Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di- GMP) is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. Current methodologies for the quantification of c-di-GMP are typically based on chemical extraction, representing end point measurements. Chemical methodologies also fail to take into consideration the physiological heterogeneity of the biofilm and thus represent an average c-di-GMP concentration across the entire biofilm. To address these problems, a ratiometric, image-based quantification method has been developed based on expression of the green fluorescence protein (GFP) under the control of the c-di-GMPresponsive cdrA promoter (Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., and Tolker-Nielsen, T. (2012) Appl. Environ. Microbiol. 78, 5060-5069). The methodology uses the cyan fluorescent protein (CFP) as a biomass indicator and the GFP as a c-di-GMP reporter. Thus, the CFP/GFP ratio gives the effective c-di-GMP per biomass. A binary mask was applied to alleviate background fluorescence, and fluorescence was calibrated against known c-di-GMP concentrations. Using flow cells for biofilm formation, c-di-GMP showed a non-uniform distribution across the biofilm, with concentrated hot spots of c-di- GMP. Additionally, c-di-GMP was found to be localized at the outer boundary of mature colonies in contrast to a uniform distribution in early stage, small colonies. These data demonstrate the application of a method for the in situ, real time quantification of c-di-GMP and show that the amount of this biofilmregulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time

Implementasi Manajemen Berbasis Sekolah (Mbs) dalam Meningkatkan Profesionalisme Guru di SD Negeri 10 Banda Aceh

Adapun latar belakang masalah dari penelitian ini adalah masih banyak guru yang kurang profesional dibidangnya, seperti adanya guru yang terlambat masuk mengajar, tidak mempunyai perangkat pembelajaran yang lengkap, kurangnya koordinasi antar kepala sekolah dan guru. Penelitian ini bertujuan untuk mengetahui profesionalisme guru melalui implementasi Manajemen Berbasis Sekolah (MBS) di SD Negeri 10 Banda Aceh.Pendekatan yang digunakan dalam penelitian ini adalah kualitatif dengan jenis penelitian deskriptif. Subjek dari penelitian ini adalah guru SD Negeri 10 Banda Aceh berjumlah 10 orang dipilih dengan menggunakan teknik purpossive sampling. Teknik pengumpulan data dengan menggunakan observasi dan wawancara.Data hasil penelitian diolah dengan mereduksi data, penyajian data, dan penarikan kesimpulanHasil penelitian ini menunjukkan bahwa kepala sekolah melakukan diskusi untuk pengambilan kebijakan yang akan diterapkan oleh sekolah, kepala sekolah melakukan evaluasi dengan memeriksa perangkat pembelajaran, kehadiran guru, prestasi belajar siswa dan keaktifan pada UKG yang dilakukan di gugus. Kelulusan siswa setiap tahunnya mencapai 100% lulus. Ini membuktikan prestasi sekolah yang diperoleh melalui keberhasilan siswa, guru-guru memanfaatkan waktu dengan sebaik-baiknya pada peroses belajar mengajar, guru menggunakan berbagai media pembelajaran. Siswa memiliki peningkatan pada hasil belajar dan dengan demikian terlihat peningkatan profesionalisme guru melalui implementasi manajemen berbasis sekolah

Is Canada really an education superpower? The impact of non-participation on results from PISA 2015

The purpose of large-scale international assessments is to compare educational achievement across countries. For such cross-national comparisons to be meaningful, the participating students must be representative of the target population. In this paper, we consider whether this is the case for Canada, a country widely recognised as high performing in the Programme for International Student Assessment (PISA). Our analysis illustrates how the PISA 2015 sample for Canada only covers around half of the 15-year-old population, compared to over 90% in countries like Finland, Estonia, Japan and South Korea. We discuss how this emerges from differences in how children with special educational needs are defined and rules for their inclusion in the study, variation in school participation rates and the comparatively high rates of pupils’ absence in Canada during the PISA study. The paper concludes by investigating how Canada’s PISA 2015 rank would change under different assumptions about how the non-participating students would have performed were they to have taken the PISA test

Article Retracted

This article has been retracted from publication on author's reques

Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by the lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. A total of 46% of patients with RDEB harbor at least one premature termination codon (PTC) mutation in COL7A1, and previous studies have shown that aminoglycosides are able to overcome RDEB PTC mutations by inducing read-through and incorporation of an amino acid at the PTC site. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in cells derived from patients with RDEB. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases more than 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense-mediated mRNA decay, suggesting that amlexanox inhibits nonsense-mediated mRNA decay in cells from patients with RDEB that respond to read-through treatment. This preclinical study demonstrates the potential of repurposing amlexanox for the treatment of patients with RDEB harboring PTC mutation in COL7A1

Disruption of ERBB2IP Is not Associated with Dystrophic Epidermolysis Bullosa in Both Father and Son Carrying a Balanced 5;13 Translocation

Mutations in the type VII collagen gene (COL7A1) cause autosomal recessive and autosomal dominant inherited dystrophic epidermolysis bullosa (DEB). We report a family with three individuals who present blistering, scarring, hypo- and hyperpigmentation, and nail dystrophy suggestive for DEB. Whereas father and son carry a 5;13 translocation, the daughter shows a normal karyotype. Segregation analysis revealed that all affected family members inherited the same COL7A1 allele. Mutation analysis disclosed a heterozygous missense mutation, c.6227G>A (p.G2076D), in COL7A1 in all affected individuals. Delineation of the translocation breakpoints showed that the ERBB2IP (erbb2 interacting protein or Erbin) gene is disrupted in 5q13.1 and GPC6 in 13q32. GPC6 encodes glypican 6 belonging to a family of cell surface heparan sulfate proteoglycans. The binding partners of Erbin, BP230 (BPAG1) and the integrin β4 subunit, both involved in hemidesmosome (HD) function, and the presence of Erbin in HD suggested that it plays a role in establishment and maintenance of cell-basement membrane adhesions. However, loss of function of one ERBB2IP copy or expression of a putative novel ERBB2IP fusion protein did not apparently modulate the DEB phenotype in both translocation patients. Nonetheless, one cannot yet exclude that ERBB2IP is a candidate for human blistering disorders such as epidermolysis bullosa

Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants.

BACKGROUND: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. RESULTS: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. CONCLUSIONS: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation

Rationale for UV-filtered clover fermions

We study the contributions Sigma_0 and Sigma_1, proportional to a^0 and a^1, to the fermion self-energy in Wilson's formulation of lattice QCD with UV-filtering in the fermion action. We derive results for m_{crit} and the renormalization factors Z_S, Z_P, Z_V, Z_A to 1-loop order in perturbation theory for several filtering recipes (APE, HYP, EXP, HEX), both with and without a clover term. The perturbative series is much better behaved with filtering, in particular tadpole resummation proves irrelevant. Our non-perturbative data for m_{crit} and Z_A/(Z_m*Z_P) show that the combination of filtering and clover improvement efficiently reduces the amount of chiral symmetry breaking -- we find residual masses am_{res}=O(10^{-2}).Comment: 25 pages, 4 figures; v2: typo in eqn. (37) fixed [agrees with published version

Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors : evidence of a novel benzodiazepine site in the α1-α1 interface

Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1β3 GABAA receptors in either the 3α1:2β3 or 2α1:3β3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a β3-β3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn2+. At the 3α1:2β3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3β3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1β3 (3α1:2β3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target