Periplakin, a novel component of cornified envelopes and desmosomes that belongs to the plakin family and forms complexes with envoplakin

The cornified envelope is a layer of transglutaminase cross-linked protein that is assembled under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We have determined the cDNA sequence of one of the proteins that becomes incorporated into the cornified envelope of cultured epidermal keratinocytes, a protein with an apparent molecular mass of 195 kD that is encoded by a mRNA with an estimated size of 6.3 kb. The protein is expressed in keratinizing and nonkeratinizing stratified squamous epithelia and in a number of other epithelia. Expression of the protein is upregulated during the terminal differentiation of epidermal keratinocytes in vivo and in culture. Immunogold electron microscopy was used to demonstrate an association of the 195-kD protein with the desmosomal plaque and with keratin filaments in the differentiated layers of the epidermis. Sequence analysis showed that the 195-kD protein is a member of the plakin family of proteins, to which envoplakin, desmoplakin, bullous pemphigoid antigen 1, and plectin belong. Envoplakin and the 195-kD protein coimmunoprecipitate. Analysis of their rod domain sequences suggests that the formation of both homodimers and heterodimers would be energetically favorable. Confocal immunofluorescent microscopy of cultured epidermal keratinocytes revealed that envoplakin and the 195-kD protein form a network radiating from desmosomes, and we speculate that the two proteins may provide a scaffolding onto which the cornified envelope is assembled. We propose to name the 195-kD protein periplakin

Family-based prevention against substance abuse and behavioral problems: culture-sensitive adaptation process for the modification of the US-American Strengthening Families Program 10–14 to German conditions

Aim: The Strengthening Families Program 10–14 (SFP 10–14) was developed in 1993 at the Iowa State University as a universal family-based prevention program against substance abuse and behavioral problems in youth aged 10 to 14 years. Its effectiveness in delaying the initiation of tobacco, alcohol and cannabis use, in decreasing the average amount consumed and in reducing adolescents’ problem behavior in school and at home has been repeatedly evaluated in randomized-controlled studies in the US. While there is a well-established system of school- and community-based prevention in Germany, there is a lack of family-based prevention. This situation would be improved by the cultural adaptation and evaluation of SFP 10–14 in Germany. Subjects and methods: Focus group meetings were held with experts from family assistance and drug prevention, as well as with parents of children within the ages of the target group, in three geographically different cities in Germany (Hamburg, Schwerin and Munich). Group members were presented the original version of the material from the US (teaching manuals and DVDs), as well as an already adapted version from the UK. Group members developed criteria in a group discussion process necessary for the adaptation of the material to the German culture. Following the newly defined criteria, new teaching DVDs and manuals were produced. Results: As a result of the focus groups meetings, several aspects concerning the adaptation of the material had to be considered. Four aspects were especially important: (1) application to the regional social structures in Germany, within the target group (risk population: migration background, socioeconomic status, family structure), (2) adaptation to the German language (colloquial language, idiomatic expressions, non-verbal language), (3) consideration of culturally dependent norms about parents’ and children’s role model behavior, as well as the problem definition for behavior that is supposed to be addressed (family, school, peer group) and (4) the program’s adequate incorporation into the conditions of the local support system. Conclusions: Neither of the two existing SFP versions (US and UK version) could serve as a matrix for the German version, extensive adaptations were necessary. Results from the adaptation process carried out earlier in the UK with the original material from the US were helpful in this process. The German version of the program (Familien stärken) will be evaluated for a target group that consists of families with low socioeconomic status. This randomized-controlled multicenter study will be carried out in different German cities (Hamburg, Hanover, Schwerin, Rostock and Munich) between 2010 and 2013

Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound‐associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE (2)) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β‐catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE (2)‐Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE (2) and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury

Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment

Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were 7.1 and 12% respectively. All seven metabolites can be measured in a perchloric acid extract of 20 μl of blood. The methods have been optimized with regard to variation in the perchloric acid content of the samples. These variations arise from the method of sample preparation used to minimize changes occurring in metabolite concentration after venepuncture

A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using air-liquid interface (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion \u3e 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions