A participatory and comprehensive intervention to improve violence prevention in two high-risk occupations: effect and process evaluation of a stepped wedge cluster randomised trial.

BACKGROUND: Work-related violence committed by clients, patients, and customers represents a major occupational health risk for employees that needs to be reduced. METHODS: We tested a comprehensive violence prevention intervention involving active participation of both employees and managers in the Prison and Probation Service (PPS) and on psychiatric wards in Denmark. We used a stepped wedge cluster randomised controlled trial design. We measured the degree of implementation of the intervention by registration of fidelity, reach, and dose and used a mixed-effects regression analysis to estimate the effects of the intervention. RESULTS: We recruited 16 work units for the intervention, but three work units dropped out. The average implementation rate was 73%. In the psychiatric wards, the intervention led to statistically significant improvements in the primary outcome (an increase in the degree to which managers and employees continuously work on violence prevention practices based on their registration and experiences), but none statistically significant improvements in any of the secondary outcomes. In the PPS units, the intervention did not lead to a statistically significant improvement in the primary outcome, but to statistically significant improvements in three secondary outcomes. CONCLUSION: Most work units were able to carry out the intervention as planned. The intervention showed mixed results regarding the primary outcome. Nevertheless, the results indicate improvements also in the sector where a change in the primary outcome was not achieved. The results point at that a participatory and comprehensive approach could be a viable way of working with violence prevention in high-risk workplaces. TRIAL REGISTRATION: ISRCTN86993466: 20/12/2017.

Visualizing tropoelastin in a long-term human elastic fibre cell culture model.

Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin-fibrillin-1 fibre network. Future investigations will allow the tracking of TE produced under various conditions. In tissue engineering applications the fluorescent fibre network can be visualized under various conditions or it serves as a tool for investigating fibre degradation processes in disease-in-a-dish-models.

Optimization of isolation and cultivation of bacterial endophytes through addition of plant extract to nutrient media.

Many endophytes have beneficial effects on plants and can be exploited in biotechnological applications. Studies hypothesize that only 0.001-1% of all plant-associated bacteria are cultivable. Moreover, even after successful isolations, many endophytic bacteria often show reduced regrowth capacity. This research aimed to optimize isolation processes and culturing these bacteria afterwards. We compared several minimal and complex media in a screening. Beside the media themselves, two gelling agents and adding plant extract to media were investigated to enhance the number and diversity of endophytes as well as the growth capacity when regrown after isolation. In this work, 869 medium delivered the highest numbers of cultivable bacteria, as well as the highest diversity. When comparing gelling agents, no differences were observed in the numbers of bacteria. Adding plant extract to the media lead to a slight increase in diversity. However, when adding plant extract to improve the regrowth capacity, sharp increases of viable bacteria occurred in both rich and minimal media.

Stimulation of skin's energy metabolism provides multiple benefits for mature human skin.

As an organism ages, there is a decline in mitochondrial function and cellular energy balance. This decline is both accelerated by and can cause the formation of reactive oxygen species (ROS) that damage nuclear and mitochondrial DNA, lipid membranes as well as structural and catalytic proteins, especially those involved in energetic pathways of cells. Further, ROS have also been linked to some of the detrimental skin changes that occur as a result of photoaging. We have previously shown that levels of Coenzyme Q10 (CoQ10), a component of the respiratory chain in mitochondria, are reduced in skin cells from aging donors, and that topical supplementation can ameliorate processes involved in skin aging. Creatine is another important component of the cellular energy system and phosphocreatine, its phosphorylated form, functions as a reservoir for high energy phosphates. Unfortunately the creatine system and thus the energy storage mechanism in skin are negatively affected by aging and conditions of oxidative stress. This article reviews some of our in vivo data about the synergistic effects of combining a stabilized form of Creatine with CoQ10 and clearly depicts their beneficial effects as active ingredients in topical formulations.

Topical activity of ascorbic acid: from in vitro optimization to in vivo efficacy.

