Cornification of nail keratinocytes requires autophagy for bulk degradation of intracellular proteins while sparing components of the cytoskeleton.

Epidermal keratinocytes undergo cornification to form the cellular building blocks of hard skin appendages such as nails and the protective layer on the surface of the skin. Cornification requires the cross-linking of structural proteins and the removal of other cellular components to form mechanically rigid and inert corneocytes. Autophagy has been proposed to contribute to this intracellular remodelling process, but its molecular targets in keratinocytes, if any, have remained elusive. Here, we deleted the essential autophagy factor Atg7 in K14-positive epithelia of mice and determined by proteomics the impact of this deletion on the abundance of individual proteins in cornified nails. The genetic suppression of autophagy in keratinocytes resulted in a significant increase in the number of proteins that survived cornification and in alterations of their abundance in the nail proteome. A broad range of enzymes and other non-structural proteins were elevated whereas the amounts of cytoskeletal proteins of the keratin and keratin-associated protein families, cytolinker proteins and desmosomal proteins were either unaltered or decreased in nails of mice lacking epithelial autophagy. Among the various types of non-cytoskeletal proteins, the subunits of the proteasome and of the TRiC/CCT chaperonin were most strongly elevated in mutant nails, indicating a particularly important role of autophagy in removing these large protein complexes during normal cornification. Taken together, the results of this study suggest that autophagy is active during nail keratinocyte cornification and its substrate specificity depends on the accessibility of proteins outside of the cytoskeleton and their presence in large complexes.

In situ labeling of DNA reveals interindividual variation in nuclear DNA breakdown in hair and may be useful to predict success of forensic genotyping of hair.

Hair fibers are formed by keratinocytes of the hair follicle in a process that involves the breakdown of the nucleus including DNA. Accordingly, DNA can be isolated with high yield from the hair bulb which contains living keratinocytes, whereas it is difficult to prepare from the distal portions of hair fibers and from shed hair. Nevertheless, forensic investigations are successful in a fraction of shed hair samples found at crime scenes. Here, we report that interindividual differences in the completeness of DNA removal from hair corneocytes are major determinants of DNA content and success rates of forensic investigations of hair. Distal hair samples were permeabilized with ammonia and incubated with the DNA-specific dye Hoechst 33258 to label DNA in situ. Residual nuclear DNA was visualized under the fluorescence microscope. Hair from some donors did not contain any stainable nuclei, whereas hair of other donors contained a variable number of DNA-positive nuclear remnants. The number of DNA-containing nuclear remnants per millimeter of hair correlated with the amount of DNA that could be extracted and amplified by quantitative PCR. When individual hairs were investigated, only hairs in which DNA could be labeled in situ gave positive results in short tandem repeat typing. This study reveals that the completeness of DNA degradation during cornification of the hair is a polymorphic trait. Furthermore, our results suggest that in situ labeling of DNA in hair may be useful for predicting the probability of success of forensic analysis of nuclear DNA in shed hair.

Challenges in Palliative Care Nursing at a University Hospital: A Qualitative Interview Study.

We examined the perception of palliative care nurses regarding challenges, coping strategies, resources, and needs when working in a university hospital in Austria. A qualitative descriptive design was applied, using semistructured interviews with 8 female and 2 male nurses. All interviews were recorded as digital audio and transcribed verbatim. We used thematic analysis and MAXQDA. In our analysis, 6 themes emerged: Four themes related to challenges: ( a ) lack of a supporting structural framework, ( b ) conflict in interdisciplinary work, ( c ) conflict with caregivers, and ( d ) dealing with death in a highly specialized university environment. One theme related to ( e ) individual solutions and coping strategies, and 1 theme comprised ( f ) needs and suggestions for improvements. Taking care of the family of a dying person, handling threatening situation, and working with inexperienced physicians were among the most important challenges reported by nurses. A supportive team, professional counseling, and training related to communication skills and to culturally specific needs of families are perceived to be necessary to provide high-quality palliative care. Addressing the needs of nurses can substantially improve their working condition and has an impact not only on the nurses themselves but also on the quality of patient care.

Identification of reptilian genes encoding hair keratin-like proteins suggests a new scenario for the evolutionary origin of hair.

The appearance of hair is one of the main evolutionary innovations in the amniote lineage leading to mammals. The main components of mammalian hair are cysteine-rich type I and type II keratins, also known as hard alpha-keratins or "hair keratins." To determine the evolutionary history of these important structural proteins, we compared the genomic loci of the human hair keratin genes with the homologous loci of the chicken and of the green anole lizard Anolis carolinenis. The genome of the chicken contained one type II hair keratin-like gene, and the lizard genome contained two type I and four type II hair keratin-like genes. Orthology of the latter genes and mammalian hair keratins was supported by gene locus synteny, conserved exon-intron organization, and amino acid sequence similarity of the encoded proteins. The lizard hair keratin-like genes were expressed most strongly in the digits, indicating a role in claw formation. In addition, we identified a novel group of reptilian cysteine-rich type I keratins that lack homologues in mammals. Our data show that cysteine-rich alpha-keratins are not restricted to mammals and suggest that the evolution of mammalian hair involved the co-option of pre-existing structural proteins.

