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Background: Macromolecules in skin cells are damaged when exposed to environmental stressors, leading to disrupted cellular function and homeostasis. While epidermal turnover can eliminate some of this damage, autophagy can rapidly remove these defective components. Niacinamide (Nam) is known to induce autophagy and optimizing formulations to maximize this response could provide improved homeostasis in stressed skin., Objective: To determine (i) whether Nam can induce autophagy related 5 (ATG5), an autophagy marker, in human keratinocytes and (ii) whether optimized low pH Nam formulations can enhance the response in 3D skin models., Methods: Human keratinocytes treated with Nam were evaluated for autophagosome accumulation and induction of ATG5 by gene expression, immunoblotting and immune-fluorescence microscopy. 3D skin equivalents were topically treated with Nam formulations at pH 5.8 and 3.8. Gene expression profiling and immunoblot analysis of ATG5 were performed., Results: Nam treatment of keratinocytes led to an accumulation of autophagosomes with a maximal signal at 48 h. Gene expression of ATG5 was induced by Nam, and immunoblots stained for ATG5 showed a significant increase after 6 h of treatment. Gene expression profiling of 3D epidermal skin equivalents treated with Nam at pH 3.8 showed stronger induction of autophagy-related genes, including ATG5, compared with pH 5.8 formulas. Enrichment for gene ontology terms on autophagy showed an increased linkage with Nam formulas at pH 3.8., Conclusions: We found that Nam induces autophagosome accumulation and ATG5 levels in keratinocytes. We also discovered that a Nam formulation at pH 3.8 can further increase levels of ATG5 in 3D skin models when compared to Nam at pH 5.8. These data support that Nam can induce autophagy in keratinocytes and formulations at pH 3.8 can enhance the impact. We hypothesize that optimized formulations at pH 3.8 can improve skin ageing appearance via autophagy induction., (© 2020 European Academy of Dermatology and Venereology.)

The electrocardiographic (ECG) diagnosis of ST-segment elevation myocardial infarction (STEMI) represents a challenge to all health care providers, particularly so for the novice ECG interpreter. We have developed--and present in this article--a 4-step algorithm that will detect STEMI in most instances in the prehospital and other nonemergency department (ED) settings. The algorithm should be used in adult patients with chest pain or equivalent presentation who are suspected of STEMI. It inquires as to the presence of ST-segment elevation as well as the presence of STEMI confounding/mimicking patterns; the algorithm also makes use of reciprocal ST-segment depression as an adjunct in the ECG diagnosis of STEMI. If STEMI is detected by this algorithm, then management decisions can be made based upon this ECG diagnosis. If STEMI is not detected using this algorithm, then we can only note that STEMI is not "ruled in"; importantly, STEMI is not "ruled out." In fact, more expert interpretation of the ECG will be possible once the patient (and/or the ECG) arrive in the ED where ECG review can be made with the more complex interpretation used by expert physician interpreters., (Copyright © 2012 Elsevier Inc. All rights reserved.)

When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5' end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic. Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting.

It has been demonstrated previously that the exposure to 7,12-dimethyl[a]benzanthracene (DMBA) and UVB radiation leads to the development of epidermal cysts, squamous cell carcinomas (SCC), melanocytic hyperplasia and melanoma in human foreskins from newborns grafted to immunodeficient mice. Improved techniques in grafting full-thickness skin from adults have enabled us to study photocarcinogenesis in human skin from different body sites and from older donors. One hundred and fifty-five normal white skin specimens from the trunk and face of 53 adult individuals were grafted onto severe combined immunodeficient (SCID) and recombinase activating gene-1 (Rag-1) knockout mice and irradiated two to three times weekly with 40 mJ/cm(2) UVB or solar-simulated UV (SSUV) over a period of up to 10 months with or without one prior topical application of DMBA. Over an observation period of 2-22 months, histopathological and immunohistochemical analyses of 134 specimens revealed actinic keratoses in 30% of the DMBA- + UV-treated grafts, in 18% of the grafts exposed to SSUV only, and in 10% of the grafts exposed to UVB only. Actinic keratoses were absent in grafts treated with DMBA only. One SCC was found in an abdominal skin graft 3 months after exposure to DMBA followed by UVB. Point mutations in codon 61 of the human Ha-ras gene were detected in the SCC, five of six analyzed actinic keratoses and in non-lesional epidermis of DMBA- and UVB-treated grafts, indicating that DMBA as well as UVB alone can induce these mutations in human skin. In contrast to the previous experience with neonatal foreskin grafts, melanocytic lesions were not found except for mild hyperplasia in few cases. The data suggest that melanocytes from young individuals are more susceptible to the transforming effects of genotoxic agents than melanocytes from adults.

