Direct assessment of individual skin barrier components by electrical impedance spectroscopy

Background: Expression of the tight junction proteins Cldn1 and 4 is altered in skin diseases such as atopic dermatitis, and Cldn1 deficiency affects skin barrier formation. Impedance spectroscopy (IS) has been proven to allow detection of alterations in the skin barrier but is currently unable to separate effects on viable epidermis (VE) and stratum corneum (SC). Methods: Effects of siRNA-mediated Cldn1 and 4 knockdown in reconstructed human epidermis (RHE) on VE and SC barrier function were investigated with Ussing chamber-based IS. Barrier components were sequentially altered, employing iron oxide nanoparticles and EGTA, to identify their contribution to the impedance spectrum. Resistance changes due to apically applied hyperosmolar electrolyte were used to identify barrier defects non-invasively. Results: IS of RHE yielded two relaxation frequencies, representing the barrier properties of the SC (similar to 1000 Hz) and VE (similar to 100 Hz). As proof of concept, it was shown that the Cldn1 knockdown-induced resistance drop arises from the impairment of both SC and VE, indicated by a shift of both relaxation frequencies. Hyperosmolar electrolyte penetration allowed non-invasive detection of Cldn1 knockdown via time-dependent frequency shifts. The absence of Cldn4 knockdown-induced changes revealed the weaknesses of transepithelial electrical resistance analysis. Conclusion: In conclusion, the present technique allows to separately measure the barrier properties of SC and VE and further evaluate the Cldn1 and 4 knockdown impact on the skin barrier. As the measurement with agarose-embedded electrolyte allowed non-invasive identification of the Cldn1 knockdown, this opens the way to detailed in vivo skin barrier assessment

The Post-Apoptotic Fate of RNAs Identified Through High-Throughput Sequencing of Human Hair

The hair of all mammals consists of terminally differentiated cells that undergo a specialized form of apoptosis called cornification. While DNA is destroyed during cornification, the extent to which RNA is lost is unknown. Here we find that multiple types of RNA are incompletely degraded after hair shaft formation in both mouse and human. Notably, mRNAs and short regulatory microRNAs (miRNAs) are stable in the hair as far as 10 cm from the scalp. To better characterize the post-apoptotic RNAs that escape degradation in the hair, we performed sequencing (RNA-seq) on RNA isolated from hair shafts pooled from several individuals. This hair shaft RNA library, which encompasses different hair types, genders, and populations, revealed 7,193 mRNAs, 449 miRNAs and thousands of unannotated transcripts that remain in the post-apoptotic hair. A comparison of the hair shaft RNA library to that of viable keratinocytes revealed surprisingly similar patterns of gene coverage and indicates that degradation of RNA is highly inefficient during apoptosis of hair lineages. The generation of a hair shaft RNA library could be used as months of accumulated transcriptional history useful for retrospective detection of disease, drug response and environmental exposure

A Search for Variability in Exoplanet Analogues and Low-Gravity Brown Dwarfs

We report the results of a $J$-band survey for photometric variability in a sample of young, low-gravity objects using the New Technology Telescope (NTT) and the United Kingdom InfraRed Telescope (UKIRT). Surface gravity is a key parameter in the atmospheric properties of brown dwarfs and this is the first large survey that aims to test the gravity dependence of variability properties. We do a full analysis of the spectral signatures of youth and assess the group membership probability of each target using membership tools from the literature. This results in a 30 object sample of young low-gravity brown dwarfs. Since we are lacking in objects with spectral types later than L9, we focus our statistical analysis on the L0-L8.5 objects. We find that the variability occurrence rate of L0-L8.5 low-gravity brown dwarfs in this survey is $30^{+16}_{-8}\%$. We reanalyse the results of Radigan 2014 and find that the field dwarfs with spectral types L0-L8.5 have a variability occurrence rate of $11^{+13}_{-4}\%$. We determine a probability of $98\%$ that the samples are drawn from different distributions. This is the first quantitative indication that the low-gravity objects are more likely to be variable than the field dwarf population. Furthermore, we present follow-up $J_S$ and $K_S$ observations of the young, planetary-mass variable object PSO 318.5-22 over three consecutive nights. We find no evidence of phase shifts between the $J_S$ and $K_S$ bands and find higher $J_S$ amplitudes. We use the $J_S$ lightcurves to measure a rotational period of $8.45\pm0.05~$hr for PSO 318.5-22.Comment: accepted for publication in MNRA

