Epidermolytic Ichthyosis Sine Epidermolysis

Epidermolytic ichthyosis (EI) is a rare disorder of cornification caused by mutations in KRT1 and KRT10, encoding two suprabasal epidermal keratins. Because of the variable clinical features and severity of the disease, histopathology is often required to correctly direct the molecular analysis. EI is characterized by hyperkeratosis and vacuolar degeneration of the upper epidermis, also known as epidermolytic hyperkeratosis, hence the name of the disease. In the current report, the authors describe members of 2 families presenting with clinical features consistent with EI. The patients were shown to carry classical mutations in KRT1 or KRT10, but did not display epidermolytic changes on histology. These observations underscore the need to remain aware of the limitations of pathological features when considering a diagnosis of EI

Loss-of-Function Mutations in SERPINB8 Linked to Exfoliative Ichthyosis with Impaired Mechanical Stability of Intercellular Adhesions

M.P. is supported by a Fellowship from the Deutsche Forschungsgemeinschaft. This work was supported in part by a generous donation of Israel and Ruthi Ram (E.S.) and from a British Heart Foundation Programme grant (D.P.K.)

Site-Specific Recombination in the Cyanobacterium Anabaena sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022▿

The integrase (Int) of the λ-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP × attB and attL × attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate PglnA-attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter PglnA. The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli

High Efficiency of a Sequential Recombinase-Mediated Cassette Exchange Reaction in Escherichia coli

A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential

Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway

SVEP1 plays a crucial role in epidermal differentiation.

SVEP1 is a recently identified multidomain cell adhesion protein, homologous to the mouse polydom protein, which has been shown to mediate cell-cell adhesion in an integrin-dependent manner in osteogenic cells. In this study, we characterized SVEP1 function in the epidermis. SVEP1 was found by qRT-PCR to be ubiquitously expressed in human tissues, including the skin. Confocal microscopy revealed that SVEP1 is normally mostly expressed in the cytoplasm of basal and suprabasal epidermal cells. Downregulation of SVEP1 expression in primary keratinocytes resulted in decreased expression of major epidermal differentiation markers. Similarly, SVEP1 downregulation was associated with disturbed differentiation and marked epidermal acanthosis in three-dimensional skin equivalents. In contrast, the dispase assay failed to demonstrate significant differences in adhesion between keratinocytes expressing normal vs low levels of SVEP1. Homozygous Svep1 knockout mice were embryonic lethal. Thus, to assess the importance of SVEP1 for normal skin homoeostasis in vivo, we downregulated SVEP1 in zebrafish embryos with a Svep1-specific splice morpholino. Scanning electron microscopy revealed a rugged epidermis with perturbed microridge formation in the centre of the keratinocytes of morphant larvae. Transmission electron microscopy analysis demonstrated abnormal epidermal cell-cell adhesion with disadhesion between cells in Svep1-deficient morphant larvae compared to controls. In summary, our results indicate that SVEP1 plays a critical role during epidermal differentiation. Exp Dermatol 2017 May; 26(5):423-430

Effect of <i>Tspear</i> down-regulation on murine hair follicles.

<p>(a) Tspear is expressed in mouse hair follicles (HFs) in the hair matrix keratinocytes, outer root sheath, inner root sheath, hair shaft and the infundibulum (scale bar = 50 μm); (b) Back skin tissue strips from <i>K14-H2B-GFP</i> mice were transfected with <i>Tspear</i> siRNA or control siRNA. RNA was extracted from transfected HFs and <i>Tspear</i> RNA expression levels were assessed by qRT-PCR. Results were normalized to <i>Gapdh</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; **p<0.01); (c-f) Z stacks optical sections of <i>K14-H2B-GFP</i> mouse HFs (c) obtained 24h following transfection with control siRNA (d) or <i>Tspear</i> siRNA (e) were used to calculate average hair bulb diameter. Three measurements were done for each HF in the bulb and proximal hair shaft (c, dashed white lines) and an average diameter was calculated accordingly. Epithelial nuclei are marked with <i>GFP</i> (scale bars = 100 μm). Data was pooled from three independent experiments (F, two sided t-test; **p<0.01); (g-i) Melanin content was assessed by quantitative Masson-Fontana histochemistry in <i>Tspear</i> siRNA treated HFs (h) compared to control (g). Data was pooled from two independent experiments (I, two sided t-test; ***p<0.001) (scale bars = 50 μm); (j-o) Apoptosis was assessed by the TUNEL assay (TUNEL, green; DAPI, blue) at the hair bulb (j-l) and infundibular (m-o) compartments of HFs downregulated for <i>Tspear</i> (k,n) compared to control siRNA treated HFs (j,m) (scale bars = 50 μm). Average number of TUNEL-positive cells in hair follicles in the respective compartments. Data were pooled from two independent experiments (l,o, two sided t-test; ***p<0.001) (scale bars = 50 μm). White dotted lines delineate the outer epidermal surface; (p) RNA was extracted from <i>Tspear</i> siRNA and control siRNA transfected HFs and <i>Notch1</i> RNA expression level was assessed by qRT-PCR. Results were normalized to <i>Rplp0</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; *p<0.05). E—epidermis; INF–Infundibulum; D—dermis; DP—dermal papilla; IRS—inner root sheath; ORS—outer root sheath; HM—hair matrix; HS—hair shaft; TUNEL—terminal deoxynucleotidyl transferase dUTP nick end labeling.</p