KNOWLEDGE AND ATTITUDE REGARDING VAGINAL RING AS A FORM OF CONTRACEPTION AMONG LEBANESE WOMEN

This study aimed to evaluate the knowledge of Lebanese women concerning the vaginal ring as a contraceptive means, and to assess their attitude regarding the usage of this form of contraception.502 participants of ages 20-49 were recruited to complete a 10-minute questionnaire to assess their contraceptive knowledge, awareness, and attitude towards the vaginal ring. Knowledge was evaluated among those who had heard about the ring by completing 16 questions. While those of no previous knowledge of the ring were directed towards a brief description about it. All participants completed 7 questions to assess their attitude towards the vaginal ring, and whether or not they might consider using it. Of all the participants 79.8 % recorded having knowledge about different forms of contraceptives, but only 29.1% knew about the ring. Of the latter, 105 had a score less than 50, and were thus considered to have poor knowledge. Whereas, 34 participants had a score of 50 and above, and were considered to have sufficient knowledge. Of the participants, 66.5% would not consider using the ring in the future. Age, level of education and enrollment in the medical field among other factors had a significant impact on the attitude of women towards the vaginal ring. Lack of advertisement, poor counselling from medical professionals, and unavailability of the product had a significant role in the inadequate awareness towards it. The ring did not seem appealing for most of the participants

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

INCENP acts as a protein scaffold that integrates the functions of two crucial mitotic kinases, Aurora B and Polo, at centromeres of mitotic chromosomes

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

INCENP acts as a protein scaffold that integrates the functions of two crucial mitotic kinases, Aurora B and Polo, at centromeres of mitotic chromosomes

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell–Trophoblast Interactions

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction

Assessment of Combination Therapy in BALB/c Mice Injected With Carbapenem-Resistant Enterobacteriaceae Strains

Monotherapeutic options for carbapenem resistant infections are limited. Studies suggest that combination therapy may be associated with better outcomes than monotherapies. However, this is still controversial. This study assessed, the efficacy of combination therapy against carbapenem resistant Enterobacteriaceae harboring singly various ESBL or carbapenemase encoding genes. Thus, four isolates harboring either blaCTXM-15, blaCTXM-15 and blaOXA-48, blaNDM-1, or blaKPC-2 genes were selected for testing. Minimal Inhibitory Concentration (MIC) was determined by broth dilution method. Gene transcript levels on single and combined treatments were done in vitro and in vivo by q RT-PCR. Assessment of treatments was done in BALB/c mice according to a specific protocol. As such, the qRT-PCR revealed a significant decrease of transcript levels in all isolates upon using rifampicin or tigecycline, singly or in combination with colistin. However, variable levels were obtained using colistin singly or in combination with meropenem or fosfomycin. In vivo assessment showed that all combinations used were effective against isolates harboring blaCTXM-15, blaOXA-48, and blaNDM-1. Conversely, the most significant combination against the isolate harboring blaKPC-2 gene was colistin with carbapenem, fosfomycin, or kanamycin. As a conclusion, combination therapy selected based on the type of carbapenemase produced, appeared to be non-toxic and might be effective in BALB/c mice. Therefore, the use of a rationally optimized combination therapy might lead to better results than monotherapy, however, clinical trials are needed for human consumption

INCENP is required for the activation of Polo kinase at the inner centromere.

<p>(A) Sequence alignment showing the conservation of Thr182 across species. (B) Immunoblot of cell lines stably expressing wild-type, T182A, or T182D Polo in the absence or presence of okadaic acid. The phosphoespecific antibody anti-Plk1^T210Ph recognises <i>Drosophila</i> Polo^T182Ph, both Myc-tagged and endogenous (e). Both endogenous and Myc-tagged Polo^T182Ph were detected as a doublet, suggesting that they can be modified at another site. The asterisk indicates a non-specific band that does not disappear after Polo RNAi. This band increases following okadaic treatment, and therefore could correspond to a non-specific phospho-epitope. (C–F) Control or RNAi-treated DMel-2 cells stably expressing Polo-GFP showing colocalization of INCENP and Polo/Polo^T182Ph. Arrows point to chromosomes shown in zoomed images. Linescans show fluorescence intensity across the kinetochores (dashed lines). (C) Control prometaphase. Polo^T182Ph is visibly enriched at the inner centromere compared to Polo (arrows). Linescans show both Polo and Polo^T182Ph are present at the inner centromere (double-ended arrows show difference in intensity with respect to background levels: green, Polo blue, Polo^T182Ph). (D) Control metaphase. Asterisks point to centrosomes, and Polo^T182Ph is virtually undetectable in the inner centromere (linescan; note that intensity drops to background level). (E,F) INCENP RNAi-treated cells. Asterisks point to Polo^T182Ph on centrosomes (note absence from kinetochores, also shown in linescans). Zoomed images show localization at the centromere/kinetochore of the indicated proteins.</p

Aurora B activity is required for the activation of Polo kinase at the inner centromere.

