomers 15a and 15b (Scheme 2) with donor/acceptor groups located farther from carboxamide group, especially in the case of the bulkier diethylamino group. Indeed, six additional isomers may be present, in addition to 15a and 15b (15c–h, see ESI^†). Column chromatography to separate these regioisomeric mixtures is shown to be quite challenging.⁸ Considering our theoretical studies, that demonstrated both syn- and anti-isomers to have similar predicted polarization values, separation of individual isomers is unnecessary.⁵Open in a separate windowScheme 2Main isomers of 15.15 was characterized with UV-vis and FTIR spectroscopic techniques and molecular weight was confirmed through mass spectrometry (see ESI^†). FTIR of 15 shows the absence of the anhydride carbonyl stretches of 14 at 1769 and 1732 cm⁻¹ respectively and the emergence of benzimidazole stretches at 1691, 1592, and 1573 cm⁻¹.⁹¹H NMR of 15 in 100% CDCl₃ initially exhibit very broad signals, but when 10% dTFA is added this aggregation is broken up (see ESI^†). The formation of the benzimidazole extends the length of conjugation considerably, this transformation can be clearly observed in the UV-vis spectra where the λ_max shifts from 523 nm for 13 to 555 nm for 14 (ESI^†). The λ_max shifts even further towards the near infrared region (752 nm) upon addition of the donor block (12) and making 15 (Fig. 2A). The drastic broadening of the band from 500 to 900 nm for 15 is caused by the extended conjugation, the formation of π–π-aggregates in the solution and the presence of a mixture of isomers,^8a as well as intramolecular charge transfer (ICT) in this push–pull system.¹⁰ The UV-vis spectrum in chloroform was predicted using a B3LYP/6-31H level of theory and compared with the experimental data (see ESI^†). The computed spectrum identifies three major bands at 409, 552, and 850 nm, respectively, in some agreement with the observed λ_max at 395, 525, and 752 nm (ESI^†). 15 was then cast into films on the Indium–Tin–Oxide (ITO)/glass substrate (see ESI^†) and charged through corona poling to test its nonlinear behaviour in the presence of an electric field.Open in a separate windowFig. 2(A) UV-vis absorption spectra of intermediates 13 (solid red), and 14 (dashed green), and final perylene-bisbenzimidazole 15 (dash-dot blue) (0.05 mg mL⁻¹ in CHCl₃). (B) Corona charging capacitor experiment of films cast from polypropylene (PP, dash black) and 15 (dash-dot blue).In corona poling, a sharp corona tip is charged up to several kilovolts until the electric breakdown of surrounding atmosphere occurs and the positive or negative ions are deposited on the film surface.¹¹ With this approach, a large electric field necessary to study the nonlinear response of the material is achieved. Once charge is deposited we used a Kelvin probe to measure the surface potential and its dependence on the amount of deposited charges, which can be determined by measuring the current from the bottom ITO electrode to the ground. This current is directly proportional to the voltage of the corona electrode, as expected, and is controlled by the corona current setting. It is then a simple calculation to convert the time under the corona charge deposition into the charge density on the top of the film. In our experiment, we use the current of 10⁻⁵ A and the area of the film is 11.5 × 6 cm².The results of the corona experiment are shown in Fig. 2B for the 2.165 μm thickness film of 15 and 6 μm polypropylene (PP) thin film for the comparison. PP is a common material used as a dielectric in many high voltage capacitors and shows a usual linear relationship between increasing charge density and a growing electric field (Fig. 2B). The dielectric constant of PP is estimated through this approach to be 2.9, close to the tabulated value.¹² When 15 is exposed to the same conditions, we observe a nonlinear voltage saturation at an electric field of 40 V μm⁻¹. We can conclude that in such nonlinear regime the deposition of additional charge leads to the increase of the polarization, not to the increase of the electric field. It should be emphasized that in PP films the electric breakdown occurs after about 10 seconds of the charge deposition, while 15 can sustain up to 5 hours at the same conditions. Correspondingly, the energy stored in the capacitor based on our molecule would be larger than that of the polypropylene-based capacitor by several orders of the magnitude.