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Aim: To explore gambling in the indigenous Sámi culture by studying thoughts, ideas and attitudes towards gambling among Sámi and people living in majority Sámi areas with knowledge of the culture. Methods: The topic was investigated in an inductive thematic analysis of semi-structured interviews with 14 people (n = 13 self-reported Sámi ethnicity). Results: The majority of the informants knew of superstitious practices that were specific to the Sámi culture, though most did not believe that these could influence gambling outcomes. Several features of the Sámi culture, including religious commitment (Laestadianism), family-oriented societies, non-materialistic ideals and self-sufficiency ideals, were presumed to protect against developing gambling issues. There were reports of reduced trust in the Norwegian healthcare system and a lack of treatment services with sufficient knowledge about Sámi culture. Conclusion: Culture-specific factors protecting against development of gambling problems could be a factor in maintaining established gambling problems by increasing associated shame and stigma, resulting in a higher threshold for help-seeking among Sámi. The findings and their potential implications with regards to the existing literature are discussed., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Genetic markers are widely applied in the study of genetic diversity for many species. The approach incorporates a Polymerase Chain Reaction (PCR) amplification of targeted sequences in the genome. Crucial for the overall success of a PCR experiment is the careful design of synthetic oligonucleotide primers. Ideally designed primer pairs will ensure the efficiency and specificity of the amplification reaction, resulting in a high yield of the desired amplicon. Important criteria such as primer-sequence, -length, and -melting temperature (T m ) are fundamental for the selection of primers and amplification of targeted nucleotide sequences from a DNA template. There are many computational tools available to assist with critical bioinformatics issues related to primer design. These resources allow the user to define parameters and criteria that need to be taken into account when designing primers. Following the initial in silico selection, a primer pair should be further tested in vivo for their amplification efficiency and robustness.Using examples taken from genetic diversity studies in a marine crustacean, this chapter provides outlines for the application of PCR technology and discusses details for the design of primers for the development and characterization of microsatellite and SNP-markers., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Metals sequestered in coastal sediments are normally considered to be stable, but this investigation shows - somewhat surprisingly - that mercury concentrations in a previously contaminated area, Harboøre Tange, Denmark, have decreased since the 1980s. Mercury concentrations were determined in sediment and benthic biota and present values were compared to values in the 1980s and values from areas without known; history of mercury contamination. Concentrations in both the upper 20 cm of the sediments and; biota are considerably lower now compared to latest monitoring (1980s). Sediment. concentrations at most locations have decreased from the 100-300 ng Hg g -1 dry weight (dw) level to levels below the Background Concentration (BC) of 50 ng Hg g -1 dw defined by Oslo-Paris Convention for the Protection of the Marine Environment of the North-East Atlantic; some stations are at the 2-10 ng Hg g -1 dw level characteristic of Danish coastal sediments with no known history of mercury contamination. Concentrations of mercury in the benthic biota along Harboøre Tange have also decreased since the 1980s but despite the lowered mercury concentrations in the sediments, concentrations in most samples of benthic invertebrate fauna still exceed those in uncontaminated coastal areas and also the Environmental Quality Standard (EQS) of 20 ng Hg g -1 wet weight (≈100 ng Hg g -1 dry weight) defined by the European Union's Water Framework Directive. Concentration ranges in selected organisms are: (Harboøre Tange l980s/Harboøre Tange now/uncontaminated areas - given in ng Hg g -1 dw): Periwinkles Littorina littorea 9000/150-450/55-77, blue mussels Mytilus edulis up to 9000/300-500/40-170, cockles Cerastoderma edule up to 8000/400-1200/200, brown shrimp Crangon crangon 700-2200/150-450/47, eelgrass Zostera marina up to 330/25-70/12. The present results - together with a literature review - show that a simple and straight forward relationship between the concentrations of mercury in sediment and benthic organisms does not necessarily exist., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nitrate has the potential to affect steroid production. Nitrate concentrations in streams in agricultural areas may exceed concentrations showing effects in laboratory studies. The effects of nitrate and/or nitrite on endocrine relevant endpoints were tested in zebrafish and brown trout. Zebrafish were exposed in two experiments to nitrate (8.8 to 89 mg NO 3 - /L) and nitrite (3.6 to 19 mg NO 2 - /L) during the period of sexual differentiation and sex ratios were determined. Vitellogenin concentrations were determined in the second experiment. The sex ratio was unaffected by the exposure to nitrate and nitrite. Vitellogenin concentrations were slightly elevated in males (but not females) in all of the groups exposed to nitrate. Juvenile brown trout were exposed to 5.7, 14, and 31 mg NO 3 - /L for 8 days and vitellogenin levels in liver were determined. Vitellogenin concentrations in the females were not affected by exposure, but in the males, there was an overall statistically significant effect of exposure to nitrate with the group exposed to 5.7 mg NO 3 - /L showing a trend of higher vitellogenin concentrations than the control group; levels in the males of the groups exposed to 14 and 31 mg NO 3 - /L were not statistically different from those of the control group. In conclusion, some marginal effect of nitrate in male fish on endocrine activity was observed but the present results for zebrafish, using environmentally relevant concentrations, do not define nitrate and nitrite as endocrine disrupting chemicals according to the generally accepted WHO/IPCS definition because no adverse effects (altered sex ratios) were demonstrated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The nanomaterial community calls for standardized in vitro assays to determine nanoparticle toxicity in the effort to reduce the number of in vivo validation experiments. We demonstrate that chip-based protein detection is suitable for assessing toxicity and may complement traditional assays to improve selection of primary hits for subsequent analysis. As nanodrug mimics, we analyzed the effect of transiently transfected siRNAs in MCF7 breast cancer cells and normal MCF12A breast cells, resembling a differential screen. As a measure of cytotoxicity, we determined cell viability as well as protein expression of glyceraldehyde-3-phosphate dehydrogenase, transferrin receptor, and the proliferation marker Ki67. The evaluation of cell lethality and protein expression unraveled cellular effects overseen by one method alone., (Copyright © 2017 Elsevier Inc. All rights reserved.)