This study demonstrates the synthesis and release of prolactin (PRL) from dermal fibroblasts (>98%) in vitro, suggesting a potential local source of PRL in skin. PRL release was first detected in confluent cultures (0.25 x 10(6) plated cells) on or before day 18 and increased to a maximal level of 2 ng/72 h by day 30. Medroxyprogesterone acetate and estradiol (E2) had no effect on PRL release, but prostaglandin E2 (PGE2) reduced the time required for PRL induction to 6-9 days. The steroids and PGE2 together were synergistic, reaching maximal values of approximately 10 ng/72 h after 2 or more weeks of treatment. Dibutyryl-cyclic AMP, a second messenger in prostaglandin signal transduction, was also synergistic with medroxyprogesterone acetate and E2, but induced significant PRL expression in the absence of the steroids (28 and 12 ng/72 h, respectively). The increase in PRL release was not a result of increased cell proliferation, because the PRL-secreting cultures had 32.2 +/- 8.8% less DNA (N = 3 individuals, 93% confidence limit) than control cultures after 3 weeks of treatment with dibutyryl-cyclic AMP, medroxyprogesterone acetate, and E2. Dermal fibroblast PRL was immunologically and electrophoretically identical to decidual and pituitary PR-Ls, and Northern blot analysis demonstrated a PRL mRNA size of 1.15 kb. Maximal PRL release from fibroblast cells was 32.0 +/- 6.1 ng/72 h (mean +/- SD at 95% confidence limit) for a donor population representing both males (n = 15) and females (n = 7) between the ages of 20-week gestation to 52 years. In contrast to term decidual fibroblast cells that also express PRL, dermal fibroblasts did not co-express insulin-like growth factor-binding protein-1.

Term decidual tissue consists of terminally differentiated decidual cells, bone marrow-derived cells, and fibroblast cells. Since undifferentiated decidual cells are fibroblast-like cells of the endometrial stroma, the possibility exists that the fibroblast cells in term decidua are undifferentiated decidual cells. To test this hypothesis, a purified population of fibroblast cells was isolated from term decidua and treated under conditions that are known to induce differentiation of endometrial stromal cells. By flow cytometry and immunocytochemistry, the fibroblast cells from term decidua were shown to be free of cells expressing bone marrow-derived cell-surface antigens and the epithelial cell marker cytokeratin. In addition, they tested positive for the cytoskeletal protein vimentin, thus establishing that they were mesenchymal cells. As with endometrial stromal cells, continuous treatment of the decidual fibroblast cells with the progesterone analog medroxyprogesterone acetate and estradiol in combination with either dibutyryl-cAMP or prostaglandin E2 induced cell aggregation and the expression of prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). When cells were plated at an initial cell density of 0.25 x 10(6) cells/well in a 24-well culture dish with medium changes every three days, PRL was first detected on Days 4-6, and the peak of averaged 24 h-PRL release (30 ng/well) occurred on Days 26-28. The mRNA for decidual fibroblast PRL followed a temporal pattern corresponding to that of the released hormone. The size of the PRL mRNA was 1.15 kb, corresponding to the alternately spliced PRL mRNA reported for decidualized endometrial stromal cells and other extrapituitary sources of PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Under appropriate conditions, protease-dispersed cytotrophoblast cells from human term placenta fuse and differentiate into syncytiotrophoblast cells that express hCG. Although human placental lactogen (hPL) is also expressed by the syncytiotrophoblast in vivo, little hPL is expressed by trophoblast cells differentiated in vitro using standard tissue culture medium or keratinocyte growth medium supplemented with fetal bovine serum. In this report, we demonstrate that cytotrophoblast cells fuse and differentiate to a phenotype that expresses large amounts of both hCG and hPL when cultured in medium containing maternal serum. When protease-dispersed cytotrophoblast cells were plated in RPMI-1640 supplemented with 10% second trimester (16-22 week) maternal serum (STMS medium), extensive cell fusion was observed by day 3 in culture. Cell fusion appeared to be essentially complete by day 6, coinciding with the peak of hCG expression and the initiation of hPL expression. The peak of hCG expression occurred between days 4-7 of an experiment; the range for maximal release of hCG was 0.1-3.7 micrograms/24 h.well when cells were plated at a density of 10(6)/well. In contrast, the peak of hPL expression occurred between days 7-11 of an experiment, and the range of maximal hPL release was 0.9-3.5 micrograms/24 h.well. Northern blot analysis indicated that the level of hPL messenger RNA (mRNA) paralleled the release of hormone. However, whereas the hCG beta-subunit mRNA paralleled the release of hCG, the hCG alpha-subunit mRNA did not, suggesting that the two genes are independently regulated. Cells cultured in nonpregnant (male or female) serum-supplemented medium also differentiated to the syncytiotrophoblast phenotype following a temporal pattern almost identical to that observed for cells cultured in STMS. However, the quantity of hormone released was at least 2-fold greater with STMS. Compared to RPMI-1640 or keratinocyte growth medium supplemented with 10% fetal bovine serum, STMS induced equal or greater expression of hCG and substantially greater expression of hPL at the level of both mRNA and protein. The extent of cell fusion, the level of hormone expression, and the regulated patterns of hormone expression suggest that cytotrophoblast cells cultured in maternal serum progress to an advanced stage of trophoblast differentiation.