We present here a new cosmetic formula system containing 3% ascorbic acid based on an optimized oil-in-water (O/W) emulsion. This formulation demonstrated a good long-term stability of the active ingredient and also of the emulsion itself. It could be deduced from in vitro release studies that this O/W emulsion enabled a better release of the hydrophilic active agent than an alternative W/O emulsion. By measuring the ultraweak photon emission, which is a well-established parameter for the oxidative stress in the skin, the high in vivo antioxidant capacity of 3% ascorbic acid was demonstrated after 1 week of product application. This placebo-controlled study also proved that ascorbic acid in an O/W cream reduced oxidative stress in human skin significantly better than the derivative sodium ascorbyl-2-phosphate, a more stable vitamin C replacement commonly used in cosmetic formulations. With increasing age, the number of papillae in the epidermal-dermal junction zone in human skin are reduced. This implies a possible consequence of reduced mechanical resistance of the skin and impaired supply of the epidermis with nutrients. In a 1-month placebo-controlled study on 25 human volunteers, a significant increase in the number of dermal papillae after application of the 3% ascorbic acid cream was demonstrated, using a confocal laser scanning microscope. Fine lines and wrinkles are a characteristic sign of aged and especially photo-aged skin. Application of 3% ascorbic acid in a 12-week placebo-controlled usage study indicated a significant reduction of facial wrinkles. Altogether, 3% ascorbic acid in a cosmetic O/W emulsion has been shown to be appropriately stable and to enable a good release of the active agent in vitro as a precondition for a high efficacy in vivo. Application in vivo resulted in a significant reduction of oxidative stress in the skin, an improvement of the epidermal-dermal microstructure and a reduction of fine lines and wrinkles in aged skin. These results were received within a relatively short period of time of product application.

Multicentre comparison of skin hydration in terms of physical-, physiological- and product-dependent parameters by the capacitive method (Corneometer CM 825).

A multicentre study for measuring skin hydration with 349 volunteers was carried out in six different laboratories. The purpose of the study was to investigate physical-, physiological- and product-dependent parameters of three test emulsions (base, base + moisturizer and base + moisturizer + lipids) in a double-blind study. A comparison between analogous and digital sensor technology of the Corneometer CM825 was examined. Here, a clear relationship between both sensor types could be highlighted. A vital point of the study was the division of the test subjects according to their skin type. To get more objective limits for three different skin types - very dry, dry and normal skin - visual expert evaluation, self-assessment and hydration measurements were analysed by means of statistical methods. The moisture-related skin types were determined as follows: very dry skin was characterized with corneometer units below 30, dry skin between 30 and 40 and normal skin higher than 40 a.u. (arbitrary units). The efficacy of the three test emulsions was examined in relation to the mentioned skin types. Analysing the measured data of all test centres, a clear dependency of skin physiology (skin type) and product efficacy became evident. The drier the skin, the higher the increase of hydration. The product performance of the three test emulsions compared to the untreated control resulted in a significant increase of skin hydration in all measuring centres. The evaluation of a product ranking showed a good differentiation between the basic emulsion and the two other products. An increase of efficacy by adding lipids could be observed in four of six centres. The important influence of the skin type of the volunteers on the degree of product performance, as demonstrated in this study, should be especially considered when drawing up guidelines for efficacy testing.

A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist.

IL-22 is an IL-10 homologue that binds to and signals through the class II cytokine receptor heterodimer IL-22RA1/CRF2-4. IL-22 is produced by T cells and induces the production of acute-phase reactants in vitro and in vivo, suggesting its involvement in inflammation. Here we report the identification of a class II cytokine receptor designated IL-22RA2 (IL-22 receptor-alpha 2) that appears to be a naturally expressed soluble receptor. IL-22RA2 shares amino acid sequence homology with IL-22RA1 (also known as IL-22R, zcytor11, and CRF2-9) and is physically adjacent to IL-20Ralpha and IFN-gammaR1 on chromosome 6q23.3-24.2. We demonstrate that IL-22RA2 binds specifically to IL-22 and neutralizes IL-22-induced proliferation of BaF3 cells expressing IL-22 receptor subunits. IL-22RA2 mRNA is highly expressed in placenta and spleen by Northern blotting. PCR analysis using RNA from various tissues and cell lines showed that IL-22RA2 was expressed in a range of tissues, including those in the digestive, female reproductive, and immune systems. In situ hybridization revealed the dominant cell types expressing IL-22RA2 were mononuclear cells and epithelium. Because IL-22 induces the expression of acute phase reactants, IL-22RA2 may play an important role as an IL-22 antagonist in the regulation of inflammatory responses.

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat.

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.

Identification of INSL5, a new member of the insulin superfamily.

