A Segment of 97 Amino Acids within the Translocation Domain of Clostridium difficile Toxin B Is Essential for Toxicity

Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 – 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP₆), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Surveillance of the Sensitivity towards Antiparasitic Bath-Treatments in the Salmon Louse (Lepeophtheirus salmonis)

The evolution of drug resistant parasitic sea lice is of major concern to the salmon farming industry worldwide and challenges sustainable growth of this enterprise. To assess current status and development of L. salmonis sensitivity towards different pesticides used for parasite control in Norwegian salmon farming, a national surveillance programme was implemented in 2013. The programme aims to summarize data on the use of different pesticides applied to control L. salmonis and to test L. salmonis sensitivity to different pesticides in farms along the Norwegian coast. Here we analyse two years of test-data from biological assays designed to detect sensitivity-levels towards the pesticides azamethiphos and deltamethrin, both among the most common pesticides used in bath-treatments of farmed salmon in Norway in later years. The focus of the analysis is on how different variables predict the binomial outcome of the bioassay tests, being whether L. salmonis are immobilized/die or survive pesticide exposure. We found that local kernel densities of bath treatments, along with a spatial geographic index of test-farm locations, were significant predictors of the binomial outcome of the tests. Furthermore, the probability of L. salmonis being immobilized/dead after test-exposure was reduced by odds-ratios of 0.60 (95% CI: 0.42–0.86) for 2014 compared to 2013 and 0.39 (95% CI: 0.36–0.42) for low concentration compared to high concentration exposure. There were also significant but more marginal effects of parasite gender and developmental stage, and a relatively large random effect of test-farm. We conclude that the present data support an association between local intensities of bath treatments along the coast and the outcome of bioassay tests where salmon lice are exposed to azamethiphos or deltamethrin. Furthermore, there is a predictable structure of L. salmonis phenotypes along the coast in the data, characterized by high susceptibility to pesticides in the far north and far south, but low susceptibility in mid Norway. The study emphasizes the need to address local susceptibility to pesticides and the need for restrictive use of pesticides to preserve treatment efficacy.     

The Association of Gum Bleeding with Respiratory Health in a Population Based Study from Northern Europe

Background

There is little knowledge about how oral and respiratory health is interrelated even though the mucosa of the oral cavity and airways constitutes a continuum and the exposures to these are partly similar.

Aims

To investigate whether gum bleeding is related to asthma, respiratory symptoms and self-reported COPD.

Methods

A postal questionnaire including questions about respiratory and oral health was sent to general population samples in seven Northern European centres. In 13,409 responders, gum bleeding when brushing teeth was reported always/often by 4% and sometimes by 20%. Logistic regressions accounted for age, smoking, educational level, centre and gender. Effects of BMI, cardio-metabolic diseases, early life factors, gastro-oesophageal reflux, dental hygiene, nasal congestion, and asthma medication were addressed.

Results

Gum bleeding always/often was significantly associated with ≥3 asthma symptoms (OR 2.58, 95% CI 2.10–3.18), asthma (1.62 [1.23–2.14]) and self-reported COPD (2.02 [1.28–3.18]). There was a dose-response relationship between respiratory outcomes and gum bleeding frequency (≥3 symptoms: gum bleeding sometimes 1.42 [1.25–1.60], often/always 2.58 [2.10–3.18]), and there was no heterogeneity between centres (p_{heterogeneity} = 0.49). None of the investigated risk factors explained the associations. The observed associations were significantly stronger among current smokers (p_interaction = 0.004).

Conclusions

A consistent link between gum bleeding and obstructive airways disease was observed, not explained by common risk factors or metabolic factors. We speculate that oral pathogens might have unfavourable impact on the airways, and that the direct continuity of the mucosa of the oral cavity and the airways reflects a pathway that might provide novel opportunities for interventions.     

Strong Interplay between Sodium and Oxygen in Kesterite Absorbers: Complex Formation,Incorporation, and Tailoring Depth Distributions

Sodium and oxygen are prevalent impurities in kesterite solar cells. Both elements are known to strongly impact performance of the kesterite devices and can be connected to efficiency improvements seen after heat treatments. The sodium distribution in the kesterite absorber is commonly reported, whereas the oxygen distribution has received less attention. Here, a direct relationship between sodium and oxygen in kesterite absorbers is established using secondary ion mass spectrometry and explained by defect analyses within the density functional theory. The calculations reveal a binding energy of 0.76 eV between the substitutional defects Na_Cu and O_S in the nearest neighbor configuration, indicating an abundance of Na? O complexes in kesterite absorbers at relevant temperatures. Oxygen incorporation is studied by introducing isotopic ¹⁸O at different stages of the Cu₂ZnSnS₄/Mo/soda‐lime glass baseline processing. It is observed that oxygen from the Mo back contact and contaminations during the sulfurization are primary contributors to the oxygen distribution. Indeed, unintentional oxygen incorporation leads to immobilization of sodium. This results in a strong correlation between sodium and oxygen, in excellent agreement with the theoretical calculations. Consequently, oxygen availability should be monitored to optimize postdeposition heat treatments to control impurities in kesterite absorbers and ultimately, the solar cell efficiency.     

Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles

Defects in lysosomal trafficking pathways lead to decreased cell viability and are associated with progressive disorders in humans. Previously we have found that loss-of-function (LOF) mutations in the Drosophila gene blue cheese (bchs) lead to reduced adult life span, increased neuronal death, and widespread CNS degeneration that is associated with the formation of ubiquitinated-protein aggregates. To identify potential genes that participate in the bchs functional pathway, we conducted a genetic modifier screen based on alterations of an eye phenotype that arises from high-level overexpression of Bchs. We found that mutations in select autophagic and endocytic trafficking genes, defects in cytoskeletal and motor proteins, as well as mutations in the SUMO and ubiquitin signaling pathways behave as modifiers of the Bchs gain-of-function (GOF) eye phenotype. Individual mutant alleles that produced viable adults were further examined for bchs-like phenotypes. Mutations in several lysosomal trafficking genes resulted in significantly decreased adult life spans and several mutants showed changes in ubiquitinated protein profiles as young adults. This work represents a novel approach to examine the role that lysosomal transport and function have on adult viability. The genes characterized in this study have direct human homologs, suggesting that similar defects in lysosomal transport may play a role in human health and age-related processes.     

Finding a common motif of RNA sequences using genetic programming: the GeRNAMo system

We focus on finding a consensus motif of a set of homologous or functionally related RNA molecules. Recent approaches to this problem have been limited to simple motifs, require sequence alignment, and make prior assumptions concerning the data set. We use genetic programming to predict RNA consensus motifs based solely on the data set. Our system -- dubbed GeRNAMo (Genetic programming of RNA Motifs) -- predicts the most common motifs without sequence alignment and is capable of dealing with any motif size. Our program only requires the maximum number of stems in the motif, and if prior knowledge is available the user can specify other attributes of the motif (e.g., the range of the motif's minimum and maximum sizes), thereby increasing both sensitivity and speed. We describe several experiments using either ferritin iron response element (IRE); signal recognition particle (SRP); or microRNA sequences, showing that the most common motif is found repeatedly, and that our system offers substantial advantages over previous methods.     

Enhanced RNA binding of dimerized aminoglycosides.

Aminoglycoside antibiotics have recently emerged as an intriguing family of RNA binding molecules and they became leading structures for the design of novel RNA ligands. The demystification of the aminoglycoside-RNA recognition phenomenon is required for the development of superior binders. To explore the existence of multiple binding sites in a large RNA molecule, we have synthesized covalently linked symmetrical and nonsymmetrical dimeric aminoglycosides. These unnatural derivatives were compared to their natural "monomeric" counterparts in their ability to inhibit the Tetrahymena ribozyme. The dimeric aminoglycosides inhibit ribozyme function 20 to 1.2 x 10(3) fold more effectively than their natural parent compounds. The inhibition curves of dimeric aminoglycosides have characteristic shapes suggesting the presence of at least two high affinity-binding sites within the ribozyme's three-dimensional fold. The interaction of a dimeric aminoglycoside with two complementary sites of the RNA molecule is proposed. This binding motif may have implications on the development of new drugs targeting pivotal RNA molecules of bacterial and viral pathogens.     

Alpha1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter

We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive (86)Rb(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine (30 microM) increased (86)Rb(+) influx. The NKCC inhibitor bumetanide (50 microM) reduced the response to phenylephrine by 45 +/- 13% (n = 12, P < 0.01). PD-98059 (10 microM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 +/- 13% (n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that alpha(1)-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after alpha(1)-AR stimulation was shown by immunoprecipitation of the phosphoprotein from (32)P(i) prelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by alpha(1)-AR stimulation through phosphorylation of this protein involving the ERK pathway.