High-throughput screening (HTS) has become an indispensable tool for the pharmaceutical industry and for biomedical research. A high degree of automation allows for experiments in the range of a few hundred up to several hundred thousand to be performed in close succession. The basis for such screens are molecular libraries, that is, microtiter plates with solubilized reagents such as siRNAs, shRNAs, miRNA inhibitors or mimics, and sgRNAs, or small compounds, that is, drugs. These reagents are typically condensed to provide enough material for covering several screens. Library plates thus need to be serially diluted before they can be used as assay plates. This process, however, leads to an explosion in the number of plates and samples to be tracked. Here, we present SAVANAH, the first tool to effectively manage molecular screening libraries across dilution series. It conveniently links (connects) sample information from the library to experimental results from the assay plates. All results can be exported to the R statistical environment or piped into HiTSeekR ( http://hitseekr.compbio.sdu.dk ) for comprehensive follow-up analyses. In summary, SAVANAH supports the HTS community in managing and analyzing HTS experiments with an emphasis on serially diluted molecular libraries.

Introduction: Since the flow diverters (FDs) have been introduced it is possible to treat aneurysms that are considered difficult or impossible to treat with usual endovascular or surgical methods. It is still uncertain which aneurysms are suitable for this new treatment. We present the periprocedural complications, immediate result, late complications, imaging follow-up at 6 and 12 months and clinical follow-up at 2-23 months., Methods: Twenty-two patients with 26 wide-necked or blister-like aneurysms had 23 treatments with implantation of a Silk stent. Eleven patients had re-canalizations, and 11 patients were either untreated or had been treated for another aneurysm., Results: Periprocedural complications were seen in four treatments (17%). However, none of these had clinical consequences. Mortality and morbidity rates were 1 of 22 (5%) and 1 of 22 (5%), respectively. Clinical outcome was unchanged in 16 patients (72%), 3 patients improved (14%) and 3 patients worsened (14%). The end-of-procedure angiography did not show complete occlusion of any of the aneurysms, but at 6 months follow-up angiography, 17 of 25 aneurysms (68%) were completely occluded, and at 12 months, 18 of 21 aneurysms (86%) were occluded., Conclusion: The effect of the Silk FD in terms of occlusion of the aneurysms seems to occur mainly during the first 6 months after placement but continues during the following time. Most delayed complications occur immediately after discontinuing the anticoagulation medication. Considering the complexity of the aneurysms treated, the rate of complications is acceptable.

