Surfactants for stabilization of dermal emulsions and their skin compatibility under UVA irradiation: Diacyl phospholipids and polysorbate 80 result in high viability rates of primary human skin cells.

Phospholipids are versatile formulation compounds with high biocompatibility. However, no data on their effect on skin in combination with UVA radiation exist. Thus, it was the aim of this work to (i) develop o/w nanoemulsions (NEs) differing in surfactant type and to investigate their physicochemical stability at different storage temperatures, (ii) establish a standardized protocol for in vitro phototoxicity testing using primary human skin cells and (iii) investigate the phototoxicity of amphoteric phospholipids (S45, S75, E80, S100, LPC80), sodium lauryl ether sulfate (SLES) and polysorbate 80 (PS80). Satisfying systems were developed with all surfactants except S100 due to low zeta potential (-21.4 mV ± 4.69). SLES and PS80-type NEs showed the highest stability after eight weeks; temperature-dependent variations in storage stability were most noticeable for phospholipid surfactants. For phospholipid-based NEs, higher phosphatidylcholine content led to unstable formulations. Phototoxicity assays with primary skin fibroblasts confirmed the lack of UVA-related phototoxicity but revealed cytotoxic effects of LPC80 and SLES, resulting in cell viability as low as 2.7 % ±0.78 and 1.9 % ±1.57 compared to the control. Our findings suggest that surfactants S45, S75 and PS80 are the most promising candidates for skin-friendly emulsifiers in sensitive applications involving exposure to UV light.

Race development and performance-determining factors in a mass-start cross-country skiing competition.

Introduction: Although five of six Olympic events in cross-country skiing involve mass-starts, those events are sparsely examined scientifically. Therefore, in this study, we investigated speed profiles, pacing strategies, group dynamics and their performance-determining impact in a cross-country skiing mass-start competition. Methods: Continuous speed and position of 57 male skiers was measured in a six-lap, 21.8 km national mass-start competition in skating style and later followed up with an online questionnaire. Skiers ranked from 1 to 40 were split into four performance-groups: R1-10 for ranks 1 to 10, R11-20 for ranks 11 to 20, R21-30 for ranks 21 to 30, and R31-40 for ranks 31 to 40. Results: All skiers moved together in one large pack for 2.3 km, after which lower-performing skiers gradually lost the leader pack and formed small, dynamic packs. A considerable accordion effect occurred during the first half of the competition that lead to additional decelerations and accelerations and a higher risk of incidents that disadvantaged skiers at the back of the pack. Overall, 31% of the skiers reported incidents, but none were in R1-10. The overall trend was that lap speed decreased after Lap 1 for all skiers and thereafter remained nearly unchanged for R1-10, while it gradually decreased for the lower-performing groups. Skiers in R31-40, R21-30, and R11-20 lost the leader pack during Lap 3, Lap 4, and Lap 5, respectively, and more than 60% of the time-loss relative to the leader pack occurred in the uphill terrain sections. Ultimately, skiers in R1-10 sprinted for the win during the last 1.2 km, in which 2.4 s separated the top five skiers, and a photo finish differentiated first from second place. Overall, a high correlation emerged between starting position and final rank. Conclusions: Our results suggest that (a) an adequate starting position, (b) the ability to avoid incidents and disadvantages from the accordion effect, (c) tolerate fluctuations in intensity, and (d) maintain speed throughout the competition, particularly in uphill terrain, as well as (e) having well-developed final sprint abilities, are key factors determining performance during skating-style mass-start cross-country skiing competitions.

How to Communicate Food Safety after Radiological Contamination: The Effectiveness of Numerical and Narrative News Messages.

Food risk and the safety of foodstuffs in the aftermath of contamination are highly sensitive issues to communicate. Food risks receive extensive attention from the news media, which requires messages to be carefully drafted to minimize harm and avoid unnecessary boycotts. Once a food risk is deemed eliminated, communication efforts must rebuild trust among consumers. The latter is a particularly difficult task after radiological contamination. This study tests whether numerical messages, narrative messages, or messages combining both elements are more effective in persuading the public to consume foodstuffs from Fukushima, a region that continues to battle stigma since the nuclear accident in 2011. We employ a survey-embedded experiment on a sample of the general Belgian population (N = 1085), during which respondents are presented with a mock news article presenting either a (1) numerical, (2) narrative, or (3) a combined message and test their subsequent evaluation of the article. We find that the numerical message leads to significantly higher perceived credibility and message acceptance than both the combined and the narrative message. Furthermore, we find that attitudes towards nuclear energy have a strong independent effect on message acceptance and evaluation, with those respondents who report a more positive stance towards nuclear energy more readily accepting the message. Food risk and safety communication may thus benefit from adopting a more detached, numerical approach for sensitive issues.

Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis.

Alzheimer's disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-ß (Aß) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aß peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aß in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.

Genetic human prion disease modelled in PrP transgenic Drosophila.

Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Increasingly, Drosophila has been used to model human neurodegenerative disease. An important unanswered question is whether genetic prion disease with concomitant spontaneous prion formation can be modelled in Drosophila We have used pUAST/PhiC31-mediated site-directed mutagenesis to generate Drosophila transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Drosophila Adult Drosophila transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient Drosophila that expressed the wild-type form of the transgene. Collectively, our novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host.

Prion strains depend on different endocytic routes for productive infection.

Prions are unconventional agents composed of misfolded prion protein that cause fatal neurodegenerative diseases in mammals. Prion strains induce specific neuropathological changes in selected brain areas. The mechanism of strain-specific cell tropism is unknown. We hypothesised that prion strains rely on different endocytic routes to invade and replicate within their target cells. Using prion permissive cells, we determined how impairment of endocytosis affects productive infection by prion strains 22L and RML. We demonstrate that early and late stages of prion infection are differentially sensitive to perturbation of clathrin- and caveolin-mediated endocytosis. Manipulation of canonical endocytic pathways only slightly influenced prion uptake. However, blocking the same routes had drastic strain-specific consequences on the establishment of infection. Our data argue that prion strains use different endocytic pathways for infection and suggest that cell type-dependent differences in prion uptake could contribute to host cell tropism.

Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie.

Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system.

Modulation of Glycosaminoglycans Affects PrPSc Metabolism but Does Not Block PrPSc Uptake.

UNLABELLED: Mammalian prions are unconventional infectious agents composed primarily of the misfolded aggregated host prion protein PrP, termed PrP(Sc). Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as the first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effects on different strains and different stages of the cell infection have not been thoroughly addressed. We examined the effects of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake, and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrP(Sc) uptake, arguing against their roles as essential prion attachment sites. However, both treatments effectively antagonized de novo prion infection independently of the prion strain and reduced PrP(Sc) formation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrP(Sc) formation independent of the prion strain. IMPORTANCE: Recently, glycosaminoglycans (GAGs) became the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formation in vitro. GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effects of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here, we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and thus might affect prion propagation by depleting intracellular cofactor pools.

Prion-induced and spontaneous formation of transmissible toxicity in PrP transgenic Drosophila.

Prion diseases are fatal transmissible neurodegenerative diseases of various mammalian species. Central to these conditions is the conversion of the normal host prion protein PrP(C) into the abnormal prion conformer PrP(Sc). Mature PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol anchor, whereas during biosynthesis and metabolism cytosolic and secreted forms of the protein may arise. The role of topological PrP(C) variants in the mechanism of prion formation and prion-induced neurotoxicity during prion disease remains undefined. In the present study we investigated whether Drosophila transgenic for ovine PrP targeted to the plasma membrane, to the cytosol or for secretion, could produce transmissible toxicity following exposure to exogenous ovine prions. Although all three topological variants of PrP were efficiently expressed in Drosophila, cytosolic PrP was conformationally distinct and required denaturation before recognition by immunobiochemical methods. Adult Drosophila transgenic for pan neuronally expressed ovine PrP targeted to the plasma membrane, to the cytosol or for secretion exhibited a decreased locomotor activity after exposure at the larval stage to ovine prions. Proteinase K-resistant PrP(Sc) was detected by protein misfolding cyclic amplification in prion-exposed Drosophila transgenic for membrane-targeted PrP. Significantly, head homogenate from all three variants of prion-exposed PrP transgenic Drosophila induced a decreased locomotor activity when transmitted to PrP recipient flies. Drosophila transgenic for PrP targeted for secretion exhibited a spontaneous locomotor defect in the absence of prion exposure that was transmissible in PrP transgenic flies. Our data are consistent with the formation of transmissible prions in PrP transgenic Drosophila.