A new member of the insulin gene superfamily (INSL5) was identified by searching EST databases for the presence of the conserved insulin B-chain cysteine motif. Human and murine INSL5 are both polypeptides of 135 amino acids, matching the classical signature of the insulin superfamily. Through the B- and A-chain regions, human INSL5 has 48% identity to shark relaxin, 40% identity to human relaxin, and 34% identity to human Leydig insulin-like factor. Northern blot analysis detected expression of human INSL5 in rectal, colon, and uterine tissue and of murine INSL5 only in thymic tissue. Using quantitative RT-PCR, expression of murine INSL5 was detected in the highest quantity in colon followed by thymus, and minimal expression was seen in testis. By radiation hybrid mapping and the use of surrounding markers, human INSL5 maps to chromosome 1 in the 1p31.1 to 1p22.3 region.

Glucose-dependent insulinotropic polypeptide stimulation of lipolysis in differentiated 3T3-L1 cells: wortmannin-sensitive inhibition by insulin.

GIP is an important insulinotropic hormone (incretin) that has also been implicated in fat metabolism. There is controversy regarding the actions of GIP on adipocytes. In the current study, the existence of GIP receptors and effects of GIP on lipolysis were studied in differentiated 3T3-L1 cells. GIP receptor messenger RNA was detected by RT-PCR and RNase protection assay. Receptors were detected in binding studies (IC50 26.7 +/- 0.7 nM). GIP stimulated glycerol release with an EC50 of 3.28 +/- 0.63 nM. GIP (10(-9)-10(-7) M) +/- IBMX increased cAMP production by 1180-2246%. The adenylyl cyclase inhibitor MDL 12330A (10(-4) M) inhibited GIP-induced glycerol production by >90%, and reduced cAMP responses to basal. Preincubation of 3T3-L1 cells with insulin inhibited glycerol responses to GIP, and the inhibitory effect of insulin was blocked by the phosphatidylinositol 3'-kinase inhibitor, wortmannin. It is concluded that GIP stimulates glycerol release in 3T3-L1 cells primarily via stimulation of cAMP production, and that insulin antagonizes GIP-induced lipolysis in a wortmannin-sensitive fashion. It is suggested that effects of GIP on fat metabolism in vivo may depend upon the circulating insulin level, and that meal-released GIP may elevate circulating fatty acids, thus optimizing pancreatic beta-cell responsiveness to stimulation by glucose and GIP.

Regulation by insulin of phosphatidylinositol 3'-kinase bound to alpha- and beta-isoforms of p85 regulatory subunit.

The roles of the alpha- and beta-isoforms of phosphatidylinositol (PI) 3'-kinase p85 regulatory subunit were studied with isoform-specific antisera in three model systems in which the insulin receptor mediates rapid phosphorylation of insulin receptor substrate-1 (IRS-1). Insulin receptor signaling stimulated the association of IRS-1 with p85 alpha protein, and p85 alpha-associated PI 3-kinase activity in 3T3-L1 adipocytes, and in transfected Chinese hamster ovary cells (CHO-T) and COS-1 cells expressing high levels of human insulin receptors. While not detectable in 3T3-L1 adipocytes, the p85 beta isoform was also found to associate with IRS-1 in response to insulin receptor activation in COS-1 and CHO-T cells. However, selective immunoprecipitation of p85 beta from unstimulated COS-1 or CHO-T cell lysates was accompanied by higher levels of PI 3-kinase activity than that associated with p85 alpha. Remarkably, the large stimulation of PI 3-kinase activity associated with p85 alpha (7.8 +/- 2.0-fold, n = 6) in insulin-treated CHO-T cells was not observed in p85 beta immunoprecipitates (1.8 +/- 0.6-fold, n = 6), and in COS-1 cells p85 beta-associated PI 3-kinase activity was completely insensitive to stimulation by the insulin receptor. These data suggest the novel hypothesis that binding of p85 beta to IRS-1 complexes in COS-1 and CHO-T cells does not mediate marked activation of PI 3-kinase activity as does p85 alpha.

Transformation of adult ventricular myocytes with the temperature sensitive A58 (tsA58) mutant of the SV40 large T antigen.

Freshly isolated ventricular myocytes have been used extensively as an adult cardiac model system. Due to their inability to undergo cytokinesis in vitro and their dedifferentiated properties in long-term culture, they can not be used for extended studies. Recent reports tell of the establishment of fetal and neonatal cardiac cell lines and the development of adult cardiomyocytes from transgenic animals. A recent report by Kirshenbaum [1], is the first to demonstrate insertion of genes in to adult ventricular myocytes using viral infection. This paper discusses the infection of primary adult differentiated cardiomyocytes with the SV40 large T antigen and subsequent proliferation under temperature sensitive control. Upon further characterization, the cells could be used as a model to study muscle differentiation and repair as well as adult cardiac cell physiology.