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.

In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different intracellular compartments and thereby mediate different responses. MEK1-activated ERK2 accumulated in the nucleus and induced proliferation. Conversely, MEK2-activated ERK2 was retained in the cytoplasm and allowed survival. Localization was a determinant for ERK2 functions since MEK1 switched from providing proliferation to be a mediator of survival when ERK2 was routed to the cytoplasm by the attachment of a nuclear export site. MEK1-mediated ERK2 nuclear translocation and proliferation were shown to depend on phosphorylation of S298 and T292 sites in the MEK1 proline-rich domain. These sites are phosphorylated on cellular adhesion in MEK1 but not MEK2. Whereas p21-activated kinase phosphorylates S298 and thus enhances the MEK1-ERK2 association, ERK2 phosphorylates T292, leading to release of active ERK2 from MEK1. On the basis of these results, we propose that the requirement of adhesion for cells to proliferate in response to growth factors, in part, may be explained by the MEK1 S298/T292 control of ERK2 nuclear translocation. In addition, we suggest that ERK2 intracellular localization determines whether growth factors mediate proliferation or survival and that the sorting occurs in an adhesion-dependent manner.

Elaborate repair pathways counteract the deleterious effects of DNA damage by mechanisms that are understood in reasonable detail. In contrast, repair of damaged RNA has not been widely explored. This may be because aberrant RNAs are generally assumed to be degraded rather than repaired. The reason for this view is well founded, since conserved surveillance mechanisms that degrade abnormal RNAs are thoroughly documented. Numerous proteins and protein-RNA complexes are involved in the metabolism of different RNA species, assuring correct transcription, splicing, posttranscriptional modifications, transport, translation and timely degradation of the molecule. However, like DNA, RNA is under constant attack of various environmental and endogenous agents that damage the molecule, such as alkylating agents, radiation and free radicals. Importantly, many DNA damaging drugs used in cancer therapy also modify RNA, presumably causing delayed or faulty translation. This may result in generation of inactive proteins, dominant negative proteins or toxic protein aggregates. Several lines of evidence indicate RNA repair as a possible cellular defence mechanism to cope with RNA damage. Thus, there are convincing examples of tRNA repair by elongation of truncated forms, and repair of cleaved tRNA by T4 phage proteins. In addition, in vitro repair of aberrant tRNA methylation by a methyl transferase has been reported. Finally, recent reports on repair of chemically methylated RNA by AlkB and a human homologue (hABH3) in vitro and in vivo strengthen the idea of RNA base repair as a cellular defence mechanism.

Protein kinase D (PKD), a family of serine/threonine kinases, can be activated by a multitude of stimuli in a protein kinase C-dependent or -independent manner. PKD is involved in signal transduction pathways controlling cell proliferation, apoptosis, motility, and protein trafficking. Despite its versatile functions, few genuine in vivo substrates for PKD have been identified. In this study we demonstrate that the transcription factor cAMP-response element-binding protein (CREB) is a direct substrate for PKD. PKD1 and CREB interact in cells, and activated PKD1 provokes CREB phosphorylation at Ser-133 both in vitro and in vivo. A constitutive active mutant of PKD1 stimulates GAL4-CREB-mediated transcription in a Ser-133-dependent manner, activates CRE-responsive promoters, and increases the expression of CREB target genes. PKD1 also enhances transcription mediated by two other members of the CREB family, ATF-1 and CREM. Our results describe a novel mechanism for PKD-induced signaling through activation of the transcription factor CREB and suggest that stimulus-induced phosphorylation of CREB, reported to be mediated by protein kinase C, may involve downstream activated PKD.