Cellular aspects of prion replication in vitro.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders in mammals that are caused by unconventional agents predominantly composed of aggregated misfolded prion protein (PrP). Prions self-propagate by recruitment of host-encoded PrP into highly ordered b-sheet rich aggregates. Prion strains differ in their clinical, pathological and biochemical characteristics and are likely to be the consequence of distinct abnormal prion protein conformers that stably replicate their alternate states in the host cell. Understanding prion cell biology is fundamental for identifying potential drug targets for disease intervention. The development of permissive cell culture models has greatly enhanced our knowledge on entry, propagation and dissemination of TSE agents. However, despite extensive research, the precise mechanism of prion infection and potential strain effects remain enigmatic. This review summarizes our current knowledge of the cell biology and propagation of prions derived from cell culture experiments. We discuss recent findings on the trafficking of cellular and pathologic PrP, the potential sites of abnormal prion protein synthesis and potential co-factors involved in prion entry and propagation.

Proteasomal dysfunction and endoplasmic reticulum stress enhance trafficking of prion protein aggregates through the secretory pathway and increase accumulation of pathologic prion protein.

A conformational change of the cellular prion protein (PrP(c)) underlies formation of PrP(Sc), which is closely associated with pathogenesis and transmission of prion diseases. The precise conformational prerequisites and the cellular environment necessary for this post-translational process remain to be completely elucidated. At steady state, glycosylated PrP(c) is found primarily at the cell surface, whereas a minor fraction of the population is disposed of by the ER-associated degradation-proteasome pathway. However, chronic ER stress conditions and proteasomal dysfunctions lead to accumulation of aggregation-prone PrP molecules in the cytosol and to neurodegeneration. In this study, we challenged different cell lines by inducing ER stress or inhibiting proteasomal activity and analyzed the subsequent repercussion on PrP metabolism, focusing on PrP in the secretory pathway. Both events led to enhanced detection of PrP aggregates and a significant increase of PrP(Sc) in persistently prion-infected cells, which could be reversed by overexpression of proteins of the cellular quality control. Remarkably, upon proteasomal impairment, an increased fraction of misfolded, fully glycosylated PrP molecules traveled through the secretory pathway and reached the plasma membrane. These findings suggest a novel pathway that possibly provides additional substrate and template necessary for prion formation when protein clearance by the proteasome is impaired.

[Assessment of drug therapy in chronic pulmonary obstructive disease]. / Leczenie chorych na przewlekla obturacyjna chorobe pluc.

UNLABELLED: Several national and international groups of experts have published guidelines for chronic obstructive pulmonary disease (COPD) diagnosis and treatment. But little is known about standards of care in real clinical practices. AIM: Of this study was to compare diagnosis and drug therapy in local medical practices against classical Polish Phthisiopneumonological Society standards for COPD management. Data has been collected in Pneumonology Dpt. Medical University of Gdansk from COPD patients referred between September 2001 and May 2002. One hundred and five consecutive cases have been included into study group. In local medical practices spirometry for COPD diagnosis and staging was used in only 42% of cases. In stable COPD theophilline was used as a first line bronchodilatator. During exacerbation treatment was much closer to guidelines recommendation. Large majority of patients, 87% of study group, was on combined inhaled bronchodilatators. CONCLUSIONS: Spirometry usage in COPD care fell below standards recommendation. Stable COPD treatment was not satisfactory. National program of guidelines implementations should be created.

[Chemotherapy of advanced non-small cell lung cancer with the combination of gemcitabine and cisplatin]. / Ocena skutecznosci gemcytabiny z cisplatyna u chorych na niedrobnokomórkowego raka pluca.

The aim of the study was to assess the efficacy of a combination of gemcitabine and cisplatin in advanced non-small cell lung cancer. Twenty-five patients were included (13--IIIB, and 12--IV stage). Gemcitabine--1000 mg/m2 was given intravenously on days 1, 8, and 15, and cisplatin--100 mg/m2 on day 2. In 13 patients partial remission was obtained, in 8--stabilisation, and in 4--progression. Median survival was 12 months (range: 1.5-32 months). Mean time to progression was 6 months. Toxicity was tolerable and included mainly thrombocytopenia, neutropenia and anemia. In 11 patients pain relief was obtained. Furthermore cough, dyspnoea and hemoptysis disappeared in a proportion of patients. These results indicate the efficacy of the combination of gemcitabine and cisplatin regimen in advanced non-small cell lung cancer, and its acceptable toxicity.