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Partition (K) and diffusion (D) coefficients are important to measure for the modelling of skin penetration of chemicals through the stratum corneum (SC). We compared the feasibility of three protocols for the testing of 50 chemicals in our main studies, using three cosmetics-relevant model chemicals with a wide range of logP values. Protocol 1: SC concentration-depth profile using tape-stripping (measures KSC/v and DSC/HSC2, where HSC is the SC thickness); Protocol 2A: incubation of isolated SC with chemical (direct measurement of KSC/v only) and Protocol 2B: diffusion through isolated SC mounted on a Franz cell (measures KSC/v and DSC/HSC2, and is based on Fick's laws). KSC/v values for caffeine and resorcinol using Protocol 1 and 2B were within 30% of each other, values using Protocol 2A were ~two-fold higher, and all values were within 10-fold of each other. Only indirect determination of KSC/v by Protocol 2B was different from the direct measurement of KSC/v by Protocol 2A and Protocol 1 for 7-EC. The variability of KSC/v for all three chemicals using Protocol 2B was higher compared to Protocol 1 and 2A. DSC/HSC2 values for the three chemicals were of the same order of magnitude using all three protocols. Additionally, using Protocol 1, there was very little difference between parameters measured in pig and human SC. In conclusion, KSC/v, and DSC values were comparable using different methods. Pig skin might be a good surrogate for human skin for the three chemicals tested. Copyright © 2017 The Authors Journal of Applied Toxicology published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Objective Cockayne syndrome is an autosomal recessive leukodystrophy and multisystem disorder characterized by mental retardation, microcephaly, severe growth failure, sensorial impairment, peripheral neuropathy and cutaneous photosensitivity. It belongs to the family of DNA repair and transcription disorders together with xeroderma pigmentosum and trichothiodystrophy. Clinical and genetic diagnosis still remains challenging. Our goal was to develop a unique and efficient mutation-screening strategy for Cockayne syndrome and related disorders and to improve our understanding of the clinical spectrum of these conditions. Methods We have designed a novel targeted high-throughput sequencing approach for the diagnosis of Cockayne syndrome and related DNA repair disorders. We have studied 30 patients presenting with the cardinal clinical features of Cockayne syndrome or related DNA repair disorders. Results We have identified the causative mutations in 14 of the 30 patients. Three patients showed biallelic mutations in the ERCC6/CSB gene, 2 patients in the ERCC8/CSA gene, 1 patient in the ERCC2/XPD gene, 1 patient in the ERCC3/XPB, 1 patient in the ERCC5/XPG, 6 in the POLH gene. We present the clinical, neurological and radiological phenotypes of these patients with regard to our experience of a well-characterized cohort of more than 100 Cockayne patients previously studied in our center and we discuss the implications for diagnosis and prognosis purposes. Conclusion This novel approach enables us to rapidly confirm the genetic diagnosis of Cockayne syndrome and related disoders and to unravel the molecular defect in atypical and overlapping clinical presentations. [ABSTRACT FROM AUTHOR]