Variability in a young, L/T transition planetary-mass object

As part of our ongoing NTT SoFI survey for variability in young free-floating planets and low-mass brown dwarfs, we detect significant variability in the young, free-floating planetary-mass object PSO J318.5-22, likely due to rotational modulation of inhomogeneous cloud cover. A member of the 23 ± 3 Myr β Pic moving group, PSO J318.5-22 has Teff = K and a mass estimate of 8.3 ± 0.5 MJup for a 23 ± 3 Myr age. PSO J318.5-22 is intermediate in mass between 51 Eri b and β Pic b, the two known exoplanet companions in the β Pic moving group. With variability amplitudes from 7% to 10% in JS at two separate epochs over 3-5 hr observations, we constrain the rotational period of this object to >5 hr. In KS, we marginally detect a variability trend of up to 3% over a 3 hr observation. This is the first detection of weather on an extrasolar planetary-mass object. Among L dwarfs surveyed at high photometric precision (<3%), this is the highest amplitude variability detection. Given the low surface gravity of this object, the high amplitude preliminarily suggests that such objects may be more variable than their high-mass counterparts, although observations of a larger sample are necessary to confirm this. Measuring similar variability for directly imaged planetary companions is possible with instruments such as SPHERE and GPI and will provide important constraints on formation. Measuring variability at multiple wavelengths can help constrain cloud structure.Peer reviewe

Contamination of personal protective equipment during COVID-19 autopsies

Confronted with an emerging infectious disease at the beginning of the COVID-19 pandemic, the medical community faced concerns regarding the safety of autopsies on those who died of the disease. This attitude has changed, and autopsies are now recognized as indispensable tools for understanding COVID-19, but the true risk of infection to autopsy staff is nevertheless still debated. To clarify the rate of SARS-CoV-2 contamination in personal protective equipment (PPE), swabs were taken at nine points in the PPE of one physician and one assistant after each of 11 full autopsies performed at four centers. Swabs were also obtained from three minimally invasive autopsies (MIAs) conducted at a fifth center. Lung/bronchus swabs of the deceased served as positive controls, and SARS-CoV-2 RNA was detected by real-time RT-PCR. In 9 of 11 full autopsies, PPE samples tested RNA positive through PCR, accounting for 41 of the 198 PPE samples taken (21%). The main contaminated items of the PPE were gloves (64% positive), aprons (50% positive), and the tops of shoes (36% positive) while the fronts of safety goggles, for example, were positive in only 4.5% of the samples, and all the face masks were negative. In MIAs, viral RNA was observed in one sample from a glove but not in other swabs. Infectious virus isolation in cell culture was performed on RNA-positive swabs from the full autopsies. Of all the RNA-positive PPE samples, 21% of the glove samples, taken in 3 of 11 full autopsies, tested positive for infectious virus. In conclusion, PPE was contaminated with viral RNA in 82% of autopsies. In 27% of autopsies, PPE was found to be contaminated even with infectious virus, representing a potential risk of infection to autopsy staff. Adequate PPE and hygiene measures, including appropriate waste deposition, are therefore essential to ensure a safe work environment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00428-021-03263-7

Investigation by Imaging Mass Spectrometry of Biomarker Candidates for Aging in the Hair Cortex

BACKGROUND: Human hair is one of the essential components that define appearance and is a useful source of samples for non-invasive biomonitoring. We describe a novel application of imaging mass spectrometry (IMS) of hair biomolecules for advanced molecular characterization and a better understanding of hair aging. As a cosmetic and biomedical application, molecules whose levels in hair altered with aging were comprehensively investigated. METHODS: Human hair was collected from 15 young (20±5 years old) and 15 older (50±5 years old) volunteers. Matrix-free laser desorption/ionization IMS was used to visualize molecular distribution in the hair sections. Hair-specific ions displaying a significant difference in the intensities between the 2 age groups were extracted as candidate markers for aging. Tissue localization of the molecules and alterations in their levels in the cortex and medulla in the young and old groups were determined. RESULTS: Among the 31 molecules detected specifically in hair sections, 2--one at m/z 153.00, tentatively assigned to be dihydrouracil, and the other at m/z 207.04, identified to be 3,4-dihydroxymandelic acid (DHMA)--exhibited a higher signal intensity in the young group than in the old, and 1 molecule at m/z 164.00, presumed to be O-phosphoethanolamine, displayed a higher intensity in the old group. Among the 3, putative O-phosphoethanolamine showed a cortex-specific distribution. The 3 molecules in cortex presented the same pattern of alteration in signal intensity with aging, whereas those in medulla did not exhibit significant alteration. CONCLUSION: Three molecules whose levels in hair altered with age were extracted. While they are all possible markers for aging, putative dihydrouracil and DHMA, are also suspected to play a role in maintaining hair properties and could be targets for cosmetic supplementation. Mapping of ion localization in hair by IMS is a powerful method to extract biomolecules in specified regions and determine their tissue distribution