Factors affecting the activation of glycogen synthase in primary culture cardiomyocytes.

The ability of insulin, IGF-1 and IGF-2 to stimulate the activation of glycogen synthase in the heart was compared under completely defined conditions using primary culture cardiomyocytes. Both insulin and IGF-1 produced similar time- and concentration-dependent activation of glycogen synthase with the maximum stimulation observed at 10-15 min following hormone administration and at > or = 10 nM insulin or IGF-1. IGF-2 was largely ineffective at physiological concentrations. When primary culture cardiomyocytes were incubated with 100 microM palmitate for 2 h and then challenged with various concentrations of insulin or IGF-1, there was a significant decrease in the ability of the cells to activate glycogen synthase. In addition, maintaining cardiomyocytes in hormone deficient culture conditions for 24 or 48 h also resulted in a reduced ability to activate glycogen synthase in response to these hormones. These results suggest that (1) both insulin and IGF-1 are potent regulators of glycogen synthesis in the heart, (2) the enzymes involved in the dephosphorylation (activation) of glycogen synthase are closely linked to both insulin and IGF-1, but not IGF-2 receptor signaling pathways, (3) glycogen synthase activation is adversely affected by the maintenance of cardiomyocytes in the presence of palmitate or for > or = 24 h in hormone deficient media which results in insulin and IGF-1 resistance, and (4) this resistance, like that found in cells from diabetic rats, is due at least in part to a decrease in glycogen synthase phosphatase activity.

Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Post-receptor defect accounts for phosphorylase hypersensitivity in cultured diabetic cardiomyocytes.

The basis for the hypersensitive response of glycogen phosphorylase to epinephrine stimulation was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. To assess potential G-protein involvement in the response, normal and diabetic derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylate cyclase activation, the cells were challenged with forskolin. After 3 hr in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hr in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes and this response which is present in alloxan-diabetic cells and is induced in vitro in normal cardiomyocytes is primarily due to a defect at a post-receptor site.

Adrenergic activation of glycogen phosphorylase in primary culture diabetic cardiomyocytes.

The basis of catecholamine-induced activation of glycogen phosphorylase was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. Cells derived from diabetic animals exhibited a hypersensitive response to epinephrine stimulation that was apparent 3 h after cell isolation and was further enhanced on maintenance of the myocytes in culture for 24 h. Normal cells initially lacked the hypersensitive response to epinephrine stimulation, although on maintenance of these cells in culture for 24 h, the hypersensitive response was acquired in vitro. To assess alpha- and beta-adrenergic mediation of the response, normal and diabetic cardiomyocytes were incubated with propranolol, a beta-blocker, before direct alpha 1-receptor stimulation with phenylephrine. Both normal and diabetic myocytes failed to undergo activation of phosphorylase in 3- or 24-h cell cultures. In addition, the effects of epinephrine on phosphorylase activation were completely inhibited by propranolol, whereas prazosin, an alpha-blocker, was unsuccessful. The present data suggest that the hypersensitive response of glycogen phosphorylase in normal and diabetic cardiomyocytes is solely mediated through beta-adrenergic receptor activation.

The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens.

The small and middle T (tumor) antigens of polyomavirus have been shown previously to associate with the 36-kDa catalytic subunit and the 63-kDa regulatory subunit of protein phosphatase type 2A, apparently substituting for a normal third 55-kDa regulatory subunit (D.C. Pallas, L.K. Shahrik, B.L. Martin, S. Jaspers, T.B. Miller, D.L. Brautigan, and T.M. Roberts, Cell 60:167-176, 1990). To facilitate a comparison of the normal regulatory subunit and T antigens, we isolated a 2.14-kb cDNA clone encoding this 55-kDa subunit from a rat liver library. Using a probe from the coding region of this gene, we detected a major 2.4-kb mRNA transcript in liver and muscle RNAs. The 55-kDa protein phosphatase 2A subunit purified from rat skeletal muscle generates multiple species when analyzed on two-dimensional gels. Transcription and translation of the clone in vitro produced a full-length protein that comigrated precisely on two-dimensional gels with three of these species, indicating that the 55-kDa protein is apparently modified similarly in vivo and in reticulocyte lysates. Additional species in the purified preparation were not found in the translate, suggesting that there are probably two or more isoforms of this protein in rat muscle. Somewhat surprisingly, there was no clear homology with T-antigen amino acid sequences.