The purpose of this study was to use chemical similarity evaluations, transcriptional profiling, in vitro toxicokinetic data, and physiologically based pharmacokinetic (PBPK) models to support read-across for a series of branched carboxylic acids using valproic acid (VPA), a known developmental toxicant, as a comparator. The chemicals included 2-propylpentanoic acid (VPA), 2-ethylbutanoic acid, 2-ethylhexanoic acid (EHA), 2-methylnonanoic acid, 2-hexyldecanoic acid, 2-propylnonanoic acid (PNA), dipentyl acetic acid or 2-pentylheptanoic acid, octanoic acid (a straight chain alkyl acid), and 2-ethylhexanol. Transcriptomics was evaluated in 4 cell types (A549, HepG2, MCF7, and iCell cardiomyocytes) 6 h after exposure to 3 concentrations of the compounds, using the L1000 platform. The transcriptional profiling data indicate that 2- or 3-carbon alkyl substituents at the alpha position of the carboxylic acid (EHA and PNA) elicit a transcriptional profile similar to the one elicited by VPA. The transcriptional profile is different for the other chemicals tested, which provides support for limiting read-across from VPA to much shorter and longer acids. Molecular docking models for histone deacetylases, the putative target of VPA, provide a possible mechanistic explanation for the activity cliff elucidated by transcriptomics. In vitro toxicokinetic data were utilized in a PBPK model to estimate internal dosimetry. The PBPK modeling data show that as the branched chain increases, predicted plasma Cmax decreases. This work demonstrates how transcriptomics and other mode of action-based methods can improve read-across., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fatty esters of 2-ethylhexanoic acid (EHA) and 2-ethylhexanol (EH) are commonly used in cosmetics. Human liver and skin S9 and human plasma were used to determine the in vitro rates of clearance (CL int ) of a series of compounds, with a range of 2-11 carbons on the acid or alcohol moiety and branching at the C2 position. The impact of carbon chain length on in vitro CL int was most prominent for the liver metabolism of esters of EH, while for in vitro skin metabolism it was greater for esters of EHA. The position of the branching also impacted the liver hydrolysis rates, especially for the C3, C4, and C5 esters with lower CL int in vitro rates for esters of EHA relative to those of EH. When the in vitro intrinsic clearance rates were scaled to in vivo rates of hepatic clearance, all compounds approximated the rate for hepatic blood flow, mitigating this dependence of metabolism on structure. This work shows how structural changes to the molecule can affect in vitro metabolism and, furthermore, allows for an estimation of the in vivo metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Adipose tissue is a key regulator of whole-body metabolic fitness because of its role in controlling insulin sensitivity. Obesity is associated with hypertrophic adipocytes with impaired glucose absorption, a phenomenon existing in the ultrarare monogenic disorder Alström syndrome consisting of severe insulin resistance. Inactivation of ALMS1 directly inhibits insulin-mediated glucose absorption in the white adipose tissue and induces severe insulin resistance, which leads to type 2 diabetes, accelerated nonalcoholic liver disease, and fibrosis. These phenotypes were reversed by specific adipocyte-ALMS1 reactivation in vivo. Subsequently, ALMS1 was found to bind to protein kinase C-α (PKCα) in the adipocyte, and upon insulin signaling, PKCα is released from ALMS1. α-Helices in the kinase domain of PKCα were therefore screened to identify a peptide sequence that interfered with the ALMS1-PKCα protein interaction. When incubated with cultured human adipocytes, the stapled peptide termed PATAS, for Peptide derived of PKC Alpha Targeting AlmS, triggered insulin-independent glucose absorption, de novo lipogenesis, and cellular glucose utilization. In vivo, PATAS reduced whole-body insulin resistance, and improved glucose intolerance, fasting glucose, liver steatosis, and fibrosis in rodents. Thus, PATAS represents a novel first-in-class peptide that targets the adipocyte to ameliorate insulin resistance and its associated comorbidities., (© 2022 by the American Diabetes Association.)

Background: Cockayne syndrome (CS) is a rare autosomal recessive disorder caused by mutations in ERCC6/CSB or ERCC8/CSA that participate in the transcription-coupled nucleotide excision repair (TC-NER) of UV-induced DNA damage. CS patients display a large heterogeneity of clinical symptoms and severities, the reason of which is not fully understood, and that cannot be anticipated in the diagnostic phase. In addition, little data is available for affected siblings, and this disease is largely undiagnosed in North Africa., Methods: We report here the clinical description as well as genetic and functional characterization of eight Tunisian CS patients, including siblings. These patients, who belonged to six unrelated families, underwent complete clinical examination and biochemical analyses. Sanger sequencing was performed for the recurrent mutation in five families, and targeted gene sequencing was done for one patient of the sixth family. We also performed Recovery RNA Synthesis (RRS) to confirm the functional impairment of DNA repair in patient-derived fibroblasts., Results: Six out of eight patients carried a homozygous indel mutation (c.598_600delinsAA) in exon 7 of ERCC8, and displayed a variable clinical spectrum including between siblings sharing the same mutation. The other two patients were siblings who carried a homozygous splice-site variant in ERCC8 (c.843+1G>C). This last pair presented more severe clinical manifestations, which are rarely associated with CSA mutations, leading to gastrostomy and hepatic damage. Impaired TC-NER was confirmed by RRS in six tested patients., Conclusions: This study provides the first deep characterization of case series of CS patients carrying CSA mutations in North Africa. These mutations have been described only in this region and in the Middle-East. We also provide the largest characterization of multiple unrelated patients, as well as siblings, carrying the same mutation, providing a framework for dissecting elusive genotype-phenotype correlations in CS., (© 2022. The Author(s).)

