Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

BACKGROUND: Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. RESULTS: Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galβ-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8–66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (>97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. CONCLUSION: The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated

A novel SOD1 splice site mutation associated with familial ALS revealed by SOD activity analysis

More than 145 mutations have been found in the gene CuZn-Superoxide dismutase (SOD1) in patients with amyotrophic lateral sclerosis (ALS). The vast majority are easily detected nucleotide mutations in the coding region. In a patient from a Swiss ALS family with half-normal erythrocyte SOD1 activity, exon flanking sequence analysis revealed a novel thymine to guanine mutation 7 bp upstream of exon 4 (c.240-7T>G). The results of splicing algorithm analyses were ambiguous, but five out of seven analysis tools suggested a potential novel splice site that would add six new base pairs to the mRNA. If translated, this mRNA would insert Ser and Ile between Glu78 and Arg79 in the SOD1 protein. In fibroblasts from the patient, the predicted mutant transcript and the mutant protein were both highly expressed, and despite the location of the insertion into the metal ion-binding loop IV, the SOD1 activity appeared high. In erythrocytes, which lack protein synthesis and are old compared with cultured fibroblasts, both SOD1 protein and enzymic activity was 50% of controls. Thus, the usage of the novel splice site is near 100%, and the mutant SOD1 shows the reduced stability typical of ALS-associated mutant SOD1s. The findings suggests that this novel intronic mutation is causing the disease and highlights the importance of wide exon-flanking sequencing and transcript analysis combined with erythrocyte SOD1 activity analysis in comprehensive search for SOD1 mutations in ALS. We find that there are potentially more SOD1 mutations than previously reporte

Kan internetbaserade quiz med fokus på formativ bedömning användas i naturkunskapsundervisningen för gymnasieelever?

Formativ bedömning har visats ge stora positiva effekter på lärande och rekommenderas användas i grundskolornas undervisning. Dock har det inte varit helt enkelt att implementera metoden i skolan. Metoden förutsätter ett tryggt klassrumsklimat och trygga elever, omständigheter som inte alltid råder. Något som kan försvåra ytterligare är om elever har dåligt självförtroende i ämnet, något som forskning visat att många elever har i de naturvetenskapliga ämnena. Ämnet naturkunskap innehåller en stor mängd kunskapsstoff, termer och begrepp. Ett självrättande internetbaserat test skulle kunna vara ett användbart redskap för inlärning i detta ämne. Några studier har indikerat att kamratbedömning, en av nyckelstrategierna i formativ bedömning, kan ge ökad oro och otrygghet hos elever och andra att bedömning som görs anonymt kan öka deras trygghetskänsla. Syftet med denna undersökning var därför att undersöka hur anonyma internetbaserade test upplevs av gymnasieelever och om de är användbara redskap för självbedömning i naturkunskap. Undersökningen gjordes med en enkät. Resultaten visade att en stor andel elever var positiva till att göra test anonymt och att de upplevde att testen ökade deras insikter om vad de kunde och inte, alltså indikerar denna studie att anonyma internetbaserade test kan vara positiva för elevers trygghet och för självreglerat lärande

Suppressor of zeste 12, a Polycomb group gene in Drosophila melanogaster; one piece in the epigenetic puzzle

In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing Drosophila embryo, the proper expression pattern of e.g. the homeotic genes Abd-B and Ubx is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression. As part of this thesis, Suppressor of zeste 12 (Su(z)12) is characterized as a Drosophila Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes Abd-B and Ubx, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. Su(z)12 mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the Su(z)12 gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. Su(z)12 was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between Su(z)12 and zeste. I also show that Su(z)12 interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3. In conclusion, results presented in this thesis show that the recently identified Pc-G gene, Su(z)12, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes

Kan internetbaserade quiz med fokus på formativ bedömning användas i naturkunskapsundervisningen för gymnasieelever?

