Splicing-dependent NMD does not require the EJC in Schizosaccharomyces pombe

Nonsense-mediated mRNA decay (NMD) is a translation-linked process that destroys mRNAs with premature translation termination codons (PTCs). In mammalian cells, NMD is also linked to pre-mRNA splicing, usually PTCs trigger strong NMD only when positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems. Here, we report that in Schizosaccharomyces pombe splicing also enhances NMD, but against the EJC model prediction, an intron stimulated NMD regardless of whether it is positioned upstream or downstream of the PTC and EJC components are not required. Still the effect of splicing seems to be direct—we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing. On the basis of these results, we propose a new model to explain how splicing could affect NMD

Determination of liposome/water partition coefficients of organic acids and bases by solid-phase microextraction

The extraction of two methylated anilines and three chlorinated phenols by solid-phase microextraction (SPME) fibers coated with polyacrylate was investigated as a function of pH. Only the neutral species of the acids and bases partitioned into the polymer. Extraction kinetics were accelerated for the hydrophobic phenols at pH values around their acidity constant. This is presumably due to a reconstitution of the neutral species in the unstirred aqueous layer adjacent to the polymer surface by the charged species through the fast acid-base equilibrium. Although the charged species is not taken up into the polymer. liposome/water distribution ratios could be measured up to a pH value, where 99% of the compounds were present as charged species. The partition coefficients of the neutral and charged species were extrapolated from the pH profiles of the liposome/water distribution ratios. The resulting values were slightly lower than those measured with equilibrium dialysis. The discrepancies are discussed with respect to differences in the experimental conditions and the possibility of matrix effects during SPME measurements

Adsorption of Streptococcus mutans on Chemically Treated Hydroxyapatite

Adsorption of Streptococcus mutans on hydroxyapatite and chemically treated hydroxyapatite was studied. Zeta potentials of the surfaces were measured. Chemically treated hydroxyapatite gave higher ζ potentials and lower S mutans adsorption.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67845/2/10.1177_00220345780570091601.pd

Identification and characterization of an inhibitory fibroblast growth factor receptor 2 (FGFR2) molecule, up-regulated in an Apert Syndrome mouse model

AS (Apert syndrome) is a congenital disease composed of skeletal, visceral and neural abnormalities, caused by dominant-acting mutations in FGFR2 [FGF (fibroblast growth factor) receptor 2]. Multiple FGFR2 splice variants are generated through alternative splicing, including PTC (premature termination codon)-containing transcripts that are normally eliminated via the NMD (nonsense-mediated decay) pathway. We have discovered that a soluble truncated FGFR2 molecule encoded by a PTC-containing transcript is up-regulated and persists in tissues of an AS mouse model. We have termed this IIIa–TM as it arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 [TM (transmembrane domain)]. IIIa–TM is glycosylated and can modulate the binding of FGF1 to FGFR2 molecules in BIAcore-binding assays. We also show that IIIa–TM can negatively regulate FGF signalling in vitro and in vivo. AS phenotypes are thought to result from gain-of-FGFR2 signalling, but our findings suggest that IIIa–TM can contribute to these through a loss-of-FGFR2 function mechanism. Moreover, our findings raise the interesting possibility that FGFR2 signalling may be a regulator of the NMD pathway

Automated Counting of Bacterial Colony Forming Units on Agar Plates

Manual counting of bacterial colony forming units (CFUs) on agar plates is laborious and error-prone. We therefore implemented a colony counting system with a novel segmentation algorithm to discriminate bacterial colonies from blood and other agar plates

Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

<p>Abstract</p> <p>Background</p> <p>Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis <it>in vivo</it>, from the start point to tumor establishment.</p> <p>Results</p> <p>We first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without <it>in vitro </it>telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants.</p> <p>Conclusions</p> <p>TERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.</p

Porphyromonas endodontalis in chronic periodontitis: a clinical and microbiological cross-sectional study

Although previous studies have shown the presence of Porphyromonas endodontalis in chronic periodontitis associated with periapical lesions, the occurrence of this pathogen in diseased periodontal sites without periapical lesions has been poorly investigated.The aims of this study were to quantify P. endodontalis in patients with chronic periodontitis without periapical lesions, to evaluate the potential correlation of P. endodontalis with Porphyromonas gingivalis and Tannerella forsythia, and to evaluate the ability of periodontal treatment to reduce these pathogens.Patients with generalized chronic periodontitis were selected by recording clinical attachment level (CAL), probing depth (PD), and bleeding on probing (BOP). Subgingival samples from 30 diseased nonadjacent sites (CAL&#x200A;&#x2265;&#x200A;5 mm, PD between 5 and 7 mm and positive BOP) and 30 healthy nonadjacent sites (PD&#x200A;&#x2264;&#x200A;3 mm and negative BOP) were collected and subjected to microbial analysis by quantitative polymerase chain reaction (qPCR) The variables of age, PD, CAL and BOP of all individuals were analyzed using the paired t-test (GrapPad Prism5&#x00AE;). Data of bacteria quantification were subjected to a normality test (D&#x0027;Agostino-Pearson Test). For bacterial correlation analysis, the Spearman correlation was used.Our results showed that diseased sites had significantly higher levels of P. endodontalis compared to healthy sites, similar to the results obtained for P. gingivalis and T. forsythia. The numbers of all bacterial species were reduced significantly after mechanical periodontal treatment. P. endodontalis was significantly correlated with the presence of T. forsythia and P. gingivalis in the diseased group.Our results suggest that there is a high prevalence of P. endodontalis, P. gingivalis and T. forsythia in periodontitis sites and that mechanical periodontal treatment is effective at reducing the pathogens studied

The Drosophila afadin homologue Canoe regulates linkage of the actin cytoskeleton to adherens junctions during apical constriction

Cadherin-based adherens junctions (AJs) mediate cell adhesion and regulate cell shape change. The nectin–afadin complex also localizes to AJs and links to the cytoskeleton. Mammalian afadin has been suggested to be essential for adhesion and polarity establishment, but its mechanism of action is unclear. In contrast, Drosophila melanogaster’s afadin homologue Canoe (Cno) has suggested roles in signal transduction during morphogenesis. We completely removed Cno from embryos, testing these hypotheses. Surprisingly, Cno is not essential for AJ assembly or for AJ maintenance in many tissues. However, morphogenesis is impaired from the start. Apical constriction of mesodermal cells initiates but is not completed. The actomyosin cytoskeleton disconnects from AJs, uncoupling actomyosin constriction and cell shape change. Cno has multiple direct interactions with AJ proteins, but is not a core part of the cadherin–catenin complex. Instead, Cno localizes to AJs by a Rap1- and actin-dependent mechanism. These data suggest that Cno regulates linkage between AJs and the actin cytoskeleton during morphogenesis