Allele-Specific Impairment of GJB2 Expression by GJB6 Deletion del(GJB6-D13S1854)

Mutations in the GJB2 gene, which encodes connexin 26, are a frequent cause of congenital non-syndromic sensorineural hearing loss. Two large deletions, del(GJB6-D13S1830) and del(GJB6-D13S1854), which truncate GJB6 (connexin 30), cause hearing loss in individuals homozygous, or compound heterozygous for these deletions or one such deletion and a mutation in GJB2. Recently, we have demonstrated that the del(GJB6-D13S1830) deletion contributes to hearing loss due to an allele-specific lack of GJB2 mRNA expression and not as a result of digenic inheritance, as was postulated earlier. In the current study we investigated the smaller del(GJB6-D13S1854) deletion, which disrupts the expression of GJB2 at the transcriptional level in a manner similar to the more common del(GJB6-D13S1830) deletion. Interestingly, in the presence of this deletion, GJB2 expression remains minimally but reproducibly present. The relative allele-specific expression of GJB2 was assessed by reverse-transcriptase PCR and restriction digestions in three probands who were compound heterozygous for a GJB2 mutation and del(GJB6-D13S1854). Each individual carried a different sequence variant in GJB2. All three individuals expressed the mutated GJB2 allele in trans with del(GJB6-D13S1854), but expression of the GJB2 allele in cis with the deletion was almost absent. Our study clearly corroborates the hypothesis that the del(GJB6-D13S1854), similar to the larger and more common del(GJB6-D13S1830), removes (a) putative cis-regulatory element(s) upstream of GJB6 and narrows down the region of location

Deficient Plakophilin-1 Expression Due to a Mutation in PKP1 Causes Ectodermal Dysplasia-Skin Fragility Syndrome in Chesapeake Bay Retriever Dogs

In humans, congenital and hereditary skin diseases associated with epidermal cell-cell separation (acantholysis) are very rare, and spontaneous animal models of these diseases are exceptional. Our objectives are to report a novel congenital acantholytic dermatosis that developed in Chesapeake Bay retriever dogs. Nine affected puppies in four different litters were born to eight closely related clinically normal dogs. The disease transmission was consistent with an autosomal recessive mode of inheritance. Clinical signs occurred immediately after birth with superficial epidermal layers sloughing upon pressure. At three month of age, dogs exhibited recurrent superficial skin sloughing and erosions at areas of friction and mucocutaneous junctions; their coat was also finer than normal and there were patches of partial hair loss. At birth, histopathology revealed severe suprabasal acantholysis, which became less severe with ageing. Electron microscopy demonstrated a reduced number of partially formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Sequencing revealed a homozygous splice donor site mutation within the first intron of PKP1 resulting in a premature stop codon, thereby explaining the inability to detect plakophilin-1 in the skin. Altogether, the clinical and pathological findings, along with the PKP1 mutation, were consistent with the diagnosis of ectodermal dysplasia-skin fragility syndrome with plakophilin-1 deficiency. This is the first occurrence of ectodermal dysplasia-skin fragility syndrome in an animal species. Controlled mating of carrier dogs would yield puppies that could, in theory, be tested for gene therapy of this rare but severe skin disease of children

Somatostatin Inhibits Cell Migration and Reduces Cell Counts of Human Keratinocytes and Delays Epidermal Wound Healing in an Ex Vivo Wound Model

The peptide hormone somatostatin (SST) and its five G protein-coupled receptors (SSTR1-5) were described to be present in the skin, but their cutaneous function(s) and skin-specific signalling mechanisms are widely unknown. By using receptor specific agonists we show here that the SSTRs expressed in keratinocytes are functionally coupled to the inhibition of adenylate cyclase. In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly influences the MAP kinase signalling pathway. As epidermal hormone receptors in general are known to regulate re-epithelialization following skin injury, we investigated the effect of SST on cell counts and migration of human keratinocytes. Our results demonstrate a significant inhibition of cell migration and reduction of cell counts by SST. We do not observe an effect on apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin inhibits cell migration independent of its effect on cAMP. Migrating keratinocytes treated with SST show altered cytoskeleton dynamics with delayed lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is diminished, providing evidence for the control of the actin cytoskeleton by somatostatin receptors in keratinocytes. While activation of all receptors leads to redundant effects on cell migration, only treatment with a SSTR5/1 specific agonist resulted in decreased cell counts. In accordance with reduced cell counts and impaired migration we observe delayed re-epithelialization in an ex vivo wound healing model. Consequently, our experiments suggest SST as a negative regulator of epidermal wound healing