Cockayne syndrome is a rare condition that encompasses a very wide spectrum of clinical severity. Mutations upstream of a transposon called PiggyBac Transposable Element Derived 3 in intron 5 of the CSB/ERCC6 gene could bring about less severe forms than mutations located downstream of that transposon insertion. Our aim was to study genotype-phenotype correlation by determining whether the position of each mutation of the CSB/ERCC6 gene has an impact on the phenotype. A hundred and forty-seven Cockayne patients, who had two pathogenic mutations in the CSB/ERCC6 gene and for whom clinical data was available, were retrospectively selected and included in the study. Data analysis was performed under the Bayesian paradigm. Analysis of the proportion of the different subtypes of Cockayne syndrome according to the position of the mutations was done using an ordinal logistic regression model. Using a vague prior, the risk of developing a more severe subtype when exposed to 2 mutations downstream compared to 2 mutations upstream was 2.0 [0.9-4.5]. Estimations varied through the sensitivity analysis. We could reasonably conclude that a relationship between the number of downstream mutations and the Cockayne syndrome clinical expression exists but it is still difficult to give a precise estimate of this relationship. The real effect could be more complex that the one described in the initial model and other genetic factors might be taken into consideration together with the mutation site to better explain clinical variability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Damaj-Fourcade, Meyer, Obringer, Le May, Calmels and Laugel.)

Cockayne syndrome (CS) is a rare disease caused by mutations in ERCC6 / CSB or ERCC8 / CSA . We report here the clinical, genetic, and functional analyses of three unrelated patients mutated in ERCC6 / CSB with a severe phenotype. After clinical examination, two patients were investigated via next generation sequencing, targeting seventeen Nucleotide Excision Repair (NER) genes. All three patients harbored a novel, c.3156dup, homozygous mutation located in exon 18 of ERCC6 / CSB that affects the C-terminal region of the protein. Sanger sequencing confirmed the mutation and the parental segregation in the three families, and Western blots showed a lack of the full-length protein. NER functional impairment was shown by reduced recovery of RNA synthesis with proficient unscheduled DNA synthesis after UV-C radiations in patient-derived fibroblasts. Despite sharing the same mutation, the clinical spectrum was heterogeneous among the three patients, and only two patients displayed clinical photosensitivity. This novel ERCC6 variant in Tunisian patients suggests a founder effect and has implications for setting-up prenatal diagnosis/genetic counselling in North Africa, where this disease is largely undiagnosed. This study reveals one of the rare cases of CS clinical heterogeneity despite the same mutation. Moreover, the occurrence of an identical homozygous mutation, which either results in clinical photosensitivity or does not, strongly suggests that this classic CS symptom relies on multiple factors.

Barded-Biedl syndrome (BBS) is a rare genetic disorder with an unmet medical need for retinal degeneration. Small-molecule drugs were previously identified to slow down the apoptosis of photoreceptors in BBS mouse models. Clinical translation was not practical due to the necessity of repetitive invasive intravitreal injections for pediatric populations. Non-invasive methods of retinal drug targeting are a prerequisite for acceptable adaptation to the targeted pediatric patient population. Here, we present the development and functional testing of a non-invasive, topical, magnetically assisted delivery system, harnessing the ability of magnetic nanoparticles (MNPs) to cargo two drugs (guanabenz and valproic acid) with anti-unfolded protein response (UPR) properties towards the retina. Using magnetic resonance imaging (MRI), we showed the MNPs' presence in the retina of Bbs wild-type mice, and their photoreceptor localization was validated using transmission electron microscopy (TEM). Subsequent electroretinogram recordings (ERGs) demonstrated that we achieved beneficial biological effects with the magnetically assisted treatment translating the maintained light detection in Bbs -/- mice (KO). To our knowledge, this is the first demonstration of efficient magnetic drug targeting in the photoreceptors in vivo after topical administration. This non-invasive, needle-free technology expands the application of SMDs for the treatment of a vast spectrum of retinal degenerations and other ocular diseases.