<p>(A) Aurora B phosphorylates Polo kinase in vitro. Bacterially expressed HIS-Polo or HIS-Polo^T182A (which is catalytically inactive and therefore unable to autophosphorylate) were incubated with (or without) Drosophila Aurora B in complex with a fragment of INCENP (residues 654–755) in presence of ³²P-g-ATP, in triplicate. Reaction products were resolved by SDS-PAGE transferred to nitrocellulose and analyzed by autoradiography (AR) and anti-Polo Western blot (WB). Quantitative measurements of signals were obtained (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001250#s4" target="_blank">Materials and Methods</a>), and the ratios were calculated for each reaction (AR/WB, A.U.: arbitrary units). Right, average values for the relative phosphorylatin of Polo^WT and Polo^T182A by Aurora B. Error bars, SEM. (B–D) DMel-2 cells stably expressing Polo-GFP treated with (B) DMSO or (C–D) Binucleine-2, immunostained for INCENP, Polo, and Polo^T182Ph (insets: zoomed images of kinetochores). In (C–D) asterisks indicate centrosomes. Merged images show INCENP/Polo/DNA. Zoomed images in (C–D) insets show examples of kinetochore pairs showing decreased levels of Polo^T182Ph. (E) Dot plot showing the quantification of INCENP/Polo/Polo^T182Ph signal intensity at the kinetochore (<i>t</i> test: *** <i>p</i><0.0001; n.s., not significant; <i>p</i> = 0.4028). Signal intensities for individual kinetochores were measured using the SoftWorx Data Inspector tool; average background was subtracted; data was plotted using KaleidaGraph software. (F) RNAi depletion of Aurora B, but not Aurora A, strongly reduces Polo^T182Ph levels in DMel-2 cells treated with okadaic acid. Cells were transfected with the indicated dsRNAs for 4 d, and 100 nM okadaic acid added for 4 h before immunoblotting to improve visualization of phosphorylated Polo. A dsRNA against the Kanamycin resistance bacterial gene was used as a negative control. Asterisks: non-specific bands. Both bulk Polo and PoloT^182ph appear as doublets. (G) RNAi depletion of Aurora B or INCENP, but not Aurora A, reduces Polo^T182Ph levels at centromeres/kinetochores. Cycling cells were treated with the indicated dsRNAs for 3 d (immunoblots are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001250#pbio.1001250.s006" target="_blank">Figure S6B</a>) and Polo^T182Ph was detected by immunofluorescence. Levels of Polo^T182Ph at centromeres/kinetochores in prometaphase and metaphase cells were measured at individual kinetochores using Image J, subtracting background (Kt-bkd). Asterisks indicate centrosomes. Error bars = S.E.M.</p

The centromeric activation of Polo in mitosis depends on INCENP and Aurora B in vivo.

<p>(A–E) Immunostaining analysis of the phosphorylation of Polo^T182 in third instar neuroblasts of (A) wild type larvae (Canton-S), (B) <i>incenp ^QA26</i> mutant larvae, and (C–D) wild type larvae treated with the Aurora B-specific inhibitor Binucleine 2. INCENP (2, green), Polo^T182Ph (3, red). Arrows point to INCENP blocks characteristic of the <i>incenp ^QA26</i> and Binucleine-2 treatment phenotype. Asterisks indicate centrosomes. (E) Dot plot showing the quantification of INCENP/Polo^T182Ph signal intensity at the kinetochore (<i>t</i> test: *** <i>p</i><0.0001; ** <i>p</i> = 0.003). Signal intensities for individual kinetochores were measured using the SoftWorx Data Inspector tool; average background was subtracted; data was plotted using KaleidaGraph software. (F–I) Levels of Polo kinase are not affected by defects in Incenp or Aurora B function (F) wild type larvae (Canton-S), (G) <i>incenp ^QA26</i> mutant larvae, and (H) wild type larvae treated with the Aurora B-specific inhibitor Binucleine 2. INCENP (5, red), Polo (6, green). (I) Dot plot showing the quantification of INCENP/Polo signal intensity at the kinetochore (<i>t</i> test: *** <i>p</i><0.0001). Signal intensities for individual kinetochores were measured using the SoftWorx Data Inspector tool; average background was subtracted; data were plotted using KaleidaGraph software.</p