The anatomy and development of the claws of Xenopus laevis (Lissamphibia: Anura) reveal alternate pathways of structural evolution in the integument of tetrapods.

Digital end organs composed of hard, modified epidermis, generally referred to as claws, are present in mammals and reptiles as well as in several non-amniote taxa such as clawed salamanders and frogs, including Xenopus laevis. So far, only the claws and nails of mammals have been characterized extensively and the question of whether claws were present in the common ancestor of all extant tetrapods is as yet unresolved. To provide a basis for comparisons between amniote and non-amniote claws, we investigated the development, growth and ultrastructure of the epidermal component of the claws of X. laevis. Histological examination of developing claws of X. laevis shows that claw formation is initiated at the tip of the toe by the appearance of superficial cornified cells that are dark brown. Subsequent accumulation of new, proximally extended claw sheath corneocyte layers increases the length of the claw. Histological studies of adult claws show that proliferation of cornifying claw sheath cells occurs along the entire length of the claw-forming epidermis. Living epidermal cells that are converting into the cornified claw sheath corneocytes undergo a form of programmed cell death that is accompanied by degradation of nuclear DNA. Subsequently, the cytoplasm and the nuclear remnants acquire a brown colour by an as-yet unknown mechanism that is likely homologous to the colouration mechanism that occurs in other hard, cornified structures of amphibians such as nuptial pads and tadpole beaks. Transmission electron microscopy revealed that the cornified claw sheath consists of parallel layers of corneocytes with interdigitations being confined to intra-layer contacts and a cementing substance filling the intercorneocyte spaces. Together with recent reports that showed the main molecular components of amniote claws are absent in Xenopus, our data support the hypothesis that claws of amphibians likely represent clade-specific innovations, non-homologous to amniote claws.

Trichohyalin-like proteins have evolutionarily conserved roles in the morphogenesis of skin appendages.

S100 fused-type proteins (SFTPs) such as filaggrin, trichohyalin, and cornulin are differentially expressed in cornifying keratinocytes of the epidermis and various skin appendages. To determine evolutionarily conserved, and thus presumably important, features of SFTPs, we characterized nonmammalian SFTPs and compared their amino acid sequences and expression patterns with those of mammalian SFTPs. We identified an ortholog of cornulin and a previously unknown SFTP, termed scaffoldin, in reptiles and birds, whereas filaggrin was confined to mammals. In contrast to mammalian SFTPs, both cornulin and scaffoldin of the chicken are expressed in the embryonic periderm. However, scaffoldin resembles mammalian trichohyalin with regard to its expression in the filiform papillae of the tongue and in the epithelium underneath the forming tips of the claws. Furthermore, scaffoldin is expressed in the epithelial sheath around growing feathers, reminiscent of trichohyalin expression in the inner root sheath of hair. The results of this study show that SFTP-positive epithelia function as scaffolds for the growth of diverse skin appendages such as claws, nails, hair, and feathers, indicating a common evolutionary origin.

Comparative anatomy of mouse and human nail units.

Recent studies of mice with hair defects have resulted in major contributions to the understanding of hair disorders. To use mouse models as a tool to study nail diseases, a basic understanding of the similarities and differences between the human and mouse nail unit is required. In this study we compare the human and mouse nail unit at the macroscopic and microscopic level and use immunohistochemistry to determine the keratin expression patterns in the mouse nail unit. Both species have a proximal nail fold, cuticle, nail matrix, nail bed, nail plate, and hyponychium. Distinguishing features are the shape of the nail and the presence of an extended hyponychium in the mouse. Expression patterns of most keratins are similar. These findings indicate that the mouse nail unit shares major characteristics with the human nail unit and overall represents a very similar structure, useful for the investigation of nail diseases and nail biology.

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis.

The claw of lizards is largely composed of beta-keratins, also referred to as keratin-associated beta-proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta-keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha-keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha-keratins resembled that of beta-keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins.

Essential role of the keratinocyte-specific endonuclease DNase1L2 in the removal of nuclear DNA from hair and nails.

Degradation of nuclear DNA is a hallmark of programmed cell death. Epidermal keratinocytes die in the course of cornification to function as the dead building blocks of the cornified layer of the epidermis, nails, and hair. Here, we investigated the mechanism and physiological function of DNA degradation during cornification in vivo. Targeted deletion of the keratinocyte-specific endonuclease DNase1-like 2 (DNase1L2) in the mouse resulted in the aberrant retention of DNA in hair and nails, as well as in epithelia of the tongue and the esophagus. In contrast to our previous studies in human keratinocytes, ablation of DNase1L2 did not compromise the cornified layer of the epidermis. Quantitative PCRs showed that the amount of nuclear DNA was dramatically increased in both hair and nails, and that mitochondrial DNA was increased in the nails of DNase1L2-deficient mice. The presence of nuclear DNA disturbed the normal arrangement of structural proteins in hair corneocytes and caused a significant decrease in the resistance of hair to mechanical stress. These data identify DNase1L2 as an essential and specific regulator of programmed cell death in skin appendages, and demonstrate that the breakdown of nuclear DNA is crucial for establishing the full mechanical stability of hair.