Parabens are antimicrobial compounds used as preservatives in cosmetics, foods, and pharmaceuticals. Paraben exposure occurs through a variety of routes including dermal absorption, ingestion, and inhalation. Ester bond hydrolysis has been shown to be the predominant biotransformation for this chemical class. Here we evaluated a series of parabens of increasing alkyl chain length and branching in addition to the aryl side chain of phenyl paraben (PhP). We evaluated the parabens under full Michaelis-Menten (MM) parameters to obtain intrinsic clearance values and found different trends between human liver and skin, which correlate with the predominant esterase enzymes in those matrices, respectively. In liver, where carboxylesterase 1 (CES1) is the predominant esterase enzyme, the shorter chain parabens were more readily metabolized, while in skin, where carboxylesterase 2 (CES2) is the predominant esterase enzyme, the longer chain parabens were more readily metabolized. Alkyl chain branching reduced the hydrolysis rates relative to those for the straight chain compounds, while the addition of a phenyl group, as in PhP, showed an increase in hydrolysis, producing the highest observed hydrolysis rate for skin. These data summarize the structure-metabolism relationship for a series of parabens and contribute to the safety assessment of this class of compounds., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Cockayne syndrome is an autosomal recessive disorder caused by biallelic mutations in ERCC6 or ERCC8 genes., Aims: To study the clinical and mutation spectrum of Cockayne syndrome., Setting and Design: Medical Genetics Outpatient Department of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow. This was a prospective study from 2007 to 2015., Materials and Methods: Clinical details were recorded, and sequencing of ERCC6 and ERCC8 were performed., Results and Conclusions: Of the six families, one family had a homozygous mutation in ERCC8 and the other five families had homozygous mutations in ERCC6. Novel variants in ERCC6 were identified in four families. Phenotypic features may vary from severe to mild, and a strong clinical suspicion is needed for diagnosis during infancy or early childhood. Hence, molecular diagnosis is needed for confirmation of diagnosis in a child with a suspicion of Cockayne syndrome. Prenatal diagnosis can be provided only if molecular diagnosis is established in the proband., Competing Interests: None

Obesity is a major risk factor for insulin resistance (IR) and its attendant complications. The pathogenic mechanisms linking them remain poorly understood, partly due to a lack of intermediary monogenic human phenotypes. Here, we report on a monogenic form of IR-prone obesity, Alström syndrome (ALMS). Twenty-three subjects with monogenic or polygenic obesity underwent hyperinsulinemic-euglycemic clamping with concomitant adipose tissue (AT) microdialysis and an in-depth analysis of subcutaneous AT histology. We have shown a relative AT failure in a monogenic obese cohort, a finding supported by observations in a novel conditional mouse model ( Alms flin/flin ) and ALMS1-silenced human primary adipocytes, whereas selective reactivation of ALMS1 gene in AT of an ALMS conditional knockdown mouse model ( Alms flin/flin ; Adipo-Cre +/- ) restores systemic insulin sensitivity and glucose tolerance. Hence, we show for the first time the relative AT failure in human obese cohorts to be a major determinant of accelerated IR without evidence of lipodystrophy. These new insights into adipocyte-driven IR may assist development of AT-targeted therapeutic strategies for diabetes., (© 2020 by the American Diabetes Association.)