Formativ bedömning har visats ge stora positiva effekter på lärande och rekommenderas användas i grundskolornas undervisning. Dock har det inte varit helt enkelt att implementera metoden i skolan. Metoden förutsätter ett tryggt klassrumsklimat och trygga elever, omständigheter som inte alltid råder. Något som kan försvåra ytterligare är om elever har dåligt självförtroende i ämnet, något som forskning visat att många elever har i de naturvetenskapliga ämnena. Ämnet naturkunskap innehåller en stor mängd kunskapsstoff, termer och begrepp. Ett självrättande internetbaserat test skulle kunna vara ett användbart redskap för inlärning i detta ämne. Några studier har indikerat att kamratbedömning, en av nyckelstrategierna i formativ bedömning, kan ge ökad oro och otrygghet hos elever och andra att bedömning som görs anonymt kan öka deras trygghetskänsla. Syftet med denna undersökning var därför att undersöka hur anonyma internetbaserade test upplevs av gymnasieelever och om de är användbara redskap för självbedömning i naturkunskap. Undersökningen gjordes med en enkät. Resultaten visade att en stor andel elever var positiva till att göra test anonymt och att de upplevde att testen ökade deras insikter om vad de kunde och inte, alltså indikerar denna studie att anonyma internetbaserade test kan vara positiva för elevers trygghet och för självreglerat lärande

The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo

A novel SOD1 splice site mutation associated with familial ALS revealed by SOD activity analysis

More than 145 mutations have been found in the gene CuZn-Superoxide dismutase (SOD1) in patients with amyotrophic lateral sclerosis (ALS). The vast majority are easily detected nucleotide mutations in the coding region. In a patient from a Swiss ALS family with half-normal erythrocyte SOD1 activity, exon flanking sequence analysis revealed a novel thymine to guanine mutation 7 bp upstream of exon 4 (c.240-7T<G). The results of splicing algorithm analyses were ambiguous, but five out of seven analysis tools suggested a potential novel splice site that would add six new base pairs to the mRNA. If translated, this mRNA would insert Ser and Ile between Glu78 and Arg79 in the SOD1 protein. In fibroblasts from the patient, the predicted mutant transcript and the mutant protein were both highly expressed, and despite the location of the insertion into the metal ion-binding loop IV, the SOD1 activity appeared high. In erythrocytes, which lack protein synthesis and are old compared with cultured fibroblasts, both SOD1 protein and enzymic activity was 50% of controls. Thus, the usage of the novel splice site is near 100%, and the mutant SOD1 shows the reduced stability typical of ALS-associated mutant SOD1s. The findings suggests that this novel intronic mutation is causing the disease and highlights the importance of wide exon-flanking sequencing and transcript analysis combined with erythrocyte SOD1 activity analysis in comprehensive search for SOD1 mutations in ALS. We find that there are potentially more SOD1 mutations than previously reported

Ectopic expression of Miple proteins does not activate Alk signaling.

<p>(<b>A-D</b>) Ectopic expression of Miple1 (C, C′) or Miple2 (D, D′) with <i>twist2xPE-Gal4</i> in stage 10 embryos fails to ectopically activate ERK (pERK) in Alk positive visceral mesoderm. This is in contrast to ectopic expression of Jeb which is sufficient to activate ERK (pERK) in all Alk positive cells of the visceral mesoderm (B′ compare with C′ and D′, arrowhead). (<b>E-H</b>) Ectopic expression of Miple1 (G, G′) or Miple2 (H, H′) with <i>twist2xPE-Gal4</i> fails to ectopically activate the <i>duf/kirre</i> enhancer trap <i>rP298-LacZ</i>. As observed with pERK above (B′), Jeb is sufficient to activate robust LacZ reporter expression in all Alk positive cells of the visceral mesoderm (F′ compare with G′ and H′, arrowhead). (<b>I-J</b>) Ectopic expression of Miple proteins does not affect pupal size during development. Expression of Jeb with the pan-neuronal driver (<i>C155-Gal4</i>) results in a reduction of pupal length (mm) in comparison to controls. In contrast, pan-neuronal expression of Miple1 or Miple2 does not affect pupal size. Representative pupae are shown in (I). Quantification is shown in (J), error bars denote S.E.M. (n = 40). All pupae analysed were female, confirmed by analysis of hatched adults.</p