Mice expressing a mutant Krt75 (K6hf) allele develop hair and nail defects resembling pachyonychia congenita.

KRT75 (formerly known as K6hf) is one of the isoforms of the keratin 6 (KRT6) family located within the type II cytokeratin gene cluster on chromosome 12 of humans and chromosome 15 of mice. KRT75 is expressed in the companion layer and upper germinative matrix region of the hair follicle, the medulla of the hair shaft, and in epithelia of the nail bed. Dominant mutations in members of the KRT6 family, such as in KRT6A and KRT6B cause pachyonychia congenita (PC) -1 and -2, respectively. To determine the function of KRT75 in skin appendages, we introduced a dominant mutation into a highly conserved residue in the helix initiation peptide of Krt75. Mice expressing this mutant form of Krt75 developed hair and nail defects resembling PC. This mouse model provides in vivo evidence for the critical roles played by Krt75 in maintaining hair shaft and nail integrity. Furthermore, the phenotypes observed in our mutant Krt75 mice suggest that KRT75 may be a candidate gene for screening PC patients who do not exhibit obvious mutations in KRT6A, KRT6B, KRT16, or KRT17, especially those with extensive hair involvement.

DNase1L2 degrades nuclear DNA during corneocyte formation.

The removal of keratinocyte (KC) nuclear DNA by deoxyribonucleases (DNases) is an important step in the formation of normal stratum corneum (SC). However, the molecular identity of the DNA-degrading enzymes has so far remained elusive. Here we show that the endonuclease DNase1-like 2 (DNase1L2) is preferentially expressed in the epidermis and that its expression correlates with terminal differentiation of KC in vitro and in vivo. In biopsies of normal skin, DNase1L2 mRNA was regularly found in suprabasal KC and DNase1L2 protein was highly abundant in the stratum granulosum. In contrast to normal skin, DNase1L2 expression was downregulated in parakeratotic epidermis such as in psoriatic lesions. When DNase1L2 gene expression was knocked down by small interfering RNA in a human skin equivalent model, nuclei were maintained through all layers of the SC. Taken together, our data demonstrate that DNase1L2 plays an essential role in DNA degradation during terminal differentiation of epidermal KC.

Caspase-14 but not caspase-3 is processed during the development of fetal mouse epidermis.

The activation of caspases is a central step in apoptosis and may also be critical for terminal differentiation of epidermal keratinocytes (KC). In particular, caspase-3 has been implicated in the differentiation of embryonic KC as well as in programmed cell death of KC, and caspase-14 has been suggested to function in the formation or homeostasis of the stratum corneum (SC). To test the putative roles of these proteases, we determined their expression level and activation status during development of fetal mouse epidermis. The level of procaspase-3 did not change significantly during epidermal development, and enzyme activation was undetectable at any timepoint investigated. Despite the lack of active caspase-3, the newly formed stratum granulosum and the regressing periderm contained cells positive in the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, indicating that nuclear DNA was degraded without activation of caspase-3, thereby arguing against a proteolytic function of caspase-3 in embryonic KC differentiation. By contrast, caspase-14 increased in abundance from embryonic day 14.5 (E14.5) onwards and consistently localized to the suprabasal layers of fetal epidermis. The caspase-14 pro-enzyme was processed into its catalytic subunits, a step required for enzyme activity, on day E17.5, coinciding with SC formation. Thus, processing of procaspase-14 is not confined to air-exposed mature skin but also occurs during epidermal development in utero. In summary, this study demonstrates that caspase-14, but not caspase-3 activation coincides temporally and spatially with embryonic KC differentiation, suggesting a role for caspase-14 in terminally differentiated KC.

Doença diverticular dos cólons / Diverticular disease of the colon

Expöem-se aspectos relativos ao diagnóstico, diagnóstico diferencial, complicaçöes e tratamento da doença Diverticular dos Cólons, tecendo breves comentários a respeito e, finalmente, colocando a experiência do Serviço de Proctologia do Hospital Säo Lucas da PUC/RS

O método psicoprofilático de preparaçäo para a maternidade / Psychoprophylatic method of the preparation to the maternity

Faz-se uma revisäo sobre a preparaçäo psicoprofilática para a maternidade. Discorrendo sobre a evoluçäo histórica e métodos, procura-se demonstrar sua importância como meio de proporcionar condiçöes saudáveis à gestante no parto e maternidade