Cockayne syndrome (CS) is a multisystem degenerative disorder divided in 3 overlapping subtypes, with a continuous phenotypic spectrum: CS2 being the most severe form, CS1 the classical form and CS3 the late-onset form. Failure to thrive and growth difficulties are among the most consistent features of CS, leaving affected individuals vulnerable to numerous medical complications, including adverse effects of undernutrition, abrupt overhydration and overfeeding. There is thus a significant need for specific growth charts. We retrospectively collected growth parameters from genetically-confirmed CS1 and CS2 patients, used the GAMLSS package to construct specific CS growth charts compared to healthy children from WHO and CDC databases. Growth data were obtained from 88 CS patients with a total of 1626 individual growth data points. 49 patients were classified as CS1 and 39 as CS2 with confirmed mutations in CSB/ERCC6, CSA/ERCC8 or ERCC1 genes. Individuals with CS1 initially have normal growth parameters; microcephaly occurs from 2 months whereas onset of weight and height restrictions appear later, between 5 and 22 months. In CS2, growth parameters are already below standard references at birth or drop below the 5th percentile before 3 months. Microcephaly is the first parameter to show a delay, appearing around 2 months in CS1 and at birth in CS2. Height and head circumference are more severely affected in CS2 compared to CS1 whereas weight curves are similar in CS1 and CS2 patients. These new growth charts will serve as a practical tool to improve the nutritional management of children with CS., (Copyright © 2020. Published by Elsevier Masson SAS.)

Nucleotide excision repair associated diseases comprise overlapping phenotypes and a wide range of outcomes. The early stages still remain under-investigated and underdiagnosed, even although an early recognition of the first symptoms is of utmost importance for appropriate care and genetic counseling. We systematically collected clinical and molecular data from the literature and from newly diagnosed NER patients with neurological impairment, presenting clinical symptoms before the age of 12 months, including foetal cases. One hundred and eighty-five patients were included, 13 with specific symptoms during foetal life. Arthrogryposis, microcephaly, cataracts, and skin anomalies are the most frequently reported signs in early subtypes. Non ERCC6/CSB or ERCC8/CSA genes are overrepresented compared to later onset cohorts: 19% patients of this cohort presented variants in ERCC1, ERCC2/XPD, ERCC3/XPB or ERCC5/XPG. ERCC5/XPG is even the most frequently involved gene in foetal cases (10/13 cases, [4/7 families]). In this cohort, the mutated gene, the age of onset, the type of disease, severe global developmental delay, IUGR and skin anomalies were associated with earlier death. This large survey focuses on specific symptoms that should attract the attention of clinicians towards early-onset NER diagnosis in foetal and neonatal period, without waiting for the completeness of classical criteria., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cockayne syndrome (CS) is a rare genetic disorder caused by mutations (dysfunction) in CSA and CSB. CS patients exhibit mild photosensitivity and severe neurological problems. Currently, CS diagnosis is based on the inefficiency of CS cells to recover RNA synthesis upon genotoxic (UV) stress. Indeed, upon genotoxic stress, ATF3, an immediate early gene is activated to repress up to 5000 genes encompassing its responsive element for a short period of time. On the contrary in CS cells, CSA and CSB dysfunction impairs the degradation of the chromatin-bound ATF3, leading to a permanent transcriptional arrest as observed by immunofluorescence and ChIP followed by RT-PCR. We analysed ChIP-seq of Pol II and ATF3 promoter occupation analysis and RNA sequencing-based gene expression profiling in CS cells, as well as performed immunofluorescence study of ATF3 protein stability and quantitative RT-PCR screening in 64 patient cell lines. We show that the analysis of few amount (as for example CDK5RAP2, NIPBL and NRG1) of ATF3 dependent genes, could serve as prominent molecular markers to discriminate between CS and non-CS patient's cells. Such assay can significantly simplify the timing and the complexity of the CS diagnostic procedure in comparison to the currently available methods.

Background: Cockayne Syndrome (CS) is a rare autosomal recessive multi-systemic disorder, characterized; by developmental delay, microcephaly, severe growth failure and sensorial impairment. Renal complications have been reported but remain underinvestigated. The objective of this study was to perform a review of renal disease in a cohort of CS patients., Methods: We retrospectively collected relevant clinical, biochemical and genetic data from a cohort of 136 genetically confirmed CS patients. Blood pressure (BP), proteinuria, albuminemia, uric acid, creatinine clearance, renal ultrasounds and renal biopsy result were analysed., Results: Thirty-two patients had a renal investigation. We found that 69% of investigated patients had a renal disorder and/or an elevated BP. Fifteen out of 21 patients (71% of investigated patients) had an increased BP, 10 out of 16 patients (62% of investigated patients) presented with proteinuria and 4 of them had a nephrotic syndrome. Thirteen patients out of 29 (45%) had a decreased Glomerular Filtration Rate (GFR), 18 out of 25 patients (72%) had a hyperuricemia. No correlation with the genetic background or clinical types of CS was found, except for the renal clearance., Conclusions: Renal disease, increased blood pressure and hyperuricemia were highly prevalent in our study. We believe that CS patients should benefit from a nephrological follow-up and that anti-uric acid drug and Angiotensin-converting enzyme (ACE) inhibitor should be discussed in these patients., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are sometimes present in foods or herbal supplements/medicines as impurities and pose potential concerns for liver genotoxicity/carcinogenicity. PAs display a strong structure toxicity relationship, however, current regulatory approaches to risk assessment take the precautionary approach of assuming all PAs display the same potency as the most toxic congeners lasiocarpine (LAS) and riddelliine (RID). Here we explore the relative potencies of a series of structurally diverse PAs by measuring DNA adduct formation in vitro in a rat sandwich culture hepatocyte (SCH) cell system. The adducts generated are consistent with those identified in vivo as biomarkers of PA exposure and potential liver-tumor formation. DNA reactive PAs require metabolic activation to form intermediates that bind DNA, therefore, adduct formation is a direct reflection of reactive metabolite formation. Since the area under the concentration versus time curve (AUC) for the depletion of parent PA from the extracellular media is a measure of PA exposure, the ratio of adducts/AUC provides a measure of hepatocyte exposure to DNA-binding metabolites corresponding to an intrinsic potency for DNA adduct formation. Intrinsic potencies relative to potencies for LAS compare well with existing relative potency data further affirming that PA toxicity varies considerably with chemical structure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cilia are highly conserved and ubiquitously expressed organelles. Ciliary defects of genetic origins lead to ciliopathies, in which retinal degeneration (RD) is one cardinal clinical feature. In order to efficiently find and design new therapeutic strategies the underlying mechanism of retinal degeneration of three murine model was compared. The rodent models correspond to three emblematic ciliopathies, namely: Bardet-Biedl Syndrome (BBS), Alström Syndrome (ALMS) and CEP290-mediated Leber Congenital Amaurosis (LCA). Scotopic rodent electroretinography (ERG) was used to test the retinal function of mice, Transmitted Electron microscopy (T.E.M) was performed to assess retinal structural defects and real-time PCR for targeted genes was used to monitor the expression levels of the major apoptotic Caspase-related pathways in retinal extracts to identify pathological pathways driving the RD in order to identify potential therapeutic targets. We found that BBS and CEP290-mediated LCA mouse models exhibit perinatal retinal degeneration associated with rhodopsin mislocalization in the photoreceptor and the induction of an Endoplasmic Reticulum (ER) stress. On the other hand, the tested ALMS mouse model, displayed a slower degeneration phenotype, with no Rhodopsin mislocalization nor ER-stress activity. Our data points out that behind the general phenotype of vision loss associated with these ciliopathies, the mechanisms and kinetics of disease progression are different., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Cockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8 / CSA or ERCC6 / CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships. We have carried out a four-centre clinical, molecular and cellular analysis of 124 patients with CS., Methods and Results: We assigned 39 patients to the ERCC8/CSA and 85 to the ERCC6/CSB genes. Most of the genetic variants were truncations. The missense variants were distributed non-randomly with concentrations in relatively short regions of the respective proteins. Our analyses revealed several hotspots and founder mutations in ERCC6/CSB. Although no unequivocal genotype-phenotype relationships could be made, patients were more likely to have severe clinical features if the mutation was downstream of the PiggyBac insertion in intron 5 of ERCC6/CSB than if it was upstream. Also a higher proportion of severely affected patients was found with mutations in ERCC6/CSB than in ERCC8/CSA ., Conclusion: By identifying >70 novel homozygous or compound heterozygous genetic variants in 124 patients with CS with different disease severity and ethnic backgrounds, we considerably broaden the CSA and CSB mutation spectrum responsible for CS. Besides providing information relevant for diagnosis of and genetic counselling for this devastating disorder, this study improves the definition of the puzzling genotype-phenotype relationships in patients with CS., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Cockayne syndrome is an autosomal recessive multisystem disorder characterized by intellectual disability, microcephaly, severe growth failure, sensory impairment, peripheral neuropathy, and cutaneous sensitivity. This rare disease is linked to disease-causing variations in the ERCC6 (CSB) and ERCC8 (CSA) genes. Various degrees of severity have been described according to age at onset and survival, without any clear genotype-phenotype correlation. All types of nucleotide changes have been observed in CS genes, including splice variations mainly affecting the splice site consensus sequences. We report here the case of two brothers from a consanguineous family presenting a severe but long-term survival phenotype of Cockayne syndrome. We identified in the patients a homozygous deep intronic nucleotide variation causing the insertion of a cryptic exon in the ERCC8 (CSA) transcript, by modifying intronic regulatory elements important for exon definition. The pathogenesis of the nucleotide variant NG_009289.1(NM_000082.3):c.173+1119G>C was validated in vitro with a reporter minigene system. To our knowledge, these are the first Cockayne patients described with this kind of disease-causing variation, though molecular mechanism underlying early onset symptoms and unexpected slow raise of progression of the disease remain to be elucidated.