Cyclic adenosine 3',5'-monophosphate (cAMP) was originally shown to induce gene transcription through activation of cAMP-dependent protein kinase (PKA), and subsequent phosphorylation of the transcription factor cAMP response element-binding protein, CREB, at serine-133. However, elevated cAMP levels may activate multiple signalling pathways with protein kinases that can phosphorylate CREB at serine-133. We analysed the pathways involved in CREB phosphorylation and activation in NIH 3T3 cells exposed to the cAMP elevating agent forskolin. PKA represented the predominant pathway during the burst phase, while the mitogen-activated protein kinase p38 pathway became activated in a PKA-dependent fashion in forskolin treated cells. The phosphorylation kinetics of p38 was delayed compared to PKA activation. Activated p38 stimulated CREB-mediated transcription and potentiated the transcriptional strength of CREB provoked by forskolin. The p38-mediated activation of CREB was inhibited by dominant negative mutants of MSK-1 and by the PKA/MSK-1 inhibitor H89, but not by dominant negative mutants of MSK-2/RSK-B and MAPKAPK2. Our results suggest that forskolin-induced CREB phosphorylation and activation in NIH 3T3 cells is mediated directly by PKA and by a time-delayed PKA-dependent p38/MSK-1 pathway. This bifurcation and time-dependent regulation of the cAMP-responsive signalling pathways may enable the cell to endure and/or enforce a cellular response provoked by a cAMP-elevating stimulus.

The transactivation domain of the cAMP response element-binding protein (CREB) consists of two major domains. The glutamine-rich Q2 domain, which interacts with the general transcription factor TAFII130/135, is sufficient for the recruitment of a functional RNA polymerase II complex and allows basal transcriptional activity. The kinase-inducible domain, however, mediates signal-induced activation of CREB-mediated transcription. It is generally believed that recruitment of the coactivators CREB-binding protein (CBP) and p300 after signal-induced phosphorylation of this domain at serine-133 strongly enhances CREB-dependent transcription. Transcriptional activity of CREB can also be potentiated by phosphoserine-133-independent mechanisms, and not all stimuli that provoke phosphorylation of serine-133 stimulate CREB-dependent transcription. This review presents an overview of the diversity of stimuli that induce CREB phosphorylation at Ser-133, focuses on phosphoserine-133-dependent and -independent mechanisms that affect CREB-mediated transcription, and discusses different models that may explain the discrepancy between CREB Ser-133 phosphorylation and activation of CREB-mediated transcription.

Recruitment of a RNA polymerase II complex by the glutamine-rich Q2 domain of cAMP response element-binding protein (CREB) allows basal transcriptional activity, while recruitment of CBP/p300 through signal-induced phosphorylation of the kinase-inducible domain at serine-133 enhances CREB-dependent transcription. Here we demonstrate that co-administration of forskolin and phorbol ester TPA to NIH3T3 cells provoked a dose-dependent increase in phosphoserine-133. CREB- and Q2-dependent transcription, as well as transcription by other glutamine-rich transcription factors, but not by transcription factors lacking glutamine-rich regions, augmented synergistically in the presence of both stimuli. Synergistic activation was abograted by specific inhibition of protein kinase C (PKC), but not of PKA. Co-stimulation increased the basal activity of a minimal, CREB-independent promoter. Therefore, Q2, which directly interacts with the RNA polymerase II initiation complex, may transmit the increased basal promoter activity provoked by these stimuli to CREB, thereby contributing to synergistic activation of CREB-mediated transcription. This synergism may have important implications on glutamine-rich transcription factor-target genes.