Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20-23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.

Janus kinases 1 and 2 regulate chemokine-mediated integrin activation and naïve T-cell homing.

Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, nonactivated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naïve T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ezrin/radixin/moesin (ERM) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated Ag-1 and very late Ag-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions.

'Saying it without words': a qualitative study of oncology staff's experiences with speaking up about safety concerns.

OBJECTIVES To explore the experiences of oncology staff with communicating safety concerns and to examine situational factors and motivations surrounding the decision whether and how to speak up using semistructured interviews. SETTING 7 oncology departments of six hospitals in Switzerland. PARTICIPANTS Diverse sample of 32 experienced oncology healthcare professionals. RESULTS Nurses and doctors commonly experience situations which raise their concerns and require questioning, clarifying and correcting. Participants often used non-verbal communication to signal safety concerns. Speaking-up behaviour was strongly related to a clinical safety issue. Most episodes of 'silence' were connected to hygiene, isolation and invasive procedures. In contrast, there seemed to exist a strong culture to communicate questions, doubts and concerns relating to medication. Nearly all interviewees were concerned with 'how' to say it and in particular those of lower hierarchical status reflected on deliberate 'voicing tactics'. CONCLUSIONS Our results indicate a widely accepted culture to discuss any concerns relating to medication safety while other issues are more difficult to voice. Clinicians devote considerable efforts to evaluate the situation and sensitively decide whether and how to speak up. Our results can serve as a starting point to develop a shared understanding of risks and appropriate communication of safety concerns among staff in oncology.

Sexual harassment over the telephone: Occupational risk at call centers

The phenomenon of sexually harassing telephone calls in the workplace has been studied only marginally. In the present study 106 employees working in call centres in Germany answered a questionnaire regarding their experiences of sexual harassment over the telephone. The following data are presented: description of the phenomenon, i.e. prevalence and characteristics, stress reactions of the victims, behavioural reactions and coping strategies, consequences and anticipated consequences; prediction of the stress reactions by characteristics of the situation; and employees' recommendations for coping with sexually harassing calls. It was found that the female employees were more often sexually harassed over the telephone at work than their male colleagues. Three out of four female employees had experienced sexually harassing telephone calls; in the majority of cases the harassers were men. Characteristic patterns of harassment included groaning, sexual insults, silence, and threats of sexual violence. Some 16% of the harassed female employees described these experiences as extremely stressful. If the harassment contained threats of sexual violence and groaning, the perceived physical response was stronger. Being subjected to sexual harassment over the telephone both at home and at work was a more severe stress than having the experience only in the workplace. In conclusion, employees' recommendations for coping with the occurrence of sexually harassing calls are described.

Taboo Language and Illness Online: The Use of Specific Emoticons and Graphic Signs to Talk about HIV/AIDS in South Africa

South Africa is one of the countries most affected by HIV/AIDS: According to 2014 UNAIDS data 6.8 million South Africans live with HIV/AIDS, which means a 18.9% prevalence rate among adults (15-49 years old). Despite this strong presence of HIV/AIDS in South African society it remains relatively stigmatized and is not openly talked about. The silence about HIV/AIDS maintained in everyday conversations and the superstitions associated with this illness have led to the creation of a taboo language. This study aims at shedding light on how South African users resort to specific emoticons and graphic signs to talk about HIV/AIDS online. For this purpose 368 Facebook status updates and comments concerning HIV/AIDS and its side effects were analysed. All participants, aged 14-48, lived at the moment of data collection in Cape Town, in the Cape Flats area. The online conversations investigated are mainly in English mixed with Afrikaans and/or Xhosa. The emoticons and graphic signs in most cases display a graphic depiction of the physical (and mental) effects of the illness. These linguistic and semiotic practices employed on Facebook provide insight into how Capetonian users, on the one hand, express solidarity and sympathy with people suffering from HIV/AIDS. On the other hand, the emoticons and graphic signs are used to label and position people affected by HIV/AIDS. Thus, in the South African context social network sites have become an important space and means for communicating HIV/AIDS issues.

Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium.

During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5'-RACE). Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and overexpression of proteins normally active in other life cycle stages will help to understand the function of the proteins investigated.