Oral absorption is a key element for safety assessments of cosmetic ingredients, including hair dye molecules. Reliable in vitro methods are needed since the European Union has banned the use of animals for the testing of cosmetic ingredients. Caco-2 cells were used to measure the intestinal permeability characteristics (Papp) of 14 aromatic amine hair dye molecules with varying chemical structures, and the data were compared with historical in vivo oral absorption rat data. The majority of the hair dyes exhibited Papp values that indicated good in vivo absorption. The moderate to high oral absorption findings, i.e. ≥60%, were confirmed in in vivo rat studies. Moreover, the compound with a very low Papp value (APB: 3-((9,10-dihydro-9,10-dioxo-4-(methylamino)-1-anthracenyl)amino)-N,N-dimethyl-N-propyl-1-propanaminium) was poorly absorbed in vivo as well (5% of the dose). This data set suggests that the Caco-2 cell model is a reliable in vitro tool for the determination of the intestinal absorption of aromatic amines with diverse chemical structures. When used in combination with other in vitro assays for metabolism and skin penetration, the Caco-2 model can contribute to the prediction and mechanistic interpretation of the absorption, metabolism and elimination properties of cosmetic ingredients without the use of animals., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS)., Methods: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies)., Results: We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes., Conclusions: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

Approaches to assess the role of absorption, metabolism and excretion of cosmetic ingredients that are based on the integration of different in vitro data are important for their safety assessment, specifically as it offers an opportunity to refine that safety assessment. In order to estimate systemic exposure (AUC) to aromatic amine hair dyes following typical product application conditions, skin penetration and epidermal and systemic metabolic conversion of the parent compound was assessed in human skin explants and human keratinocyte (HaCaT) and hepatocyte cultures. To estimate the amount of the aromatic amine that can reach the general circulation unchanged after passage through the skin the following toxicokinetically relevant parameters were applied: a) Michaelis-Menten kinetics to quantify the epidermal metabolism; b) the estimated keratinocyte cell abundance in the viable epidermis; c) the skin penetration rate; d) the calculated Mean Residence Time in the viable epidermis; e) the viable epidermis thickness and f) the skin permeability coefficient. In a next step, in vitro hepatocyte Km and Vmax values and whole liver mass and cell abundance were used to calculate the scaled intrinsic clearance, which was combined with liver blood flow and fraction of compound unbound in the blood to give hepatic clearance. The systemic exposure in the general circulation (AUC) was extrapolated using internal dose and hepatic clearance, and Cmax was extrapolated (conservative overestimation) using internal dose and volume of distribution, indicating that appropriate toxicokinetic information can be generated based solely on in vitro data. For the hair dye, p-phenylenediamine, these data were found to be in the same order of magnitude as those published for human volunteers., (Copyright © 2015. Published by Elsevier Inc.)

Background: Bardet-Biedl Syndrome (BBS) is a genetically heterogeneous ciliopathy with clinical cardinal features including retinal degeneration, obesity and renal dysfunction. To date, 20 BBS genes have been identified with BBS10 being a major BBS gene found to be mutated in almost 20 percent of all BBS patients worldwide. It codes for the BBS10 protein which forms part of a chaperone complex localized at the basal body of the primary cilium. Renal dysfunction in BBS patients is one of the major causes of morbidity in human patients and is associated initially with urinary concentration defects related to water reabsorption impairment in renal epithelial cells. The aim of this study was to study and compare the impact of a total Bbs10 inactivation (Bbs10 (-/-)) with that of a specific renal epithelial cells inactivation (Bbs10  (fl/fl) ; Cdh16-Cre (+/-))., Results: We generated the Bbs10 (-/-) and Bbs10  (fl/fl) ; Cadh16-Cre (+/-) mouse model and characterized them. Bbs10 (-/-) mice developed obesity, retinal degeneration, structural defects in the glomeruli, polyuria associated with high circulating arginine vasopressin (AVP) concentrations, and vacuolated, yet ciliated, renal epithelial cells. On the other hand, the Bbs10  (fl/fl) ; Cadh16-Cre (+/-)mice displayed no detectable impairment., Conclusions: These data highlight the importance of a systemic Bbs10 inactivation to trigger averted renal dysfunction whereas a targeted absence of BBS10 in the renal epithelium is seemingly non-deleterious.

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Ciliopathies, a class of rare genetic disorders, present often with retinal degeneration caused by protein transport defects between the inner segment and the outer segment of the photoreceptors. Bardet-Biedl syndrome is one such ciliopathy, genetically heterogeneous with 17 BBS genes identified to date, presenting early onset retinitis pigmentosa. By investigating BBS12-deprived retinal explants and the Bbs12(-/-) murine model, we show that the impaired intraciliary transport results in protein retention in the endoplasmic reticulum. The protein overload activates a proapoptotic unfolded protein response leading to a specific Caspase12-mediated death of the photoreceptors. Having identified a therapeutic window in the early postnatal retinal development and through optimized pharmacological modulation of the unfolded protein response, combining three specific compounds, namely valproic acid, guanabenz, and a specific Caspase12 inhibitor, achieved efficient photoreceptor protection, thereby maintaining light detection ability in vivo.

Studying ciliopathies, like the Bardet-Biedl syndrome (BBS), allow the identification of signaling pathways potentially involved in common diseases, sharing phenotypic features like obesity or type 2 diabetes. Given the close association between obesity and insulin resistance, obese BBS patients would be expected to be insulin resistant. Surprisingly, we found that a majority of obese BBS patients retained normal glucose tolerance and insulin sensitivity. Patient's adipose tissue biopsies revealed upregulation of adipogenic genes and decrease of inflammatory mediators. In vitro studies on human primary mesenchymal stem cells (MSCs) showed that BBS12 inactivation facilitated adipogenesis, increased insulin sensitivity, and glucose utilization. We generated a Bbs12(-/-) mouse model to assess the impact of Bbs12 inactivation on adipocyte biology. Despite increased obesity, glucose tolerance was increased with specific enhanced insulin sensitivity in the fat. This correlated with an active recruitment of MSCs resulting in adipose tissue hyperplasia and decreased in inflammation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This research examines the potential for coherent anti-Stokes Raman scattering (CARS) to provide reliable gas temperature measurements in the presence of liquid droplets. The droplets cause dielectric breakdown by focusing the CARS laser beams. This breakdown produces a plasma that can disrupt or obscure the CARS signal. Specifically, we examine the influence of laser induced breakdown on the CARS signal, and we determine the importance of droplet position relative to the CARS focal volume and droplet concentration on the reliability of CARS temperature measurements in droplet-laden flows. In addition, we propose a reliable data reduction procedure to minimize the disruptive influence of laser induced breakdown on CARS temperatures.